AU2015200950A1 - ActRII receptor polypeptides, methods and compositions - Google Patents

Abstract

In certain aspects, the present invention provides compositions and methods for modulating (promoting or inhibiting) growth of a tissue, such as bone, cartilage, muscle, fat, and/or neuron. The present invention also provides methods of screening compounds that modulate activity of an ActRII protein and/or an ActRII ligand. The compositions and methods provided herein are useful in treating diseases associated with abnormal activity of an ActRII protein and/or an ActRII ligand.

Description

ACTRII RECEPTOR POLYPEPTIDES, METHODS AND COMPOSITIONS CROSS-REFERENCE TO RELATED APPLICATIONS The present application is a divisional application of Australian Application No. 2012244215, which is incorporated in its entirety herein by reference. 5 This application claims the benefit of U.S. Provisional Application Serial No. 60/590,765, filed July 23, 2004. All the teachings of the above-referenced application is incorporated herein by reference. BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be 10 considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. The transforming growth factor-beta (TGF-beta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a large variety of cell types in both 15 vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. The family is divided into two general branches: the BMP/GDF and the 20 TGF-beta/Activin/BMPlO branches, whose members have diverse, often complementary effects. By manipulating the activity of a member of the TGF-beta family, it is often possible to cause significant physiological changes in an organism. For example, the Piedmontese and Belgian Blue cattle breeds carry a loss-of-function mutation in the GDF8 (also called myostatin) gene that causes a marked increase in muscle mass. 25 Grobet et al., Nat Genet. 1997, 17(l):71-4. Furthermore, in humans, inactive alleles of GDF8 are associated with increased muscle mass and, reportedly, exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8. Changes in muscle, bone, cartilage and other tissues may be achieved by agonizing or antagonizing signaling that is mediated by an appropriate TGF-beta family - 1 member. Thus, there is a need for agents that function as potent regulators of TGF-beta signaling. SUMMARY OF THE INVENTION In certain aspects, the present disclosure provides ActRII polypeptides. Such 5 ActRII polypeptides may be used for the treatment of a variety of disorders or conditions, in - la particular, muscle and neuromuscular disorders (eg., muscular dystrophy, amyotrophic lateral sclerosis (ALS), and muscle atrophy), undesired bone/cartilage growth, adipose tissue disorders (e.g., obesity), metabolic disorders (eg type 2 diabetes), and neurodegenerative disorders. In specific embodimentsAciRRi polypeptides (e.g.. oluble ActRs polypeptides) S can antagonize an AcmtR receptor (e g., ActRIIA or ActRIIB) generady in any process associated with ActRI activity Optionally, AcURI polypeptides of the invention may be designed to preferentially antagonize one or more ligands of ActRi receptors, such as GDF8 (also called myostatin), GDYl l, activin, Nodal, and BMP? (also called PM), and may therefore be useful in the treatment of additional disorders Examples of ActRH polypeptides 10 include the naturally occurring ActRI polypeptides as well as functional variants thereof. 1 certain aspects the disclosure provides pharmaceutical preparations comprising a soluble ActRi (e.g., ActRIA or ActRIB) polypeptid. that binds to an ActZii ligand such as GDF8, Gi.E L activin, BMP7 or nodal, and a phamaceuticalky acceptable carrier Optionally, the soluble ActRil polypeptide binds to an ActRE ligand with a Kd less than 10 15 micromolar or less than I inncromolar, 100, 10 or I nanomolar Optionally, the souble ActRil polypeptide inhibits ActRlI signaling such as intracellular signal transduction events triggered by an ActR11 ligand. A soluble ActRii polypeptide for use in such a preparation may be any of those dis closed herein, such as a polypeptide having an ammio acid sequence selected from 1EQ ID NOs: 1-2 and 912 or having an amino acid sequence that is at least 20 80%, 85%, 90%, 95%, 97% or 99% identical to an amino acid sequecwe selected fron SEQ ID NOs: 1-2 and 912 A soluble ActRII polypeptide may include a funcdonal fragment of a natural ActR polypeptide such as one comprising at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs; 14 and 942 or a sequence of SEQ ID Ns: I or 2, lacking the C-termainal 10 to 15 amino acids (the "taif A soluble ActRH polypeptide may 25 include one or aore a iterations in the amino acid sequence (eg, in the higand-binding domain) relative to a naturally occurring ActRi polypeptideT he aiteratiorin the amno acid sequence may, fbr example, alter glycosyation of the poypeptide when produced in a mammalian, insect or other eukaryotic cell or alter proteolytic cleavage of the po.ypeptide relative to the naturaly occurring ActRUI polypeptide. A soluble ActRII polypeptide nay be 30 a fusion protein t has, as one doman, an ActRl polypeptide (eg. a ligand-binding domain of an Actd!.) and one or more additional domains that provide a desirable property, such as improved pharrnacokinetics, easier purificatioMn, targeting to particuar tissues etc. -2- For example, a domain of a fusion protein may enhance one or more of i vivo stability, in vivo half life, uptake/administration, tissue localization or distribution, formation of protein complexes, multimerization of the fusion protein, and/or purification A soluble ActRHl fusion protein may include an immunoglobulin Fe domain(wild-ype or mutant) or a seum 5 aibumin in a prferred embodiment, an ActRiLFe fusion comprises a relatively unstructured linker positioned between the Fc domain and the extracellular ActRil domain. This unstructured linker may correspond to the roughly 1 5 amino acid unstuctured region at the terminal end of the extracellular domain of ActRiA or ActRUB (the "tail") or it may be an artificial sequence of between S and 15, 20, 30, 50 or more amino acids that are relatively 10 free of secondary structure, A linker may be rich in glycine and proline residues and may, for example, contain repeating sequences of threonine/serine and glycines (e gTG4 or SG4 repeats). A fusion protein may include a purification subsequence, such as an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion, Optionally, a soluble AcMiU polypeptide includes one or more modified amino acid residues selected from glycosylated 15 anno acid, a PEGyIated amino acid, a famesylated amino acid, an acetylated amino acid, a bionvlated amino acid, an amino acid conjugated to a lipid moietyand an amino acid conjugated to an organic derivatizing agent. A pharmaceutical preparation may also include one or more additional compounds such as a compound that is used to treat an ActRii associated disorder. Preferably a pharmnaceutical preparation is substantially pyrogen free 20 In general, it is preibrable that an ActRil protein be expressed in a mammalian cell line that mediates suitably natural glycosylation of the ActRIl protein so sto diminish the likelihood of an unfavorable immune response in a patient Human and CHO cell lines have been used successfully, and it is expected that other common mammalian expression vectors will be useful 25 in certain aspects, the disclosure provides packaged pharmaceuticals comprising a pharmaceutical preparation described herein and labeled for use in promoting growth of a tissue or diminishing or preventing a loss of a tissue in a human. Exemplary tissues include bone, cartilage, muscle, fat, and neuron. In certain aspects, the disclosure provides soluble ActRI polypeptides comprising an altered 30 ligand-binding (eg, GDF-binding) domain of an AcIRIt Such altered ligand-binding domains of an ActRt receptor comprise one or more mutations at amino acid residues such as E37, E39, R40, K55, RS6, Y60, A64, K74, W78, 179, D80, F82 and F101 of human -3 - ActRlIB. Such altered ligand-binding domains of an ActRUH receptor comprise one or more mutations at amino aid resides such as E38, E40 R41, K56, R57,6 K6$, KYS 79, L80, D81 183 and F102 of human ActRIIA. Optionany, the altered ligand-binding domain can have increased selectivity for a ligand such as GDF8/GDE I relative to a wild-type 5 igand-binding domain of an ActRI receptor. To illustrate, these mutations are demonstrated herein to increase he selectivity of the altered ligand-binding domain for ODF11 (and therefore, presumably OD8) over activin presentedd with respect to ActRIB), K74Y, K7F K741 and D80 The following mutations have the reverse effect, increasing the ratio of activin binding over GDFl 1: D$4A, KS$A, L79A and F82A The overall (GDF II and 10 activin) binding activity can be increased by inclusion of the tail" region or, presumably, a unstructured linked region and also by use of a mutation such as A64R (which occurs naturnily) or K74A, Other mutations that caused an overall decrease inligand binding affity, include: R40A E37A, RS6A, W78A, D80K, D80R, D80AD80G, DSOF, D80M and DSON. Mutations may be combined to achieve desired effects. For example, many of the 15 mutations that affiet the rado of GDl I:Activin binding have an overall negative effect on ligand binding, and therefore, these may be coined with mutations that generally increase ligand binding to produce an improved binding protein with ligand selectvity. Optionallythe altered 1igandbinding domain has a ratio of K, for activin binding to Kj for GDF8 binding that is at last 2, 5, 10,. or even 100 fold greater relative to the ratio for 20 the wild-type ligand-binding domain. Optionally, the altered ligand-binding domain has a ratio of lCo for inhibiting activin to IC 0 for inhibiting GDF8/GDF I that is at least 2, 5, 10, or even 100 fold greater relative to the wild-typeligand-binding domain, Optionally, the altered ligand-binding domain inhibits ODF8/ODI I with an Its at least 2, 5, 10, or even 100 tioes less than the ICs for inhibiting activin These soluble ActRIH polypeptides can be 2.5 fusion proteins that include an immunoglobulin Fc domain (either wild-ype or mutant). in certain cases, the scM.n polypeptides are antagonists i itors) of GDF8/GDF Ii In certain aspects, the disclosure provides nucleic acids encoding a solble ActRfu polypeptide, which do not encode a complete ActRil polypeptide. An isolated 30 polyrucleotide may comprise a coding sequence for a soluble ActRIl polypeptide, such as described above. For example, an isolated nucleic acid may include a seqence coding for an extracellular domain (e,g. ligand-binding domain) of an ActRII and a sequence that would -4code for part or all of the transmembrane domain and/or the cytoplasmic domain of an ActRI, but for a stop codon positioned within the transmembrane domain or the cytoplasmic domain, or positioned between the extreellular domain and the tranamembrane domain or cytoplasmic domain. For example, an isolated polynucleotide may comprise a fdulength 5 AetRR polynucleoide sequence such as SEQ ID NO 7 or 8, or a partially tmncated version, said isolated polynaucleotide further comprising a transcription termination codon at least six hundred nucleotides before the 3Merminus or otherwise positioned such that translation of the polynucleotide gives rise to an extracellular domain optionally fused, to a truncated portion of a full-ength ActRIL Nucleic acids disclosed herein may be operably linked to a 10 promoter for expressionand the disclosure provides cells transformed with such recombinant polynucleotides, Preferably the cell is a mammalian cell such as a CEI-O cell in ertain aspectsthe disclosure provides methods for making a soluble ActPi polypeptide. Sue ,a method may include expressing any of the nucleic acids (egSEQID NO: 5 or 6) disclosed herein in a suitable cell, such as a Chinese hamster ovary (CHO) cell. I 5 Such a method may comprise: a) culturing a cell under conditions suitable for expression of the soluble ActRIr polypeptide, wherein said cell is transformed with a soluble ActRIl expression construct; and b) recovering the soluble ActRE polypeptide so expressed. Soluble AciRI polypeptides may be recovered as crude, partially purified or highly purified fractions using any of the well known techniques fur obtaining protein from cell cultures, 20 In certain aspects, a soluble ActR polypeptide disclosed herein may be used in a method for treating a subject having a disorder associated with muscle loss or insufficient muscle growth Such disorders include muscle atrophy, muscular dystrophy amyotrophic later sclerosis (ALS), and a muscle wasting disorder (eg cachexia, anorexiaDMD syndrome, BMD syndrome, AIDS vasing syndrome, muscular dystrophies neumuscular 25 diseases, motor neurn diseases, diseases of the neuromuscular junction, and inflammatory myopathies). A method may comprise administering to a subject in need thereof an effective amount of a soluble ActRR polypeptide, In certain aspects,a soluble ActRil polypeptide disclosed herein may be used in, a method for treating a subject having a disorder associated vih neurodegeneration. Such 30 disorders include Alzheimer's Disease (AD) Parkinson's Disease (PD), Amyotrophic Lteral Sclerosis (ALS), Huntington's disease (RD), A method may comprise administering to a subject in need thereof an effectiveamount ofa sohible ActRil polypeptide. -5 - In certain aspects, a soluble ActRil polypeptide disclosed herein may be used in a method for treating a subject having a disorder associated with abnormal cell growth and diffrentiation. Such disorders include inflammation, allergy, autoimmune diseases. infectious diseases, and tumors. A method may comprise administering to a subject in need 5 thereof an effective amount of a soluble ActRI polypeptide. A selective activin binding ActIR proein may be particularly useful for treating an activin-dependent cancer, such as ovarian cancer, In certain aspects, a soluble ActRJ polypeptide disclosed herein may be used in a method for decreasing the body fat content or reducing the rate of increase in body fat 10 content; and for treating a disorder associated wnih undesirable body weight gain, such as obesity, nominsulin dependent diabetes mefitus (NIDDM), cardiovascular disease, cancer h"ypertension., osteoarthritis, stroke respiratory problems, and gal bladder disease. These methods may comprise admainistering to a subject in need thereof an effective amount of a soluble ActkrI polypeptide. 15 In certain specific aspects, a soluble ActRiI polypeptide disclosed herein may be used in a method for treating a disorder associated with abnormal activity of GDF8 Such disorders include metabolic disorders such as typ e 2 diabetes, impaired glucose tolerance, metab olic syndrome (eige, sndrome X), and insulin resistance induced by trauma (eig. burn or nitrogen imbalarce) adipose tissue disorders (eOg, obesity); niuscular dystrophy 20 (including Duchenme's muscular dystrophy); anyotopthic lateral sclerosis (ALS); muscle atrophy; organ atrophy; frailty; carpal tunnel syndrome; congestive obstmctive pu hnonary disease , sacopenia, cachexia and other muscle wasting syndromes; osteoporosis; glucocort icoid induced osteoporosis; osteopenia; osteoarthri tis; 0steoporosis-related fracture low bone mass due to chronic glucocorticoid therapy, premature gonadal failure, 2$ androgensuppression vitamin D deficiency secondary hyperparathytoidis.mu, ri tritiona deficiencies, and anoreia nervosa, The method may comprise administering to a subject in need thereof an effective amnount of a soluble ActRi polypeptide, In certain aspects, the disclosure provides a method for identifying an agent that stimulates growth of a tissue such as bone, cartilage, muscle, fit, and neuron. The method 30 comprises: a) identifying a test agent that binds to a ligand-binding domain of an ActRi polypeptide competitively with a soluble ActRif polypeptide; and 11) evaluating the effect of the agent on growth of the tissue. -6- In certain aspects the disclosure provides methods for antagonizing activity of an ActRI polypeptide or an ActRII ligand (eg, GDFS, GDFI l, activin BMP7, and Nodal) ina cell The methods comprise contacting the cell with a soluble ActRI polypeptide. Optionally, the activity of the ActRI polypeptide or the ActRil ligand is monitor by a 5 signaling iransduction mediated by the ActRWI/AcRil ligand complex, for example, by monitoring cell proiferation, The cells of the methods include an osteoblast, a chondrocyte, a myocyte, an adipocyte, a muscle cell, and a neuronal cell In certain aspects, the disclosure provides uses of a soluble ActRU polypeptide for making a nmedicament for the treatment of a disorder or condition as described herein 10 Brief Description of the Drawings Figure 1 shows a human ActRIIA soluble (extracellular) polypeptide sequence (SEQ ID NO: Y ) The C-terminal "tail" is undelined. Figure 2 shows a human ActRIB soluble (extracellular) polypeptide sequence (SEQ 15 ID NO 2). The C-terminal "tail" is underlined, Figure 3 shows human ActRPIA precursor protein sequence (SEQ ID NO: 3), The signal pepide is underined; the extmcellular domain is in bold (also referred to as SBQ ID NO: ); and the potential N-Iinked glycosylation sites are boxed, Figure 4 shows human ActRIIB precursor protein sequence (SEQ ID NO: 4). The 20 signal peptide is underlined; the extracellular domain is in bold (also refrnred to as SPQ ID NO); and the ptenialN-inked glycosylaion sites are boxed. Figure 5 shows a nucleic acid sequence encoding a human ActRilA solubte extracellularr) polypeptide, designed as SEQ ID NO: 5. Figure 6 shows a nucleic acid sequence encoding a human ActRITB soluble 25 (extracellu a polypeptide designed as SEQ ID NO: 6, Figure 7 shows a nucleic acid sequence encoding human ActRUIA precursor protein, designed as SEQ ID NO: 7. Figure 8 shows a nucleic acid sequence encoding huian ActRTIB precursor protein, designed as SEQ ID NO: 8 -7- Figure 9 shows expression of the extracellular (soluble) domains of ActRHIA or ActRilB. &onstrets expressing human extracellular domains of ActRIIA or ActRUB were made with all three signal sequences. Figure 10 shows three soluble ActRiB polypeptides with various signal sequences, 5 SEQ ID NOs: 9-1 Figure 11 sIhows one soluble ActRIlA polypeptide with its native sigai sequence. SEQ ID NO: 12. Figure 12 shows design of the Fe fusions of ActRUA or ActRU-B polypeptides The flexible linker sequence and the Fe sequence (SEQ ID NO: 13) are shown. Mutations can be 10 made at one more amino acid residues of the F" sequce, Examuples of such residue for mutations are underlined, and referred to as Asp265, lysine-322 and Asn434. Figure 13 shows thehgand-binding pocket of an AetRUB polypeptide Lxamples of amino acid residues in the ligand-hinding pocket are sh own as E39, K55, Y60, K74, W78 D, andY 4 11. ActRlB polypeptides of the invention may comprise mutations at one or 15 more of these amino acid residues. Figure 14 shows ar alignment of the extaceflular domains of ActiRA and ActRIB, with the positions of mutations that, in AcIRiM, are demonstrated herein to affect ligand binding. The alignment shows that the position of these mutations is conserved in ActRIA Figure 15 shows a scmatic for the A204 Reporter Gene Assay, The figuA shows 20 the Reporter vector pG,3(CAGA)12 (described in Dennier et al, 1998, EMBO 17,3091 3100,) The CAGA 12 motif is presen in TGF-Beta responsive genes ( PA-I gene),so this vector is of general use for factors signaling through Smad12 and 3, Figure 16 shows the effects of various stations in ActRIUB-Fe on a (iDF- I A-204 Reporter Gene Assay, The background A64 .onsiruct showed theleast effect on GDFI I 2$ activity. The A64K mutation (also a naturaly occurring frnn) caused a substantial increase in GDF-I ! inhibition, and a combination of the A64K mutation with the addition of the 15 C terminal amino acids of the extracelhdar domain (the 1$ amino acid "tail") produced an ev en more potent inhibitor of iDF-i I activity Figure ? Shows the effects of various mutations in ActRiIB-Fc on an Activin A, A 30 204 Reporter Gene Assay, he background A64 construct showed the least effect on Activin -8- A activity The K74A mutation caused a substantial increase in Activin A inhibition. A control sample lacking Activin A showed no activity. 5 1 vee Int~e 'ecito ftheIvetn Int certain aspects the present invention relates to ActiR polypeptides, As used herein, the term "ActRII" refers to a tmilny of activin receptor type I (ActRI) proteins and ActRII-related proteins, derived from any species. Reference to ActRII herein is understood to be a rekrence to any one of the cun-ently identified frms, including ActRUTA (also known 10 as ActRI) and ActRB. Members of the ActRif family are genendly all transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich regIon a transmembrane domain, and a cytoplasmic domain wih predicted serineithreonine kinase specifiity: Amino acid sequences of human ActRIIA precrsor protein and ActRUB precursor protein are illustrated in Figure 3 (SEQ ID NO 3) and Figure 4 (SEQ ID NO: 4), 15 respectively. The term "ActRiI polypepide" s used to refer to polypeptides compris'g any naturally occurring polypeptide of an ActRII family member as wecll as any variants hereof (including mutants, fragments, fMsionstand peptidomimetic fonns) that retain a useful activity, For example, ActRII polypeptdes include polypeptides derived from the sequence 20 of any known ActRI having a sequewe at least about 80% identical to the sequence of an ActRi polypept deand preferably at least 855%, 90% 95%, 9*7 99% or greater Identity, In a specific embodiment, the inventikn relates to soluble ActRIl polypeptides As described herein, the tern "soluble ActRJ polypeptide" generafly refers to polypeptides comprising an extracellular domain of an ActRl protein, The term "soluble ActRII 25 polypeptde; as used herein, includes any naturally occurnmg extracelhdar domain of an ActRH Protein as well as any variants thereof (including mutants. fragments and peptidomimeic forms) that retain a usefi activity. For example, the extracellular domain of an ActRIl protein binds to aligand and is generally solubbe, Examples of soluble ActRJJ polypeptides include AcetRiIA and ActRilB soluble polypeptides illustrated in Figure 1 (SEQ 30 ID NO: I and FigureS2 ISEQ ID NO: 2), respectively, Other exagles of soluble ActRU polypeptides comprise a signal sequence in addition to the extraeldular domain of an ActRI -9protein, for example, the sequences 'lhustrated in Figure 10 (SEQ ID NOs: 911) and Figure II (SEQ ID NO 12). The signal sequence can be a native signal sequence of an ActARJI Lt a signal sequence from another protein such as a tssue plasmainogen activator (TPA) signal sequence or a honey bee melatin (HBM) signal sequence, 5 T-GFl signals are mediated by eteromeric compkxes of type I and type U serin/ threonine kinase receptors, which phosphorylate and activate downstream Smad proteins upon ligand stinulation (Massaga 2000, Nat Rev. MoL Cell Biot 1:169-178), These type I and type I receptors are all transmembrane proteins, composed of a Nganinding extracelllar domain with cysteline-rich region, a transmembrane domain, and a cytopiasmic If) domain with predicted serine/threonine specificity. Type I receptors are essential for signaling: a.nd type 11 receptors are required fr binding ligatids and for expression of type I recept ors, Type I and U activin receptors form a stable complex after ligand binding, resuming in phosphorylation of type I receptors by type receptors. Two related type H receptors, ActRHA and ActRUB, have been identified as the type 15 11 receptors for activist (Mathews and Vak, 1991, Cell 65,973-982; Attisanoet al, 1992, Cell 68' 97-108), Besides activins, ActRilA and ActRRB can biochemically interact with several other TGFp-f family proteins, including BMP7, Noda), GDF, and ODEl I (Yamashita et a, 1995, Cell BioL 130:217-226; Lee and McPherron, 2001, Proc. Nat. ad. Si. 989306-9311; Yeo and Whitman 2 01, Mol Cell 7; 949-957; Oh et at. 2002, Genes Dev; 20 16:2749-54) In certain embodiments, the present invention relates to antagonizing a igand of ActRI receptors (also referred to as an ActRligand) with a subject ActRiR polypeptde (e g., a soluble ActfRu polypeptide) Thus, composions and methods of the present invention are useful for treating disorders associated with abnormal activity of one or more 25 ligands of ActRiI receptors. Exemplary ligands ot ACtRI receptors include some TGF-rl family members, such as activinNodal, DF8 GDP and BMP7. Tiesebigands of ActRI receptors are described in more detail below Activins are dimeric polypeptide growth factors and belong to the TGF-beta superfamily. There are three activins (A, B, and AB) that are homo/heterodimers of two 30 closely related fi subunits (pap 3 A, p 3 14ijB and PA, in the TGF-beta superfamiy, activins are unique and multifunctional factors that can stimulte hormone production in ovarian and - 10 placental cells support neuronal cell survival influence cenlcycle progress positively or negatively depending om cell type, and induce mesodermal differentiation at least in amphibian embryos (DePaolo et al. 1991 Proc SocEp Bioi Med. 1,98,500-512- Dyson etal 1997, Curr Rioid 84; Woodruff, 1998,Biochen Pharmacob 55:953-963), Moreover, 5 erythroid differentiation factor (EDF) isolated fion' the stimulated human monocytic leukemic cells was found to be identical to activin A (Murata et at 1988, PNAS, 85:2434), it was Iuggested that activin A acts as a natural regulator of erythropoiesis in the bone marrow In several tissues activin signaling is antagonized by its related heterodiner, inhibit, For example, during the release of follicle-stimulating hormone (FSN) from the 10 pituitary. activin promotes ES secretion and synthesis while inhibin prevents FSH secretion and synthesis, Other proteins that may regulate aetivi bioactivity and/or bind to activin include follistain (ES), follistatin-related protein (FSRP), a macroglobuhin, Gerberus, and endoglin, which are described below, Nodal proteins have functions in mesodermn and endoderm induction and formation, 15 as well as subsequent organization of axial structures such as heart and stomach in early embryogenesis, it has been demonstrated that dorsal tissue in a developing vertebrate embryo contributes predominantly tote axial stmetures of the notochord and pre-chordal plate while it recruits surrounding cAs to iorm non-axial embryonic structures, Nodal appears to signal through both type I a type 11 receptors and intracelluar etfectors known 20 as Smad proteins, Recent studies support the idea that ActRLA and AcRIUB serve as type 1 receptors for Nodal (Sakuna et al Genes Cells. 2002 7401~12). It is suggested that Nodal ligands interact with their co-factors (e.g, cripto) to activate activin type I and type 11 receptors., which phosphorylate Smad2, Nodal proteins are implicated in many events critical to the early vertebrate embryo, ncudingmesodemanaon anterior patterning, and left 25 right axis specification. Experimental evidence has demonstrated that Nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and 'G-Fbeta. However. Nodal is unable to induce p.lx2-tux, a reporter specifically responsive to bone morphogenetic proteins. Recent results provide direct biochemicaI evidence that Nodal signaling is mediated by both activi-n-TGF-beta pathway Smads, Smad2 30 and Smad3. Further evidence has shown that the extacellular cripto protein is required for Nodal signaling, making it distinct from activin or TOF-beta signaling, - 11 - Growth and Differentiation Factor-8 (GD8) is also known as mryostatin GDFS is a negative regulator of skeletal muscle mass. GDF8 is highly expressed in the developing and adult skeletal muscle. The GDF8 null mutation in transgenic mice is characterized by a marked hypertrophy and hyperplasia ofthe skeletal muscle (McPherron ct al., Nature, 1997, 5 387:83-90), Similar increases in skeletalnuscle mass are evident in naturally occurring mutations of ODES in cattle (Ashmore et at1974. Growth,8:501-507; Swatland and Kieffer, J. Anim Sci. 1994, 3852-757; McPherrou and Lee. Proc Natd Acad Sci. USA, 1997, 94:12457-12461; and Kambadur et al, Genoine Res, 1997, 7:910-915) and strikingly. in humans (Schuelke at, N Engl Med 2004;350:2682-8). Studies have also show that 10 muscle wasting associated with HfViufection in humans is accompanied by increases in ODFS protein expression (OonzaleCadavid et al PINAS 9 95: i 4938-43), In addition, GDFS can modulatu the production of muscle-specific enzymes (e.g, creatine kinase) and modulate myobhast cell proliferation ( WO 00/43781). The GDFS propeptide can noncovalently hind to the mature GODF domain dimerinactvating its biological activity 15 (Miyazono et at ( 988) RJot Chem., 263: 64074,415; Wakefield et at (1988I J. bloL Chem., 263; 764647654; and Brown et al. (1990) Growth Factors 3: 35-43). Other proteins which bind to GD8 or structurally related proteas and inhibit their biological activity include flistain' and potentially; foistatin-related proteins (Gamer et at (1999) Dv. Biot, 208' 222-232), 20 Growth and Differentiation Factor-Il (GDI 1), also known as BMP1 1, is a secreted protein (McPherron et aL, 1999, Nat. eet 22:260-264) GDF 11 is expressed in the 'al bud limb bud, maxillay and mandibular arches, and dorsal root ganglia during mouse development (Nakashima et at, 1999; Mech, Dv 80; 185 9. ODIF I plays a uniqe role in patterning both mesoderalru and neural tissues (Gamer et al., 1999, Dev BioL 208:222 25 32), ODl I was shown to be a negative regulator of chondmrtgenesis and myogenesis in developing chick limb (Gamer et al., 2001, Dev iot 2291407-20) The expression of GDF1 I in muscle als suggest its role in regulating muscle growth in a similar way to DFS. In addition, the expression of GDM I in brain sugeests that ODF may- also possess activities that relate to the fAution of the nervous system Interestingly, OP1 I was found 30 to inhibit neurogenesis in the olfactory epithelium (Wu et al, 2003, Neuron 37:197n207): Hence, GDF I may have in vitro and in vivo applications in the treatment of diseases such as muscle diseases and neurodegenerative diseases (eg. amyotrophic lateral sclerosis). - 12 - Bone morphogeneic protein (BMP7), also called osteogenic protein I (OP- I) is well known to induce cartilage and bone formation, In addition, BIEMP7 regulates a wide atray of physiological processes, For example, B3MP7 may be the osteoinductive factor responsible for the phenomenon of ep elial osteogenesis, t is also fund that BMP plays a role in 5 calcium regulation and bone homeostasis. Like activin, BMP7 binds to type 1 receptors; ActRIIA and FIB. However, BMP7 and activin recnhit distinct type I receptors into heteromeric receptor complexes The major BMPtype I receptor observed was ALK2 while activin bound exclusively to ALK4 (ActRlB BMP7 and activin elicited distinct biolkgical responses and activated different Smad pathways (Macias-Silva et a1 1998,JIii 1,0 Chem, 2725628',36), In certain aspects, the present invention relates to the use of certain ActRII polypeptides (e.g soluble ActRIT polypeptides) to antagonize ActRU receptors general, in any process associated with ActRl a c tiv, Optionally, ActRi polypepdes of the invention may antagonize one or more ligands of ActRIl receptors, such as activin, Nodal, GDF8, 15 GDF I and BMP7, and may therefore be useful in the treatment of additional disorders Therefore, the present invention contemplates using ActRUl polypeptides in treating or preventag diseases or conditions that are associated with abnormal activity of an ActRIl or an ActRI ligand. ActRUl or ActRil ligand are involved in the regulation of many critical biological processes, Due to their key functions in these processes, they may be desirable 20 targets for therapeutic intervention, For example, ActR1 polypeptides (e g, e .g, soluble AcRII polypeptides) may be used to treat human or animal disorders or conditions- Example of such disorders or conditions include, but are not limited to, metabolic disorers such as type 2 diabetes,nripaired glucose tolerance, metabolic syndrome (e.g, syndrome X and insulin resistance induced by trauma (e gj burns or nitrogen imbalance); adipose tissue 25 disorders (eig, obesity); muscle and neuromuscular disorders such as muscular dystrophy (including Duchenne's muscular dystrophy); amyotrophic lateral sclerosis (ALS) muscle atrophy; organ atrophy; fLraiity; carpal tunnel syndrome; congestive obstructive pulmonary disease; and sarcoperuiacachexia and other muscle wasting syndromes. Other examples include osteoporosis, especially in the elderly and/or postnenopausal wonen; glucocorticoid 30 induced osteoporosis osteopenia;osteoarthritis;and osteporosis-reated fracturs Yet further examples include low bone mass due tohronic glucocorticoid therapy, premature gonadal faihare androgen suppression vitamin deficiency, secondary hyperparathyroidism, - 13 nutritional deficiencies, and anorexia nervosa. These disorders and condition are discussed below under emplary Therapeutic Uses" The terms used in this speciicaion generally have their ordinary meanings in the art, within the context of this inventon and in the specific context where each term is used, 5 Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them, The scope or meaning of any use of a term will he apparent from the specific context in which the term is used. "About" and "approxmateiy" shall generally nean an acceptable degree of error for 10 the quantity measured given the nature or precision of the measurements. Typically. exemplary degrees of error are within 20 percent (%) preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systemsthe terms "about" and " approximateIy" may mean values that are within al order of magnitude, preferably within 5 15 fold and more preferably within 2-fold of a gi ev-aIe. Numerical quartities given her are approximate unless stated otherwise, meaning th.at the term "about" or approximately" can be infe md when not expressly stated, The methods of the invention may include steps of comparing sequences to each other, includig wild-type sequence to one or more mutants (sequence variants). Such 20 comparisons typically comprise aiigmnnents of polymer sequences, e g.using sequence alignment programs andor algorithms that are well known in the art (for example. BLAST, FASTA and MEGALGN, to name a few). The skilled artsan can readily appreciate that, in such alignments, where a mutation contains a residue insernon or deltion, the sequence ig-mnent will introduce a "gap" (typicaiy represented by a dash, or "A") in the polymer 25 sequence not containing the inserted or deleted residue, "Homologous" in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a "common evolutionary orign tchiding proteins from superfHmilies in the sante species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nuclei aids) have 30 sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or mxotifs and conserved positions, - 14 - The term "sequence similarity" in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin, However in common usage and in the instant application, the term "'homologous" 5 when modified with an adverb such as "highly," may refer to sequence similarity and may or may not relate to a common evolutionary origin 2, Actilj Polypetide In certain aspects, the invntion relates to Ac-tRll polypeptides (e g soluble ActII 10 polypeptides), Preferably, the fragments, fmctional varian s and modified forms have similar or the same biological activities of their corresponding wild-type ActRII polypeptides. For example, an ActRrpolypeptide of' the invenion may bind to and inhbit function of an ActRiI protein and/or art ActRli ligand protein (ag actiin Nodal GEF8 GDEI I or BhMP7). Optionally an ActRH1 polypeptide modulates growth of tissues such as bone, 15 cartilage, musc 1, fiat and/or neuron, Examplek of ActRTi polypeptides include human ActRIA precursor polypeptide (SEQ ID NO: 3),human ActRIB precursor polypeptide (SEQ I D NO: 4) soluble human ActRIA polypeptides (e g, SEQ ID NOs: I and 12), soluble human ActRIlB polypeptides (eg., SEQ ID NOs: 2 and 9-11), In certain embodiments, isolated fragments of the ActR: polypeptides can be 20 obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding an ActRil polypeptide (eng, one of SEQ ID NOs 1-2 and 9-12), In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrield solid phase f-Moc or t-Boc chemistry, The fragments can be produced (recombinanty or by chemical synthesis) and tested to identify those 25 peptidyl fragments that can function, for example, as antagonists (inhibitors) or agonists (activators) of an ActRIl protein or an ActRil ligand. in certain embodimens, a fanctional variant of the ActRII polypeptides hasan amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 1 -2 and 9'2. In certain aes, the functional variant has an amino acid sequence at 30 least 80% 85%,90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1-2 and 9-12 - 15 - In certain embodiments, the present invention contemplates making functional variants by modifying the structure of an ActRil polypeptide for such purposes as enhancing therapeutic efficacy, or stabilty (egex viv shelf life and resistance to proteolytic degradation in vivo). Such modified ActRSl polypeptides when designed to retain at least 5 one activity of the naturally-occurring form of the ActRil polypeptides. are considered function equivalents of the natural-occurring ActRIH polypeptides. Modified ActRIl. polypeptides can also he produced, for instance,/by amino acid substitution, deletion, or addition For instance, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with glutanatfe, a threonine with a serne, or a 10 similar replacement of amino acid with a stnrucraly related amino acid (e.g conservative mutations) will not have a major effect on the biological acnvity of the resulting molecule, Conservative replacements are those that take place within a family of amino acids that are related in their side chains, Whether a change in the amino acid sequence of an ActRIH polypeptide results in a functional homolog can be readily determined by assessing the ability 15 of the variant ActRil polypeptide to produce a response in cells in a fashion similar to the wild{ype ActRIl polypeptide, In certain specific embodiments, the present invention contemplates making mutations in the extracehulaT domain (also referred to as ligand-binding domain) of an ActRil polypeptide such that the variant (or mutant) ActRl polypeptide has altered ligand 20 binding activities (e.g., binding affinity or hindirng specificity). In certain cases, such variant ActRU polypeptides have altered (elevated or reduced) binding affinity for a specific ligand. In other case, the variant AcRUT polypeptides have altered binding specificity for their ligands. For example, the variant ActII polypeptide preferentialy binds to a specific li'gand 2S (e GD F 8). For example, amino acid residues of the ActRlB protein, such as E39, K55, Y60, K74, W78, D850, and F10 (shown 'n figure 13), are in the igand-binding pocket and mediate binding to its ligands such as activin and GDFS Thus, the present inventicOn provides an altered ligand-binding domain (e g., GDFS-binding don-ain) of an ActRI receptor, which comprises one or more mutations at those amino acid residues, Optionally, 30 the altered ligandt>inding domain can have increased selectivity for a ligand such as GDS relative to a wild-type ligand binding domain of an ActRi receptor To illustrate, these mutations increase the selectivity of the altered ligand-binding domain for GDFS over - 16 activin. Optionaly, the altered ligand-binding domain has a ratio of Kj for acvin binding to Ks for GDF8 binding that is at least 2, 5, 10, or even 100 fold greater relative to the ratio for the wildtype ligand-binding domain, Optionally, the altered ligand-binding domain has a ratio of IC, for inhibiting activin to C for inhibiting GDF8 that is at least 2, 5, 10, or even 5 100 fold greater relative to the wildtype ligand-tinding domain Optionally, the altered igantdibinding domain inhibits GDES with an iCso atleast 2/5. 10, or even 100 timeless than the iCs for inhibiting activin As an specific example, the positively-charged amino acid residue Asp (D80) of the ligand binding domain of ActRIB can be mutated to a different amino acid residue such that 10 the variant ActRJI polypeptide preferentially binds to GDF8, but not activin Preferably, the D60 residue is changed to an amino acid residue selected front the group consisting of a uncharged amino acid residue, a negative amin acid residueand a hydrophobic amino acid residue. As will be recognized by one of skill in the art, most of the described mutations. vaiants or modifications may be made at the nucleic acid level or, in some cases, by post 15 translational modification o r chemical synthesis, Such techniques re well known in. the art i certain embodiments. Lhe present invention contemplates specific mutations of the ActRil polypeptides so as to ater the glycosylation of the polypeptide Exemplary glycosylation sites in ActdIlA and ActRIIB polypeptides are iilustrate.d in Figures 3 and 4 respectively. Such mutations may be selected so as to introduce or eliminate one or more 20 glycosylation sites, such as Olinked or Nlinked glycosylation sites. Aparaginelinked glycosylatior recognition sites generally comprise a tripeptidc sequence, asparagine~-X. threoninIe (where "X" is any amiri acidqohich is specifically recognied by appropriate cellular gycosylation enzymes. Tlhe alteration may also be made by the addition oi or substitution by. one or more scene or threonine residues to the sequence of the wild-type 25 ActRII polypeptide (for 0-inked glycosylation sites). A variety of amino acid substitutions or deletions at one or both of the first or third amno acid positions of a glycosylation recognition ite (and/or amino acid deletion at the second position) results in non glycosylation at the modified tripeptide sequence. Another means or increasing the number of carbohydrate moieties on an ActRII polypeptide is by chemical or enzymatic coupling of 30 glycosides to the ActRil polypeptide Depending on the coupling mode used, the sugars) may be attach ed to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryI groups such as those of cysteine; (d) free hydroxyl groups such as those of shrine, threonine, - 17 or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f the araide group of glutamine. These methods are described in VO 87/05330 published Sep, I, 1987, and in Aplin and Wriston (981 CRC Crit Rev Biochen,, pp 259-306, incorporated by refetece Ierein. Removal of one or more 5 carbon ydrate moieties present on an ActRI polypepride may be accomplished chemically and/or enzymatically Chemical deglycosylation may involve, for example, exposure of the ActRHl polypeptide to the compound trifiuoromiethanesuifordc acid, or arn eqivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acety ghcosamine or Nwacetylgalactosamine)vile leavig the amino acid 10 sequence intact. Chemical deglycosylation is further described by Hakimaddin et al. (1987) Arch, B iochen. Bioph ys. 25932 and by Edge et at (1981) Anal, Biochem 118:131. Enzymatic cleavage of carbohydrate moieties on ActRil polypeptides can he achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et at (1987) Meth Enzymoi 138: 350. The sequence of ai ActRi1 polypeptide may be adjusted, as appropriate, 15 depending on the type of expression system usedas mammalian, yeast, insect and plant cells may all introduce differing glycosylation pattens that can be affected by the amino acid sequence of the peptide, In general ActRiI proteins -fo use in humans will be expressed in a mamm-alian cell line that provides proper glycosylation, such as HEK293 or CHO cel lines, although other mammalian expression cell lines are expected to be usefias well 20 This discosure further contemplates a method of generatng mutants, particularly sets of combinatorial mutants of an ActRH polypeptide, as well a- truncation mutantspools of combinatorial mutants are especially usefid for identifying functional variant sequences. The purpose of screening such combinatorial libraries may be to generate, fBm example ActRil polypeptide variants which can act as either agonists or antagonist, or altenatielyv which 25 possess novel activities adl together A variety of screening assays are provided below and such assaystnay be used to evaluate variants. For example, an ActRI polypeptide Variant may be screened for ability t bind to an AczRf polypeptide, to prevent binding of an AeRUI ligand to an ActRl polypxptide. The activity of an ActRIl polypeptide or its variants may also be tested in a cell-based 30 or in vivo assay, For example, the effect of an ActRit polypeptide variant on the expression of genes involved in bone production in an osteoblast or precursor may be assessed This may, as needed, be performed in the presence of one or more recombinant ActRH1 ligand - 18 protein (eg, BMP7h and cells may be transfected so as to produce an ActRfl polypeptide and/or variants thereof and optionally, an ActRil ligand. Likewise, an ActRil polypeptide may be admrinisterd to a mouse or other animal, and one or more bone properties, such as density or volume may be assessed, The healing rate for bone fractures may also be 5 evaluated, Similarly, the activity of an ActRIi polypeptide or its variants may be tested. in muscle cells, adipocytes, ad neuron cels for any effect on growth of these cells fbr example, by the assays as described below, Such assays are well known and routine in the art, CombinatorialIy-derived variants can be generated Which have a selective potency 10 relative to a naturally ccurrmng ActR>I polypeptide, Such variant proteins, when expressed from recombinant DNA constructs, can be used in gene therapy protocols. Likewise mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding a wild-type AcdR polypeptide, For example, the altered protein can be rendered either more tab less stble to proteolytic degradation or other cellular 15 processes which result in destution of, or othenwise ntactivation of a native ActRIl. polypeptide. Such variants and the genes which eneode them, can be utlized to alter ActRll polypeptide levels by rodulang the hailite of the ActRIl polypeptides. For instance, a short haif life can give rise to more transient biological effects and, when pan of an inducible expression system, can allow tighter control of recombinant ActRIl polypeptide levels within 20 the cell. in a preferred embodimentthe combinatoral library is produced by way of a degenerate library of genes encoding a librwry of polypeptides which each include at least a portion potential ActRil polypeptide sequences For instance, a mixture of synthetic oligonucleotides can be enzymaticaly ligated into gene sequences such that the degenerate 25 set of potential ActR11 polypeptide nocleotde sequences are expressible as individual polypeptidesor atenatively, as a set of larger fusion pro t eins (c g fir phage displays There are many ways by which the library of potential homologs can be generated rinm a degenerate oligonucleotide sequence Chemicalsynbthesis of a degenerate geei sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be 30 ligated into an appropriate vector for expression The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, &A (1983) Tetrahedron 39; Itakura et al, (1981) Recombinant ON A, Proc 3rd Cleveland Sympos. - 19 - Macromolecules, eed, AG Walton, Amsterdam: Elsevier pp273-289; Itakura e a,(1984) Ann. Rev Biochen. 53:323; Itakura etal. (1984) Science 198:1056; Ike etat (1983) Nucleic Acid Res. 11:477). Such techniques have been employed in the directed evolution of other proteins (see, ft example, Scott et al., (1990) Science 249:386390; Roberts et al, $ (1992) PNAS USA 89%429-2433; Devlin et aL (990) Science 249: 4t4-06; Cwirla etat, (1990) PNAS USA 87: 6378-6382; as well as US Patent Nos: 5,223,409, 5198,346, and 5.096,815). Alternatively, other forms of mutiagenesis can be utilized to generate a conmbinatorial library. For example' ActRif polypeptide variants (both agonist and antagonist forms) can be 10 generated and isolated from a library by screening using, for example, alanine scanning mutageresis and the like (Ruf et al, (1994) Biochemistry 33:15672; WangetaL (1994) S, Biod, Chemn. 269:3095-3099; Balint eta, (1993) Gene 137:109- 18; Grodbergeta, (1993) Eur Biochem. 218:97461; Nagashima et at. (1993) J, iat Cheat 26:288 2892; Lowman et al. (1991) Biochemistry 30:1083210838; and Cnnningham eal (1989) 15 Science 24410814085), by linker scanning mutagenesis (Gustin et al, (993) Virology 193:653-660; Brown etal., (1992) Molt Cell Biot 12:2644-2652; McKnight et a, (1982) Science 232:316); by saturation mutagenesis (Meyers et al. (1986) Science 232:613); by PCR mutagenesis (Ieung et al, (1989) Method Cell Mol Diol 1_1 149); or by random mu'tagenesis, including chemical mutagenesis, etc, (Miller et al, (1992) A Short Course in 20 Bacterial Genetics, CSHL Press, Cold Spring Harbor NTY and Greener et aL, (1994) Strategies in Mdo Biol 7:32-34), Linker scanning mutagenesis, particularly in a combinatorial setting, is an attractive method for identifying trucated (bioactive) frms of ActRU polypeptides. in a specific embodiment, snilar methods can be used for making soluble fomrs of ActRil polypeptides. which can act as agonists or antagonists of ActRH1 fimctions. 25 A wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and tunmcations, and, for that matter fhr screening cDNA libraries fhr gene products having a certain property, Such techniques vill be generally adaptable fr raplid screening of the gene libraries generated by the combinatorial mutagen of AcIi polypeptides. The most widely used techniques for 30 screening large gene libraries typically comprises ekOning the gene library into replicable expression vectors, transfoning app ropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in wich detection of a desired activity - 20 facilitates relatively easy isolation of the vector encoding the gene whose product was detected, Each of the illustrative assays deserved below are amenable to high through-put analysis as necessary to screen large numbers of degenerate sequences created by combinatorial niutagenesis techniques, 5 In certain enbodiments, the ActRil polypeptides of the present invention include pepddomimetics. As used herein, the term "peptidomnietic" includes chemically modified peptides and peptidolike molecules that contain nom-naturally occurring anmino acids, pepisids, and tie liko Peptidomimeics provide various advantages over a peptide including enhanced stability when admrinistered to a subject Methods tbr identifying a peptidonnietic 10 are well known in the art and include the scrmening of databases that contain libraries of potential peptidomhnetics, For example, the Cambridge Structural Database contains a collection of greater than 300,000 compoids that have known crystal structures (Allen et aL, Acta Crystallogr. Section B, 35231 (19?9)) Whev no crystal strucaire of a target molecule is available, a structure can be generated using, for example, the program 1 5 CONCORD (Rusinko et at, I Chem inf Comput Sci. 29:251 (1989)) Another database, the Available Chemicals Directory (Molecular Design Limited, Informimos Systems; San Leandro Calif, contains about 100,0 compounds that are comnmircaly available and also can be searched to identiy potential peptidominnetics of the ActRJI polypoeptides. To illustrate, by employing scanning mutagenesis to map the anmino acid residues of 20 an ActRI polypeptide which are involved in binding to another protein, peptidomimetic compounds can be genertd which mnnc those rsidues involved in binding. For instance, nomhydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e, see Freidinger eI a, in Peptides: Chemisty and Biolog, G R Marshal td, ESCOM Publisher: Leiden Netherlands, 1988), azepine (e,g.. see I-uffman et al in Peptides; 25 Chenistry and Biology, G Marshall ed,, ESCOM Puhisher: Leiden Netherlands, 1988), substituted gammna lactam rings (Garvey et a in Peptides;Chemistry and Biology R R Marshall td., ESCOM Publisher: Leiden Nethedands; 1988), keto-methylene pseudopeptides (Ewenson et aL, (1986) . Med. Chem 29:295; and Ewenson et al in Pepddes: Structure and Function (Proceedings of the 9th American Peptide Synposium) Pierce Chemical Co, 30 Rockland, IL, 1985), b-tum dipeptide core. (Nagai et aL (1985) Tetrahedron Lett 26647; and Sa to et at (1986) J Chem Soc Perkin Trans 1,12 31, and b-arninoalcohols (3ordon et aL, - 21 - (1985) Biochem Biophys Res Commun 126;419; and Damn et al,986) Bochem Biophys Res Commuri 134,1) In certain embodimentsthe ActRil polypeptides of the invention may funter comprise pos4translational modifications in addition to any that are naturally present in the 5 ActRlF polypeptides Such modifcations include, but are not limited tu, acetylation, carboxylation, glycosylation. phosphorylaion, lipidation, and acylation, As a result the modified ActRlH polypeptides may contain nonwamino acid elements, such as polyethylene gycols lipids, poly or mono-saccharide, and phosphates. Effects of such non-amin acid dements on the functionality of a ActRHiT polypeptide may be tested as descrbed herein for 10 other ActRil polypeptide variants. When an ActRil polypeptide is produced in cells by cleaving a nascent frn of the ActRIl polypeptide, post-translational processing rmay also be important for correct folding andlor function of the protein, Different cells (such as CHO, HeLa, MDCK, 293 WZ8, NIH3T3 or HEK293) have specific cellular machinery and characteristic mechanisms for such posttranslational activists and may be chosen to ensure 15 the correct modification and processing of the ActRll polypeptides, In certain aspects, functional variants or modified forms of the ActRid polypeptides include fusion proteins having at least a portion of the ActRI polypeptidcs and one or more fusion domains, Well known examples of such fusion domains include, but are not limited to, poiyhistidine, alu, glutatione S transfeuse (GST), thioredoxin protein Aprotein G, 20 an inmmunogobulin h chain constant region (Fc maltose binding protein (MB). or human serum albumin, A fusion domain may be selected so as to confer a desired property, For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. For the pupose of affinity purification relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- orcobalt- conjugated 25 resins are used, Many of such matrices are available in kit" forim, such as the Phamnacia GST purification system and the QiAexpress' system (Qiagen) useful with (HIst) fusion partners. As another example, a fusion domain may be selected so as to facilitate detection of the ActRiI polypepides. Examples of such detection domains include the various fluorescent proteins (e.gGFP) as well as "epitope tagswhich are usualy hort peptide sequences for 30 which a specific antibody is available Well known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virais haemagglutinin (HA), and c-myc tags, In some cases, the fusion domains have a protease cleavage site such - 22 as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated pmoteins can ten be isolated from the fusion domain by subsequent chromatographic separation. in certain preferred embodiments.an ActRil polypeptide is fused with a domain that stabilizes 5 the ActRHi polypeptide in vivo (a.stabiizer domain) By "stabilizing" is meant anything that increases serum half ife regardless of whe ter this is because of decreased destution, decreased clearance bythe kidney, or other pharmacokdnetic effbet, Fusions with the Fe portion of an inrnunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins, Likewise., fusons to human serum albumin can confer desirable 10 properties. Other types of fusion domains that may be selected include mudtimerizing (e g. dimeris:ing, tetramerizing) domains and functional domains (that confer an additional biological function, such as further stimulation of muscle growth) As a specific example the present invention provides a fusion protein as a GDFS antagonist which comprises an extracellular (e gGDFCh.binding) domain fused to an Fo 15 domain (e g. SEQ ID NO, I3 THiTC P Pd APELLc;GPSVFLFP PKPKDTLM:. IaPEreVTOCXV (A} VSNHEIJ VKFtWVDG VVhAKTKPREEQYNS TYRVVSVET VIHl UWLNTGIKEYKCK (N)VSN KAL PVPKI K S KAK GQPRE PQVYTLPE SRE MTKN QVS LTCL VKtGFY PSD AVE WESNGQPE&N NLS GTPPL D _3 PYFLYSK>LAVDKSR QQGN S CSVMEAN A I qKSLSS PGK* 20 Preferably the Fe domain has one more mutationis at residues such as Asp-265, lysine 322, and Asn-434 (see figure 12). In certain cases, the mutant Fe domain having one or more of these mutations (e.g Asp'265 mutation) has reduced ability of binding to the Fcy receptor relative to a widtype k domainm in other cases the nmtant E domain having one or more of these mutaions {e g, As-34 mutation) has increased ility binding to the 2$ MHC class I-related Fcreceptor (FcRN) relative to a wilttype Fe domain. it is understood that different elements of the fusion proteins may be arranged in any manner that is consistent withe desired functionality, For example, an ActRil polypeptide may be placed C-termiral to a heterologous domain, or, alternatively, a heterologous domain may be placed Cterminial to an AciRII polypeptide. The ActRi polypeptide domain and the 30 heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains - 23 - In certain emboditnents, the ActRi polypeptides of the present inventon contain one or more modifications that are capable of stabilizing the ActRI polypeptides For example, such modifications enhance the in vitro half life of the ActRU polypeptides, enhance circulatory half life of the ActRlI polypeptides or reducing proteolytic degradation. of the 5 AcIRI polypepides Such stabilizing modifications include., but are not ignited to, fusion proteins including for example" fusion proteins comprising an ActRil polypeptide and a stabilizer domain) modifications of a glycosylation site (including, for example, addition of a glycosylation site to an ActRH poypeptide), and nodifications of carbohydrate moiety (inchiding, for examnpie remiroval of carbohydrate mnojettes trin an AetRil polypeptide). Ir 10 the case of fusion proteins an ActII polypeptide is fused to a stabilizer domain such as an IgG molecule (eg an Fc domain), As used herein, the term "stabilizer domain" not only refers to a fusion domain (e g Fc as in the case of fusion proteins, but also includes nonproteinaceous modifications such as a carbohydrate noicty, or nonproteinaceous polymer. such as polyethylene glycoL. 1$ In certain embodiinents, the present invenion makes available isolated and/or purified forms of the ActRRI polypeptides, which are isolated from, or otherwise substantially free of, other proteins. In certainembodiments, ActRil polypeptides (unmodified or modified) of the invention can be produced by a variety of art-known techniques, For exaniple, such ActRil 20 polypeptides can be snthesized using standard protein chemistry techniques such as those described in Bodansky, Principles of Peptide Synthesis; Springer Verlag, Berli (i993) and Grant G A, (pd), Synthetic Peptides: A U sers Guide W 1 Freeman and Company, New York (1992). In addition, automated peptide synthewizers are commercially available (e.g., Advanced CherTech Model 396; Miigen/Biosearch 9600) Altemativel the ActRi 25 polypeptides, fragniC.as or variants thereof may be recombinantly produced using various expression systems (Eg, E; colt Chinese RIster Ovary cells, COS cells, bacalovirus) as is well known in the art (also see below In a further embodiment, the modified or unmodified ActRIl polypeptides may be produced by digestion of naturally occuring or recombinantly produced fulllength ActRIl polypeptides by using, for example, a protease, e g., trypsin, 30 thermolysin, cbymotrypsn, pepsin, or paired basic amino acid converting enzyne (PACE). Computer analysis (usig a commercially avaiLable software, e~g., MacVector, Omega, PCGene, Molecular Simulation, Inc,} can be used to identify proteolytic ceavage sites - 24 - Alternatively, such Actil polypeptides may be produced from naturally occurring or tecombnantiy produced full-Iength Act po peptides such as standard techniques known in the at such as by chemical cleavage (e.g., cyanogen honiide; hydroxylanine> ' , Nucleic Acids Encodinv ActRj Polypides In certain aspects, the invention provides isolated and/or recobinint nucleic acids encoding any of the AcuRi polypeptides (e.g soluble AtRU poulypeptides), including fragments functional variants and fusion proeins discosed herein. For example, SEQ ID NOs: 7-8 encode naturally occurring ActRI precursor polypeptides, while SEQD NOsD 5-6 10 encode soluble ActRi poiypeptdes. The subject nucleic acids may be singlestranded or double stranded, Such nucleic acids mnay be DNA or RNA molecules, These nucleic acids are may be used, for example, in methods for making ActRu polypeptides or as direct therapeutic agents (eig, in a gene therapy approach) In certain aspects, the subject nuclec acids encoding ActRi polypeptides are further 15 understood to include nucleic acids that are variants of SEQ ID NO: 7 or 8, Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions such as allelic variants, and ill therefore, include coding sequences that differ from the nucleodde sequence of the coding sequence designated in SEQ ID NO: 7 or a. 20 in certain embodiments, the invenon provides isolated or recombinant nucleic acid sequences that are atleast 80% 85%, 90% 95% 97%; 98%, 99% or 100% identical to SEQ ID NO: 5 or 6. One of ordinary skill in the art will appreciate that nucleic acid sequences complementary to SEQ ID NO: 5 or 6, and variants of SEQ ID NO: 5 or 6 are also within the scope of this invention In further enbodimenrs, the nucleic acid sequences of the invendon 25 can be isolated, recombinant and/or fused with a heterologous nucleotide sequence, or in a DNA library In other emrbodments, nucleic acids of the invention also include nucleotide sequences uhat hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ ID NO: 5 or 6, complement sequence of SEQ ID NO: 5 or 6, or fragments 30 thereof As discussed above, one of ordinary skill in the art will understand readily that appropriate stringency conditions which promote DNA hybridization can be varied, One of - 25 ordinary skill in the art will understand readily tat appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perfonn the hybridization at 6:0 x sodium chloride/sodium citnate (SSC) at about 45 C, followed by a wash of'2.0 x SSC at 50 IC. For example, the salt concentration irI the wash step can be 5 selected from a low stringency of about 2.0 x SSC at 50'C to a high stringency of about 0.2 x SSC at 50 "C In addition, the temperature in the wash step can be increased horn low strngency conditions at room temperature about 22 *C to high stringency conditions at about 65 *C( Both temperature and salt may be: varied, or temperature or salt concentration may be held constant while the other variable is changed In one enibodiment, the invention 10 provides nuceie acids which hybridize under low stringency conditions of 6 x SSC at room temperature fobowed by a wash at 2 a SSC at room temperature Isolated nuclei acids vhich differ from the nucleic acids as set forth in SEQ ID NOs ,-6 due to degeneracy in the genetic code are also within the scope of the invention, For example, a number of amino acids are designated by more than one triplt Codons that 15 specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent mutations which do not affect the amino acid sequence of the protein, However, it is expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject proteins will exist among maninalian cells, One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 20 3-5% of the nucleotides) of the nucleic acids encoding a particular protein may exist among individuals of a given species due to natural alelic variation, Any and all such nucleotide variations and resulting amino acid polymorphisrms ame within the scope of this invention. In certain embodimnents, the recombinant nucleic acids of the invention may be operably linked to one or more regulatory nucleotide sequences in an expression construct 25 Regulatory nucleotide sequences wil generally be appropriate to the host cell used for expression, Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typicallyssaid one or mtore regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional, start and termination 30 sequences, translational start and termination sequences, and enhancer or activator sequences Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that - 26 combine elemtents of mow than one promoter, An expression construct may be present in a cell on an episode, such as a plasmid, or the expression constnuct may be inserted in a chrorrosome. In a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well $ known in the art and will vary with the host cell used. in certain aspects of the invention the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding an ActRIl polypeptide and operably linked to at least one regulatory sequence Regulatory sequences are art-recogpized and are selected to diect expression of the ActRil polypeptide Acc&rdingly, the term 10 regulatory sequence includes promotes, enhancers, and other expression control elements. Exemplary regulatory sequences are described in G3oeddel, Gene Expression Technology: Methds in tynolohgy, Acadein Press,San Diego CA (1990'. For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence wben operatively linked to it may be used in these vectors to express DNA sequences encoding an 15 ActRII polypeptide. Such useful expression control sequencesinclude, for examplethe early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus inunediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RN A polymerase, the major operator and promoter regions of phage lambda , the control regions for fd coat protein, the promoter for 20 3-phosphoglycerate kinase or other glycoiytic enzymes, the promoters of acid phosphatasa eyg., Pho, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cels or their viruses and various combinations thereof, It should be understood that the design oft he expression vector may depend on such factors as the 25 choice of the host cell to be transformed and/or the type of protein desired to be expressed Moreover, the vector's copy number, the ability to control that copy nmnber and the expression of any other protein encoded by the vector, such as antibiotic markers, should also he considered. A recombinant nucleic acid of the invention can be produced by lighting the cloned 30 gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cels(yeast, avian, insect or namnmalian), or both. Expression vehicles for production of a recombinant ActRhl polypeptide include plasrmids and other vectors. For - 27 instance, suitable veetors include phasmids of the types: pBR3224redved plasmids,pEMBI derived plasnids, pEX-derived plasmids, pSTac-derived plasmids and pUC-derived plasmids tor expression in prokaryotfic els, suchls Es cohl Some maman exression vectors contain both prokaryotic sequences to facilitate 5 the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells The pcDNAilamp, pcDNA1/neo, pRWCMV, pSV2gpt, pS~neo, pSV2dhfr, pTk2 pRSVneo, pMSO, pSVT, pko-neo and pl-yg derived vectors ar examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacteria, plasmids; such as pBR322, 1.0 to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells, Altenmatively, derivatives of viruses such as the bovine papilloma virus (BPV-l), or Epstein Barr virus (pHFFBo pREP-derived and p205) can be used for transient expression of protens in eukaiyotic cells, Examples of other viral (including retrovra) expression systems can be found below in the description of gene therapy delivery systems. The various methods 15 employed in the preparation of the plasmids and in transfornation of host organisms are well .nown in the art, For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual.2nd Ed, ed. by Sambrook,Fritsch and Maniatis(Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17, In some instances, it may he desirable to 20 express the recombinant polypeptides by the use of a baculovirus expression system Examples of such baculovirus expression systems include pVLderived vectors (such as pVL1392, pVLI393 and pVI-941) pAcUMderived vectors (such as pAcUWI), and pBlueBac-derived vectors (such as the 3-gal containing pilueBac TII) In a preferred embodimetnt avector will be designed for production of the subject 25 ActRT polypeptides in C1HO cells such as a Pcnv-Script vector (Stratagene, La Jolla, Calif.), pcDNAA vectors (Iwvitrogen, Carsbad, Calif,) and pCTIneo vectors (Promega, Madison, Wisc). As will be apparent; the subject gene constructs can be used to cause expression of the subject ActR! poiypeptides in cells propagated in culture, e.g, to produce proteins including fusion proteinsa or variant proteins, for purification 30 This invention also pertains to a host cell trarsfected with a recombinant gene including a coding sequence (eg, SEQ ID NO: 7 or 8) for one or more of the subject ActRil polypeptides The host ell may be any prokaryotic or eukaryoic cell. For example, an - 28 - ActRUI polypeptide of the invention may be expressed in bacterial cells such as. clt, insect cells (eg..sing a baculovirus expression systemm, yeast, or mammalian cells. Other stable host cells are known to those skilled in the art, Accordingly the present invention further pertains to methods of producing the 5 subject ActRi polypeptides. For example, a nost cell transfected with an expression vector encoding an ActRil polypcptide can be culared under appropriate ond'tions to allow expressing of the ActRf polypeptide to occur. The ActRi polypeptide may be secreted and isolated from a mixurre of cells and medium containing the ActRII polpyeptide. Alternatively, the ActRII polypeptide may be retained cytoplasmically or in a membrane 10 fraction and the cells harvested, lysed and the protein isolated, A cell cunre id udes host cells, media and other byproducts Suitable media for cell culture arc well known in the art The subject ActRil polypeptides can be isolated from cell culture medium, host cels or both, using techniques known in the art for purifyng proteins, including ion-exchange chromatography, gel fit-a Ion chromatography, ultrafiltration, electrophoresisu, n d 15 imniunoaffinity purifieation wth antibodies specfe for particular epitopes of the ActRH1 polypeptides. In a preferred iubodiment, the ActRU polypeptide is a fusion protein containing a domain which facilitates its prification. In another embodiment, a fusion gene coding for a purifcation leader sequencesuch as a poly-fHis)/enterokinase claavage site sequence at the N-terminus of the desired portion 20 of the recombinant ActI polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni metal resin The purification leader sequence can then be subsequent removed by treatment with enterokinase to provide the purified ActRTl polypeptide (e g'iC lochuli et at., (19S7) Chromatography 411:17; and Janknecht et alP AAS USA 88:8972) 25 Techniques for making fusion genes are wei known Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended ternnmi for iga tion, restriction enzyme digestion to provide for appropriate termini filling-in of cohesive ends as appropriate, alkline phosphatase treatment to avoid undesirable join ing, 30 and enzymatic ligation. In another embodiment, the Asion gene can be synthesized by conventional techniques including automated YNA synthesizers. Afternatively PCR amplification of gene fragments can be carried out using anchor primers which give rise to - 29 complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequTence (see, fo example, CurntProtocolsm Mviecuhr Biology, e4s, Ausubel et aL John Wiley &: Sons: 1992), ) 4, Aniboe Another aspect of the invendon pertains to antibodies An antibody that is specifically eactive wih anActRil polypeptide (et a soluble ActRl polypeptide and which bind.s competitively with the ActRIl polypeptide may be used as an aragonist of ActRRI polypeptide activities. For example, by using imnnmogens derived from an ActlUl 10 polypeptde antiprotein/antipeptde antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press' 1988)) A mamaimal, such as a mouse, a hamster or rabbit can be immunized with an imunogpiee trfm of the AcaRII polypeptide, an antigenic fragment which is capixble of eliciting an anybody response, or a fusion protein, Techniques 15 for conferring imaunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art An iminunogenlic portion of an ActRil polypeptide can be administered in the presence of adjuvant, 'The progress of ununizaton can be monitored by detection of antibody titers in plasma or serum Standard ELISA or other immunoassays can be used with the rnnunogen as antigen to assess the levels of antibodies, 2Folowing inmunization of an animal with an antigenic preparation of an AtRI polypeptide, antisera can be obtained and, if desired, polyckmal antibodies can be isolated fronm the serun, 'T produce monocIona) antibodies, antibody-producing cells (lymphocytes) can be harvested from an iuinized animal and fused by standard sonmiatic cell fusion procedures with immortalizing cels such asmyloma cells to yield hybridoma cells: Such 25 techniques are well known in the at, and :nc'udefor example, the hybridona technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Koabar et al., (1983) immnology Today, 4: 72) and tChe E.BV hybridorna technique to produce human monociona antibodies (Cole et al, (1985) Monoclonal Antibodies and Cancer Therapy Alan R. LissInc pp. ??6). Hybridma cels 30 can be screened immunocheimically for production of antibodies specifically reactive with an - 30 - ActRI polypeptde and monoclonal antibodies isolated from a culture comprising such hybridoma cells The term "antibody" as used herein is intended to include fragments thereof which are also specifically reactive with a subjec ActRi polypeptide. Antibodies can be fragmented 5 using conventional techniques and the fragments screened for utility in the same manner as described above for whole aetMbo)dies, For example, F(ab fragments can be generated by treating antibody with peprin, The resulting Flab) fragment can be treated to reduce disu lide bridges to produce tab fragments The antibody of the present invention is further intended to include bispecific, sngle-chain, and chieoric and humanized molecules having 10 affinity for an ActRU polypeptide conferred by at least one CDR region of the antibody, in preferred embodhnents, the antibody further composes a label attached thereto and able to be detected (e g the label can be a radioisotope, fluorescent compound,ezyme or enzyme co factor), in certain preferred embodiments an antibody of the invention is a manoclonal IS antibody and in certain embodiments, the invention makes available methods for generating novel antodies For example a method for generating a monoclonal antibody that binds specificaly to an ActRi polypepde may comprise adminserinag to a mouse an amount of an imnmogenic composition comprising the ActR1 polypeptide active to stinulate a detectable immune response, obtaining antibodyproducing cells (e,g, cells from the spleen) 20 from the mouse and fusing the anobodyproducing cells with myeloma cells to obtain antibadysproducing hybridomnat and testing the antbodyproducing hybridomas to identify a hybridomna that produces a monocolnal antibody that binds specifically to the ActRif polypeptide, Once obtained, a hybridoma can e propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that 25 binds specifically to the ActRIT polypeptide. The monoclonal antibody may be purified from the cell culture. The adjective specifically reactive widh as used in reference to an antibody is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (e~g, an ActRII polypeptide) and other antigens that 30 are not of interest that the antibody is useful for, at minimum, detecting the presence of the antigen ofinterest in a particular type of biological sample In certain methods employing the -31 antibody, such as therapeutic applications higher degree of specificity in binding may be desirable. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate effectively between the desired antigens and crosse reacting polypeptides: One characteristic that infences the specificity of an aibody,,antigen iteraction is the affinity of the antibody fior the antge, Although th desired specitcity may be reached wvith u -rangte of different affinities, generally preferred antibodies -wii have an affinity (a dissociation onstantj of about l "t 1 0 4, 109 or less. In addition, the techniques used to screui antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained, For example if an antibody 10 is to be used for binding an antigen in solution, it may be desirable to test solution binding, A variety of different techniques are available for testing interaction between antibodies and antigens to identify particularly desirable antibodies; Such teconles include ELISAs. surface plasnmn resonance binding assays (eeg, the Biacore binding assay, Bia-core AB, Uppsala, Sweden), andwich assays (eg, the paramagnetic bead system of IGEN 15 International Inct Gaithersburg, Maryland westem blots immunoprecipitation assays and immiunohistochnemistrys In certain aspects, the disclosure provides antibodies that bind to a soluble Ac tRI polypeptide. Such antibodies may be generated much as described above using a soluble ActRIl polypeptide or fragment thereof as an antigen. Antibodies of this type can e used., 20 eig., to detect ActRil polypeptides in biological samples and/or to monitor sloicbe AcRl polypeptide levels in an individual, In certain cases, an antibody that specifically binds to a soluble ActRil polypeptide can be used to modulate activity of an ActRi polypeptide and/or an ActRil ligand, thereby regulating promotingg or inhibiting) growth of tissues, such as bone cartilage, muscle, fat, and neurons 25 5, Sgjreen~Ass In certain aspects; the present invention relates to the use of the bAdect ActRi polypeptides (e.g, soluble ActRU polypeptides) to identify compounds (agents) which are agonist or antagonists of the ActRiI polypeptides, Compounds identified through this 30 screening can be tested in tissues such as bone, cartlage.muscle, fat; and/or neurors, to - 32 assess their ability to modulate tissue growth in vitro. Optionally, these compounds can further be tested in animal models to assess their ability to modulate tissue growth in vivo, There are numerous approaches to screning for therapeutic agents for modulating tissue growth by targeting the ActRil polypepides, In certain embodiments, bigh-throughput 5 screening of compounds can be carried out to identify agents that perturb ActRIl-mediated effects on growth of bone cartilage, muscle fat, and/or neuron In certain embodiments, the assay is carried out to screen and identify compounds that specifically inhibit or reduce binding of an AcIRi polypeptide to its binding partner, such as an ActR11 ligand (eg., activin, Nodal, GDF8, GDF I or BMP7 Alternatively, the assay can be used to identify 10 compounds that enhance binding of an ActRi polypeptide to its binding protein such as an ActRIl ligand, In a further embodiment, the compounds can be identified by their ability to interact with an AcRil polypeptide. A variety of assay formats will suffice and, in light of the present disclosure, those not expressly described herein will nevertheless be comprehended by one of ordinary skill in the 15 art. As described herein, the test compounds (agents) of the invention may be created by any combinatorial chemical method. Altenativel, the subject compounds may be naturally occurring bionmolecuies synthesized in vivo or in vitro. Conmpounds (agents) to be tested for their ability to act as modulators of tissue growth can be produced, for example by bacteria, yeast plants or other organsms (eg., natural products), produced chemically (.g, small 20 moleculesincluding peptidomnmenzis), or produced recombinantly Test compounds contemplated by the present inv mention include nonpeptidyl organic molecles, peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleic acid molecules. In a specific embodiment, the test agent is a small organic molecule having a molecular weight of less than about 2,000 daltons. 25 The test compounds of the invention can be provided as single, discre entities, or provided in libraries of greater complexity, such as made by combinatorial chemistry These libraries can comprise, for example, alcohols, aikyl halides, amines, amides, esi t ers, aldhydes, ethers and other classes of organic compounds, Presentation of test compounds to the test system can be in either an isolated Ormn or as mixtures of compounds especially in 30 initial screening steps, Optionally, the compounds may be optionally derivatized with other compounds and have derivatizing groups that facilitate isolation of the compounds: Noni limiting examples of derivatizing groups include biotin, fluorescein, digoxygenin, green - 33 tluoresent protein, isotopes, polyhistidine, mantcads, giutathione S transferase (GS) photoactivatible crosslinkers or any combinations thereof n many drug screening programs w, hich test hbraries of compounds and natural tracts, high thr ughpuassays are desirable in order to maximize the munber of compounds 5 surveyed in a given period of ime Assays which are performed in cell-free systems, such as may be derived with purified or sen-ilpurified proteins, are often preferred as "primary" screens in that they can be generated to penit rapid development and vdatively easy detection of an aeration in a molecular target which is mediated by a test conp ound, Moreover, the effects of ceiular toxicity or bioavailability of the test compound can be 10 generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an aeration of binding affinity between an ActRl polypeptide and its binding protein (e-g an ActRI ligand). Merely to illustrate, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified ActIi polypeptide which is 15 ordinarily capable of binding to an AcIRd igand as appropriate for the intention of the assay. To the mixture of the compound and ActRil polypeptide is then added a composition containing an ActRil ligand; Detection and quantification of ActRIX/ActRIIl ligand complexcs provides a means for determining the compound's efficacy at inhibiting (or potentiating) complex formation between the ActRJ polypeptide and its binding protein. 20 The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay car also be performed to provide a baseline for comparison, For example, in a control assay, isolated and purifed ActR1l ligand is added to a composition containing the ActRI! polypeptide, and the fmioation of ActRIFAcTRRI ligand complex is quiantitated in the absence 25 of the test compound, It will be understood that, in general the order in which the reactants may be admixed can be varied and can be admixed sinultaneously. Moreover n place Of purified proteins, cellular extracts and lysates may be used to render a suitable cell- free assay system. Complex formation between the AcR! polypeptide and its binding protein ay be 30 detected by a variety of t For instance, modulation of the formation of complexes can be quanituated using, for example, detectably labeled proteins such as radiolabeled (e g., - 34 - 8s.-, 14to rR1), rcendy labeled (e g VTC), or enzymaticallybeleeled ActRil polypeptide or its binding protein, by immunoassay, or by chromatographic detection in certain eibodiments, the present invention contempt es the use of fluorescence polarization assays and thurescence resonance energy transfer (FRET) assays in measuring, 5 either directly or indirecdy, the degree of intemaction between an ActRA polypeptide and its binding protein. Further, other modes of detection, such as those based on optical waveguides (POT Publication WO 96/26432 and UiS. Pat.,% No5.67,1%), surface plasmon resonance (SPR), surface charge sensors, and source force sensors, are compatible with many embodiments of the invention, 10 Moreover, the present invention contemplates the use of an interaction trap assay also known as the "two hybrid assay; fbr identifying agents that disrupt or potentiate interaction between an AcaRU poypeptide and its binding protein See ft example, U-S Pat. No, 5283317ervos et A (1993) Cell 72,223232; Madura et al. (1993) J Biol Chem. 2681204612054; Bartel eW.a (1993) Biotechniques 14:920-924; and ivabuohi et at. 11993) 5 Oncogene 81693.16%), In a specific embodiment, the present invention contemplates the use of reverse two hybrid systems to dentiy compounds (eg., small molecules or peptides) that dissociate interactions between an ActRH polypeptide and its binding protein See for example, Vidal and Legrain, (1999) Nucleic Acds Res 27919-29; Vidal and Legrain, (1999) Trends Biotechnol 1;374-81; andU Pat. Nos. 2 5,52-5,490 5955,280; and 5,965,368, 20 in certain embodiments, the subject compounds are identified by their ability to interact with an ActRl polyeptide of the invention, The interaction between the compound and the ActRIl polypeptide may be covalent or ncoatent. For exainple, such interactionT can be identified at the protein lcelusing in. vitro biochemical methods, including photo crosslinking, radiolabeled ligand binding, and affinity chromaography Jakoby WB etat 25 1974, Methods in Enzymology 46:1). In certain cases, the compounds may be screened in a mechanism based assaysuch as an assay to detect compounds which bind to an ActRU polypeptide. This ma; include a solid phase or fluid phase binding even. Alternatively, the gene encoding an ActRlH polypeptide can be transfected with a reporter system (e.gf galactosidase, hciferase, or green luorescent protein) into a cell and screened against the 30 1brary preferably by a h1gh throughput screening or with individual members of the library Other mechanism based binding assays may be used, -for example, binding assays which detect changes in free energy. Binding assays can be performed with the target fixed to a - 35 wel, bead or chip or captured by an iunobilied antibody or resolved by capillary electrophoresis The bound compounds may be detected usually using colorimetric or fluorescence orsurface plasrnon rsonance. In certain aspects, the present invention provides methods and agents for stimulating 5 muscle growth and increasing uscie mass, for example by antagonizing functions of an ActRU polypeptide and/or an ActRKI ligand, Thee fore, any compound identified can be tested in whole cells or tissues, in vitro Or indivo, to confirm their ability to modulate muscle growth Various methods known in the art can be utilized for this purrpose For example metchods of the invention are performed such that the sign transduction through an ActRI 10 potein activated by bindingto an ActRiJ Ligand (e.g, GDFS) has been reduced or inhibited, it will be recognized that the growth of muscle tissue in the organism would result in an increased muscle mass in me organs as compared to the muscle mass of a conesponding organism (or population of organisms) in which the signal transduction through an ActRIli protein had not been so effected 15 For example, the effect of the ActRil polypeptides or test compounds on muscle cell growth/proliferation can be determined by measuring gene expression of Pax2 and Myf-5 which are associated with proliferation of myogenio cells, and gene expression of MyoD which is associated with muscle differentiation (e g, Amthor et al, Dev lBiol. 2002, 251:241 57), It is known that GDFS down-regulates gene expression of Pax-3 and Myf5, and 20 prevents gene expression of MyoD. The ActRII polypeptides or test compotds are expected to antagonize this activity of GDF& Anoher example of cell-based assays includes measuring the proliferation of myoblasts such as C(2)C1 2) myoblasts in the presence of the ActRH poiypeptides or test compounds (eg Thomas it aL J Blo Clhem 2000, 275:40235 43), 25 The present invention also contemplates in vio assays to measure muscle mass and strength For example, Whittemore et al (Biochem Biophys Res Commun. 2003, 300:965 71) discloses a method of measuring increased skeletal muscle mass and increased grip strength in mice. Optionally, this method can be used to determine therapeutic effects of test compounds (e.g., ActRI polypeptides) on muscle diseases or conditions, for example those 30 diseases for which muscle mass is limiting - 36 in certain aspects, the present invention provides rnethods and agents for modulating (stimulating or inhibiting) bone formation and increasing bone mass, Therefore.any compound identified can be tested in whole ceits or tissues, in vitro or inivo, to confirm their ability to modulate bone or cartilage growth. Various methods known in the art can be 5 utilized Br this purpose, For example, the effect of the ActRit polypeptides or test compounds on bone or cartilage growth can be determined by neasuring iduction of Msx2 or differentiation of osteoprogenitor cells into osteoblasts in cell based assays (see, eg, Daluisk ei at, Nat GeneL 2001, 27(4)84t; Hino et al, Front Biosci. 2004, 9:15209). Another example ot celi-ased 10 assays inc iudes analyzing the osteogenic activity of the subject Acti1 polypeptides and test compounds in mesenchytnal progenitor and osteoblastic cells. To illustrate, recorbiant adenoiruses expressing g an ActRip olypeptide were constructed to infect pluripotent mesenchyma progenitor Cfi 0 T'cells, preosteobiastic C201 2 cells, and osteobasde T 85 cells. Osteogenic activityithen deterned by measuring he induction of alkaline 1$ phosphatase, osteocalcinh and matrix mieraiation (se, e.g, Cheng et aL, J bone Joint Surg Am. 2003, 8-A(8'i54452). The present invention also contemplates in vivo assays to measure bone or cartilage growth. For example, Namkunrg~Manhai et a, Bone, 28: 6-S (2001) discloses a rat osteoporotic model in which bne repair during the early period after fracture is studied. 20 Kubo et al, Steroid Biocheristry & Molecular Biology 68:19202 (1999) also discloses a rat osteoporotic model in which bone repair during the late period after fracture is studied. These references are incorporated by reference herein in their entirety for their disclosure of rat model for study on osteoporatic bone fracture; in certain aspects, the present invention makes use of fracture healing assays that are know in ithe art These assays include fracture 25 technique, histologtcal analysis, and biomechanical analysis, which are described in, for example, U.S Pat. No. 6,521,750,vhich is incorporated by reference in its entirety for its disclosure ofexpenrental protocols for causing as well as measuring the extent of fractures and the repair process. in certain aspects, the present Invention provides methods and agents for controling 30 weight gain and obesity. At the cellular level, adipocyte proliferation and differentiation is critical in the development of obesity, which leads to the generation of additional fat cells (adipocytes). Therefore, any compound identified can be tested in whole cells or tissues, in - 37 vitro or in vivo; to confirm their ability to modulate adipogenesis by neasuring adipocyte pro liferation or differeniation. Various methods known in the art can be utilized for this purpose, For example, the Affect of an ActRi polypeptide (e.g a soluble ActRlU polypeptide) or test compounds on adipogenesis "an be determied by measuring 5 differentiation of 3T31 preadipocytes to mature adipocytes in cell based assays, such as, by observing the accunadation of tricylglycerolin Oil Red O staining vesicles and by the appearance of certain adipocyte. markers such as FABP (aP2/422) and PPAR2 See, for example, Reusch et aLt 2000 Mol Cell BioL 20: 1008-20; Deng et aL 2000, Endocrinology: 141:2370-6, Bell et at 2000, Obes Res. 8:249-54. Another exanple of ceilbased assays 10 includes analyzing the role of ActRit polypeptides and test compounds in prolieration of adipocytes or adipocyte precursor cells (e g, 3T3-Li cells), such as, by monitorig bromodeoxyuridine (Brdtpositive cells. See, fbr example, Fico et at, 1998., Mal Cell Biochem. 189:17 Masuno et a, 2003 Toxicoi Sci, 75:314-20. It s understood that the screening assays of the present invention apply to not only the 15 subject ActRil polypeptides and variants of the ActRU polypeptides but also an test compous including agonists and antagonist of the ActRH polypeptides, Further, these screening assays are usefid fbi' drug target verification and quality control purposes. 6, Uemla liR tig 20 In certainembodiments compositions (&g.. ActRu polypeptides) of the present invention can be used for treating or preventing a disease or condition that is asseolated with abnormal activity of an ActRit polypeptide and/or an ActRil ligand (eig., CDF8). These diseases, disorders or conditions are generally referred to herein as "AciRl-associated conditionsS in certain embodinients, the present invention provides methods of treating or 25 preventing an individual iz need thereof through adnardstering to the individual a therapeutically effective around of an ActRI polypeptide as described above. These methods are particularly aimed at therapenaad prup hylactic treatments of annals and more particularly humans, As used herein, a therapeutic that "prevents" a disorder or condition refers to a 30 compound that, in a statistical samplereduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the - 38 severity of one or more symptoms of the disorder or condition relative to the unreated controlsample. The term "treating" as used herein includes prophylaxis of the named condition or amelioration or elimination of the condition once it has been established. ActR/ActRf ligand complexes play essential roles in tissue growth as welt as early 5 developmental peses such as the correct formation of various atruclures or in one or Imore post-developmenta' capacities including sexual development, pituitary hormone production, and creation of bone and cartilage. Thus, AciRI ssociated conditions include abnonral tissue growth and developmental defects in addition, ActRIassociated conditions include, but are not limited to, disorders ofcell growth and differentiation uch as infan mation 10 allergy, autoiimune diseases infetious diseases, and tunors Exemplary ActRI-bassociated conditions include neuromuscular disorders (eg. muscular dystrophy and muscle atrophy, congestive obstuctive pulmonary disease, muscle wasting syndrome sarcopenia, cacheva, adipose tissue disorders (eg> -obesity), type 2 diabetes, and bone degenerative disease (e osteoporous), Other exemplary ActRii 15 associated conditions include usculodegenerative and neuromuscular disorders, tissue repair (eg., wound h dealing) neuro generaive diseases (c.g anyotrophic lateral sclerosis) inununolog disorders (eg disorders related to abnormal proliferation or function of y and obesity or disorders related to abnomnal proliferation of adipocytes, in certain embodiennts, composinons (eg, soluble ActRil polypeprides) of the 20 invention are used as part of a treatment for a muscular dystrophy: The term "muscular dystrophy" refers to a group of general tivc muscee diseases characterized by gradual weakening and deterioration of skeletal muscles and sometimes the heart and respiratory muscles, Muscular dystrophies are genetic disorders characterized by progressive muscle wasting and weakness that begin with microscopic changes in the muscle As muscles 25 degenerate over time, the person's muscle strength declines Exemplary muscular dystophies that can be treated with a regimen including the subject ActRil polypeptides include, Duchenne Muscular Dystrophy (DMD); Becker Muscular Dystrophy (13MD), Emery-Dreifuss Muscular Dyt ophy (EDMI)), Limb-Girdle Muscular Dystrophy (LUMD), Pacioscapulohumeral Musular Dystrophy (FSl or FSHD) (also known as Landouzy 30 Dejerine), Myotonic Dystrophy (MMD) (also known as Steinerts, Disease), Oculopharyngeal Muscular Dystrophy (OPMD), Distal Muscular Dystrophy (DD), Congenital Muscular Dystrophy (CMDP) - 39 - Duchenne Muscular Dystrophy (DMD) was first described by the French neurologist Guilaunme Benjamin Amand Duchene in the 1860s. Becker Muscular Dystrophy (BMD) is named after the German doctor Peter Eml Becker, who first described this variant of DM1D in the 1950s DMD is one of the most frequent inherited diseases in males,affecting one in 5 3,500 boys, DMD occurs when the dystrophin genelocated on the short arm of the X chromosome is broken Sime maies only carry one Copy of the X chromosomne, thy only have one copy of the dystrophin goene without the dystrophin protein, muscle is easily damaged during cycles of contraction and relaxation. While eady in t he disease muscle compensates by regeneration Wter on muscle progenitor cells cannot keep ip with the 10 ongoing damage and healthy muscle is replaced by nonftnctional fibrolfattv tissue, BMD results from different rmtations in the dystmphin gene BMD patients have some dystrophin, but it is either insufficient in quantity or poor in quality. Having some dystrophin protects the muscles ofhose with BMD 1om degenerating as badly or as quickly as those of people with DMDW 1$ For example. recent researches demonstrate that blocking or eliminating function of GDF8 (an AtRft ligand) in vivo can dfectively treat at least certain symptonts in DMD and BMD patients (Bogdanovich et al, supra; Wagner et al, supra), Thus, the subject ActRi polypeptides may act as GDFS inhibitors (antagonists)and constitute an ater active means of blocking the functions of GDF8 and/or ActRil in vivo in DMD and PMD patients. 20 ~Similarly, Che su bject ActRf J tmlyreotide2' provide an effctive irians- to increase muscle mass in other disease conons that are in need of muscle growth For example, GonzaiezCadavid et al (supra) reported that that GDF8 expression coneiatesinverselv with fat-free inass in humans and that increased expression of the GDF$ gene is associated with weight loss in men with AIDS wasting syndrome By inhibiting the function ofjGDF8 in 2$ AIDS patents, atleast certain synptonus of AIDS may be alleviated, if not completely eliminated, thus significantly improving quality of life in AIDS patients. Since loss of GDF8 (an ActlkU ligand) function is also associated with fat loss without diinution of nutrient intake (Zimmers et a, supra; McPherron and Lee, supra), the subject ActRi polypeptides may further be used as a therapeutic agent for slowing or 30 preventing the development of obesity and type 'i diabetes, - 40 - The cancer anorexia-cachexia syndrome is among the most debiitating and life threateing' aspects of cancer, Progressive weight loss in cancer anorexia-cachexia syndrome is a common feature of many types of cancer and is responsible not only for a poor quality of life and poor response to chemotherapy but also a shorter survival time than is found in 5 patients with comparable tumors without weight loss. Associated with anorexia, fat and muscle tissue wasting psychological distress and a lower quality of life cachexia arses from a complex interaction between the cancer and the host P is one f the most common causes of death among cancer patients and is present in 80% at death, t is a omplex example of metabolic chaos effecting proteincarbohydrate, and fat nmetabolisnm Tumors produce both 10 direct and indirect abnormalities, resulting in anorexia and weight loss. Currently, there is no treatment to control or reverse the process Cancer anorexia-cachexia syndrome affects cytokihe production, release of lipid-mobilizing and proteolysis-inducing factors, and alterations in intermediary metabolism, Although anorexia is common, a decreased food imake alone is unahle to account for the changes in body composition seen in cancer patients, 15 and increasing nutrient intake is unable to reverse the wasting syndrome. Cachexia should be suspected in patients with cancer ifan involuntary weightloss of greater than five percent of premorbid weight occurs within a six-month period. Since systemic overexpression of GDF8 in adult mice was found to induce profound nmse and at loss analogous to that seen inhuman cachexia syndromes (Zimmers et al., 20 supra) the subject ActRil polypeptides as pharmaceutical compositions can be beneficially used to prevent, treat or alleviate the symptoms of the cachexia syndrome, where muscle growth is desired In other embodiments the present invention provides methods of inducing bone and/or cartilage formation, preventing hone loss, increasing bone mineralization or 25 preventing the demineralization of bone. For exampethe subject ActRif polypeptides and compounds identified in the present invention have application treating Osteoporosis and the healing of bone fractures and cartiage defect in humans and other animals ActRl polypeptides may be usefa in patients that are diagosed with subchnical low bone density, as a protective measure against the development of osteoporosis. 30 In one specific embodiment, methods and compositions of the present invention may find medical utility in the healing of bone fractures and cartilage defects in humans and other animals. The subject methods and compositions may also have prophylactic use in closed as - 41 well as open fracture reduction and also in the improved Fixation of artificial joints, De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma-induced, or oncologic rejection induced craniofacial defects. and also is useful in cosmetic plasic surgery. Further, methods and compositions of the invention may be used in 5 the treatment of periodontal disease, and in other tooth repair processes, in certain cases, the su bjct ActRil polypeptides may provide an en vronimkenIt to attract bone-rmiing cells; stimulate growth of bonefrmiing cells or induce difkrentiation of progenitors of bone forcing cells. ActRI polypeptides of the invention may also be usefuI in the treatment of osteoporosis. FurtherActl polypeptides may be used in cartilage defect repair and 10 prvyention/reversal of osteoarthritis. In another specific embodiment, the invention provides a therapeutic method and composition for repairing fractures and other conditions related to cartilage and/or bone defects or periodontal diseases, The invention further provides therapeutic methods and compositions for wound healng and tissue mpair, The types of wounds include, but are not 15 limited to bums, incisions and ulcers See e, PCT Publication No, W084/0 1106, Such compositions comprise a therapeutically effective amount of at least one of the ActRil polypeptides of the invention in admixture with a phrmaceutically acceptable vehicle, carier or matrix, In another specific embodiment, methods and compositions, of the invention can be 20 applied to conditions causing bone loss such as osteoporosis, hyperparathyroidism., Cushing's disease, thyrotoxicosis, chronic diarrheal state or malabsorption, renal tubular acidosis, or anorexia nervosa, Many people know that being female, having a low body weight, and leading a sedentary lifestyle are risk factors for osteoporosis (loss of bone mineral density, leading to fracture risk) However, osteoporosis can also result from the iong-terM use of 25 certain medications, Osteoporosis resulting from drgs or another medical condition is known as secondary ostporosis I a condition known as Cushing's disease, the excess amount of cortisol produced by the body results in osteoporosis and fractures, The inost common medications associated with secondary osteoporosis are the corticosteroids a class of drugs that act like cortisol, hormone produced naturally by the adrenal glands, Although 30 adequate levels of thyroid hormones (which are produced by the thyroid gland) are needed for the development of the skeleton, excess thyroid hormone can decrease bone mass over time. Antacids that contain aluminum can lead to bone loss when taken in high doses by - 42 people with kidney problems, particularly those undergoing dialysis. Other medications that can cause secondary osteoporosis include phenytoin (Dilantin) and barbiturates that are used to prevent seizures; methotrexate (Rheumatrex, Imnnmex, Folex PFS), a drug for some forms of arthritis, cancer, and immune disorders; cyclosporine (Sandimamune, Neoral), a drug used 5 to treat some autoimmune diseases and to suppress the immune system in organ transplant patients;luteinizing hormone-releasing hormone agonists (Lupron, Zoladex}) used to treat prostate cancer and endometriosis; heparin (Calcipauine Liquamrin), an ant'cletting medication; and ch olestyramine (Questran) and colestip'1 (Cole'stdi used to treat bigh cholesterol, Gum disease causes bone loss because thes amful bacteria in our mouths 10 force our bodies to defend against them The bacteria produce toxins and enzymes under the gumbine causing a chronic infction. In a further embodiment, the present invention provides methods and therapeutic agents for treating diseases or disorders associated with abnormal or unwanted bone growth., For example, patients having the disease known as Fibrodysplasia Ossificans Progressiva 15 {FOP) grow an abnormal "second skeleton" that prevents any movement, Additionally, abnormal bone growth can occur after hip replacement surgery and thus ruin the Sergical outcome. This is a more common example of pathological bone growth and a situation in which the subject methods and compositions may be therapeutically useful The same methods and compositions may also be useful for treating other forms of abnormal bone 20 growth (eg., pathological growth of bone following trauma, bums or spinal cord injury),and for treating or preventing the undesirable conditions associated with the abnornal bone growth seen in connection wih metastatic prostate cancer or osteosarcoma, Eixamples of these therapeutic agents include, but are not limited to, ActRIl polypeptides that antagonize functions of an ActR1 ligand (e g, BMP^), compounds that disrupt interaction between an 25 ActRU and its ligand (eg, BMP7), and antibodies that specifically bind to an Ac-Ri1 receptor such that an ActRII ligand (e.g., BMP7) cannot bind to the ActRi receptor In other embodiments, the present invention provides compositions and methods for regulating body fat content in an animal and for reatirg or preventing conditions related thereto, and particularly, heahcompromising conditions related thereto.According to the 30 present invention, to regulate (control) body weight can refer to reducing or increasing body weight. redwing or increasing the rate of weight gain, or increasing or reucing the rate of weight lossand also includes actively maintaining, or not signifcanly changing body weight - 43 - (eig, against external or internal influences which may otherwise increase or decrease body weight). One embodiment of the present invention relates to regulating body weight by administering to an animal (e.g, a human) in need thereof an ActRII polypeptide. In one specific embodiment, the present invention relates to methods and compounds $ fbrreducing body weight and/or reducing weight gain in an animal, and more particularly, for treating or ameliorating obesity in patients at risk for or suffering from obesity, in another specific embodiment the present invention is directed to methods and compounds for treating an animal that isunible to gain or retain weight (eg. an animal with a wasting Syndrome) Such methods are effective to icLrease body weight and/or mass, or to reduce weight nd/or 10 mass loss or to improve conditions associated with or caused by undesirably low (e g., unhealthy) body weight and/or mass. In other embi dimentst the subject ActRlI polypeptides can be used to form pharmaceutical compositions that can be benetlialiy used to prevent> treater alleviate symptoms of a host of diseases involving neurodegeneration. While not wishing to be bound 15 by any particular theorythe subject ActRfl polypeptides may antagonize the inhibitory feedback mechanism mediated through the type I receptor ALIK? thus allowing nww neuronal growth and differentiation The subject ActRIT polypeptides as pharnnaceutical compositions can be beneficially used to prevent, treat, or alleviate symptoms of diseases with neurodegcneratin, including Aheimer's Disease (AD), Parkinson's Disease (PD), 20 Amyotrophic Lateral Sclerosis (ALS), and Huntington's disease (H4D), AD is a chronic incurable, and unstoppable central nervous system (CNS) disorder that occurs gradually, resuding in memory loss, unusual behavior personality changesand a decline in thinking abilities These losses are related to the death of specific types of brain cels and the breakdown of connections between then AD has been described as cbildhood 2$Sdeveopmentin reverse i most people with ADsymptoms appear after the age 60 The earliest symptoms include loss of recent memory, faulty judgment, and changes in personality. Later in the disease, those with AD may forget how to do simple tasks like washing their hands. Eventually people with AD ose all reasoning abilities and become dependent on other people for their everyday care. Finally the disease becomes so 30 debilitating that patients are bedridden and typically develop coexisting illnesses AD patients most commonly die from pneumonia,8 to 20 years from disease onset, - 44 - PD is a chronic, incurable, and unstoppable CNS disorder that occurs gradually and results in uncontrolled body movements, rigidity, tremor, and gait difficulties These motor system problems are related to the death of brain cells in an area of the brain that produces dopamine, a chemical that helps control muscle activity. In most people with PD, symptoms 5 appear after age 50, The initial symptoms of PD are a pronounced tremor affecting te extremities, notably in the hands or lips, Subsequent characteristic symptoms of PD are stiffness or slowness of movement, a shufflng walk, stooped posture, and inmpaired balance. There are wide ranging secondary symptoms such as memory loss dementia, depression, emotional changes, swallowing difficulties, abnormal speech, sexual dysfunction, and 10 bladder and bowel problems. These symptoms wil begin to interfere with routine activities, such as holding a fork or reading a newspaper. Finally, people with PD become so profoundly disabled that they are bedridden, People with PD usually die from pneumonia, ALS, also called Lou Gehrig's disease (motor neuron disease) is a chronic, incurable, and unstoppable C NS disorder that attacks Ie motor neurons. components of the CNS that 15 connect the brai to the skeletal muscles. in ALS e motor neurons deteriorate and eventually die, and though a person' bain normally remains flly functioning and alert, the command to move never reaches the muscles. Most people who get ALS are between 40 and 70 years old, The first motor neurons that weaken are those leading to the arms or legs, Those with ALS may have trouble walking, they may drop things, fil slur their speech, and 20 laugh or cry uncontrollably. Eventually the muscles in the limbs begin to atrophy from disuse. This muscleeakness will become debilitating and a person wil need a wheel chair or become unable to function out of bed, |Most ALS patients die from respiratory failure or from complications of ventilator assistance like pneumonia, 3-5 years from disease onset. The causes of these neurological diseases have renmained large y unknown, They are 25 conventionally defined as distinct diseases, yet clearly show extraordinary similarities in basic processes and coimonly demonstrate overlapping symptoms far greater than would be expected by chance alone. Current disease defimions fail to properly deal with the issue of overlap and a new classification of the neurodegenerative disorders has been called for. HD is another neurodegenerative disease resulting from genetically programmed 30 degeneration of neurons in certain areas of the brain, This degeneration causes uncontrolled movements, loss of intellectual faculties, and emotional disturbance. HD is a familial disease passed from parent to child through a dominant: mutation in the wild--type gene. - 45 - Some cary symptoms of HfD are mood swings depression irritability or trouble driving, leaning new things, remembering a fact, or making a decisim As the disease progress, concentration on Intelectual tasks becomes increasingly difficult and the patient may have difficulty feeding himnsefor nerse'f and swallowing. The rate of de progression and the 55 agvt of onset vary from person to person. Tay-Sachs disease and Sandhoffdisease are gly coiipid storage diseases caused by the lack of lysosomal Whexosaminidase (Gravel et at, in The Metabolic Basis of Inherited Disease. eds. Scriver et at. McGraw{iliNew York pp 2839-2879, 1995 both disordersM2 gagnioside and related glycolipid substrates for hexosaminidese 10 accumulate in the nervous sys t em and trigger acute neurodegeneration In the most severe forms, the onset of symptoms begins in early infancy. A precipitous neurodegrenerative course then ensues, wrth affected infants eihibiting motif dysfunction. seizure, visual loss, and deafness. Death usually occurs by 2-$ years of age Neuronal loss through an apoptolic mechanism has been demonstrated (Huang at a, Hum, Mol. Genet 6: 18791885, 1997), 15 It is well known that apoptosis plays a role in AIDS pathogenesis in the immune system. However HIX/I also induces neurologiai disease. Si et alt (. Clin. Invest, 98, 1979-1990, 199 examined apoptosis induced by HMIVy infection of the central nervous system (CNS) in an in vitro modal and in bain tissue from AIDS patients, and found that HIW 1 inffedtion of primary brain culures induced apoptosis in neurons and astrocytes in 20 vitro. Apoptosis of neurons and astrocytes was also detected in brain tissue from 10/ 1 AIDS patients, including 5/$ patients with HIWI dementia and 4/5 nundanented patients Neuronal loss is also a salient feature of prion diseases, such as Creuzfeidt-akob disease in human. BE in cafte (m ad cow disease)>Scrapie Disease in sheep and goats, and feline spongiftbn encephalopathy (FS$) in cats, 2 The subject ActRI' polpeptides are also useful to prevent, treat, and alleviate symptoms of various PNS dsorders, such as the ones described betow. The PNS is composed of the nerves that lead to or branch off fom the CNS. The peripheral nerves handle a diverse arrayof fuctvions in the bod-y, including sensory, motor; and autonomic functions, When an individualhas a peripheral neuropathy, nerves of the PNS have been 30 damaged. Nerve damage can aise from a number of causes, such as disease, physical injury, poisoning or malnutrition. These agents may affect either afferent or efferent nerves. - 46 - Depending on the cause of damage, the nerve cel axon, its protective myehin sheah, or both may be injured or destroyed. The term peripheral neuropaty" encompasses a wide range of disorders in which the nerves outside of the brain and spinal cord-peripheral nervesa-have been damaged, 5 Pei pheral neIropathy may also be referred to as peripheral neuritis, or if many nerves are involved, the t eis polyneuropathy or polyneuritis may be used, Peripheral neuropathy is a widespread disorder, and there are many underlying causes. Some of these causes are commonmsuch as diabetesand others are extremely rare such as acrylamide poisoning and certain inherited disorders, The most common worldwide 10 cause of peripheral neuropathy is leprosy, Leprosy is caused by the bacterium Mycobacterium leprae, which attacks the peripheral nerves of affected people, According to statistics gathered by the Wodd Health Organization, an estimated 1.15 million people have leprosy worldwide, Leprosy is extremely rare in the United States, where diabetes is the most corminonly .15 known cause of peripheral neuropathy. It has been estimated that more than 17 million people in the United States and Europe have diabetes-related polyneuropathy. Many neuropathies are idiopathic - no known cause cn be found, Trhe most common of the inherited peripheial neuropathies in the United States is Charco-Marie -Tooth disease, which affects approximnatcly125,000 persons 20 Anothkr of the better known peripheral neuropathics is Guillain-Barr. syndrome, which arises from complications associated with viral illnesses., such as cytomegalovirus, E.pstein Barr virus, and human immunodefreency virus (HIV) or bacterial infection, including Capylobacter jeuni and Lyme disease.. The worldwide incidence rate is approximatly 1.7 cases per 100,000 people annually Other wellknown causes of peripheral 25 neuropathies include chronic alcoholism. infection of the varicella-zoster virus, botulism, and poliomnyliis. Perpheral neuropathy may develop as a primary symptom, or it may be due to another disease. For example, peripheral neuropathy is only one symptom of diseases such as amyloid neuropathy, certain cancers, or inherited neurologic disorders. Such diseases may affect the peripheral nervous system (PNS) and the centra nervous system (CNS) as wellas 30 other body tissues. - 47 - Other PNS diseases treatable with the subject ActR poiypeptides include; Brrachial Plexus Neuropathies (diseases of the cervical and first thoracic roots, nerve trunks, cords, and peripheral nerve components of the brachial plexus. Clinical maniibstationxs iclude regional pain, paresthesia; muscle weakness, and deteased sensation in the upper extrmnty These 5 disorders may be assolated with trauma, including birth injuries; thoracic outlet syndrome neoplasms, neuritis, radiotheTapy; and other conditions, See Adams et ak Principles of Neurology, 6th ed, pp 1351-2); Diabetic Neuropathies (peripheral, autonomic, and cranial nerve disorders that are asociated with diabetes meilitu4 These conditions usually result fromn diabedi microvascular injury involving sall blood vesses that supply nerves vasaa 10 nervorum) Relatively common conditions which may be associated with diabetic neuropathy inckde third nerve palsy; nmononeuropathy; mononeuropathy multiplex, diabetic amyotrophy; a painful polyneuropathy; autonomic neuropathy; and thoracoabdominal neuropathy (see Adams et aL, Principles of Neurology, 6th ed, p13 2 5); mononeuropathies (disease or trauma involving a single peripheral nerve in isolation, or out of proportion to 15 evidence of diffise peripheral nerve dysfunction Mononeuropathy muliplex refers to a condition characterized by multiple isolated nerve injuries, Mononeuropathies nay result from a wide variety of causes, including ischemia; traumatic injury; compression; connective tissue diseases; cumulative trauma disorders; and other conditions); Neuralgia (intense or aching pain that occurs along the course or distribution of a peripheral or cranial nerve); 20 Periphera Nervous System Neopasms (neoplasis which arise from peripheral nerve tissue. This inchides neurofibromas; Schwannomas; granular cell tumors; and malignant peripheral nerve sheath tumors, See Deita Jr et al., Cancer Principles aid Practie of Oncology, 5th ed, ppl 750-4); Nerve Compression Syndromes (mechanical compression of nerves or nerve roots from intemal or extemal causes, These may result in a conduction block to nerve 25 impises, due to, for example, myelin sheath dysfAnction, or axonal loss. The nerve and nerve sheath injuries may be caused by ischemia; inflammation; a direct mechanical effect; or Neuritis (a general term indicating inflammation of a perpheral or cranial nerve) Clinical manifestation may include pain; parsthesias; paresis; or hyperthesia; Polyneuropaties (diseases of multiple peripheral nerves), The various torms are categorized b. the type of 30 nerve affected (e g. sensorymotor or autonomic), by the distribution of nerve in',jury (e g. distal vs. proximal), by nerve component primarily affected (e,demyclinating vs axonal), by etiology, or by pattern of inherine. - 48 - 7. Pharrnaceu dclCmoion In certain embodiments.compounds (e.g. ActRil polypeptides) of the present invention are formulated with a pharmaceutical acceptable carrier. For example an ActR1l 5 polypeptide can be administered aone or as a component of a pharmaceutical formulation (therapeutic composition) The subject compounds may be formulated for administration in any convenient way for use in human or veterinary medicine. In certan embodimense the therapeutic m ethod of the invention includes administering the composition topically, systemically, or locally as an implant or device. 10 When administered the therapeutic composition for use in this invention is, of course, in a pyrogenfree, physiologicallacceptable form Furthe, the composition may desirably be encapsulated or injeced in a viscous form fio delivery to a target tissue site (e. one, cartinge, muscle, fat or neuron) tbr example a site having a tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful 15 agents other than the ActRi polypeptides which may also optionally be included in the composition as described above. may alternatively or additionally, be administered simultaneously or sequentially with the subject compounds (esg, ActRII polypeptides) in the methods of the invention. In certain embodiments compositions of the present invention may rincude a matrix 20 capable of delivering one or more therapeutic compounds (e.g. ActRI polypeptides) to a target tissue site (e.g, bone, cartilage, muscle, fat or neuron), providing a structure for the developing tissue -ad optinally capable of being restored into the body, For example, the matrix may provide slow release of the AcIR1i polypeptides. Such matrices may be formed of materials presently in use for other implnted medical applications. 25 The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties The particular application of the subject compositions will define the appropriate folrulation. Potential matrices tor the compositions may be biodegradable and chemically defined calcium sulfate, ticRalciumphosphate, hydroxyapatite polylactic acid and polyanhydrides. Other potential 30 materials are biodegradable and biologically well defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracelhuar matix components. Other - 49 potential matrices are non-biodegradable and chemically defined, such as sintered bhydroxyapatite. biogiassa laminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The blocerrnicamay be altered in 5 composition, such as in calciumahuminatephosphate and processing to alter pore size' particle size, particle shape. and biodegradability In certain embodiments, methods of the invention can be administered for orally, eg, in the form of capsules caches, pils, tabes lozenges (using a flavored basis, suaRy sucro a a a u fl powders, granules, or as a soltion or a suspension in an 10 aqueous or non-aqueous liquid, or as an oilinwater or water-in.oil liqid emsionor as an elixir or syrup, or as pastilles (using an Inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient An agent may also be administered as a bolus, electuay or paste. 15 In solid dosage forms for oral administraton (capsules, tablets pills, dragees, powders granules and the like), one a m re therapeutic compounds of the present invention may be mixed with one or more pharnaceuticaly acceptable carriers, such as sodium citate or dicalcium phosphate, and/or any of the following- (1) fwers or extenders, such as starches, lactose, sucrose, glucose manntoi and/orsilicic acid; (2) binds such as for example, 20 carboxymethylcellulose alginates, gelatin polyvinyl pyrrolidone sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcumn carbonate, potato or tapioca starch, alginic acid, certain silicates, sd odium carbonate; (5) sohuion retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for examplecetyl alo hol and glycerol 25 nonostearate; (8) absorbents, sucIh as kaolin and bentnite Jay 9) lubricants, such a tale, calcium steaarae, magnesium snearate.solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof- and (10) coioing agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise offering agents. Solid compositions of a similar type may also be employed as fillers in soft and hardiied gelatin capsules using 30 such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycois and the like. - 50 - Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions suspensions syrups, and ei xirs, In addition to the active ingedient, the liquid dosageorms may conain ine diluents commonly used in the an, such as water or other solvents, sobilidig agents and emuhsners, such as ethyl alcohol 5 isopropyl alcohol, ethyl carbonate, ethyl acetate! benzyl alcohol, benzyl benzoate propylene glycol, I hutycene glycot oils (in particular, cottonseed, groundnut corn, ge n, olive, castor, and sesame oils), glycerol, tetrahydrofbrylalcoho polyethylene ghycos and fatty acid esters of sorbitanI and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvats such as wettng agents, emulsifying and suspending agentssweetening 10 flavoring, coloring perfnting, and preservative agents, Suspensions, in addition to the active compounds, may contain suspending agency such as ethoxylated isostearyl alcohlIs, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mi-xtures thereof 15 Certain co(positions disclosed herein may be administered topically, either to skin or to mucosal membranes, The topical formulationsmay further include one or more of the wide variety of agents known to be effective as skin or stratum cormeum penetration enhancers. Examples of these are 2-pyrrolidone, N-nethyl-2-pyrroiidone, dimnethylacetamide, dimethylformamide propyene glycol, methyl or isopropyl alcohol, 20 dimethyl sulfoxide, and zone Additional agents may further be included to rake the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surface active agents. Keratolyic agents such as those known in the art may also be included. Examples are salicylic acid and sulfur. Dosage forms for the topical or transdermal administration include powders, sprays, 25 ointments, pastes, creams, lotions gels, solutions patches, and inhalants, The active compound may be mixed u nder sterile conditions with pharmaceutically acceptable carrier, and With any preservatives, buffers, or propellants which may be required, The ointments, pastes, creams and gels may conta, in adaition t a subject compound of the invention (e.g., an ActRil polypeptide), recipients, such as animal and vegetable fats, oils, waxes, paraffins, 30 starch, tragacanth, celulose derivatives, polyethylene glycols, sificones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof - 51 - Powders and sprays can contain, in addition to a subjec compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or nxtures of these substances Sprays can additionally contain customary propellants, such as cblorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and 5 propane. In certain embodiments, pharmaceutical compositions suitable for parenteral administration may conprise one or more ActRil polypeptides in coimbinatin with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, aspersions suspensions or e-tulsions. or sterile powders which may be reconstituted into 10 sterle injectable solutions or dispersions just prior to use, which May contain antioxidants, buifers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents Eixamples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene 15 glycl and the like), and suitable mixtures thereof vegetable oils, such as olive ol, and injedable organic ester such as ethyl oleate Proper fluidity can be maintained, for eamnpieby the use of coating materials, such as lecithin by the maintenance of the required Particle size in the case of dispersions, and by the use of surfactants. The compositions of th invention may also contain adjuvants, sheb as preservatives, 20 wetting agents, emulsifying agents and dispersing agents, Preention of the action of microorganisms may be emsured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanot phenol sorbic acid, and the like, It rnav also be desirable to include isotonic agents, such as sugars sodium chloride and the like into the compositions In addition prolonged absorption of the injectable pharmaceutical form nmay 25 be brought about by the inclusion of agents which delay absorption, such as aluminum mnonostearate and gelatin t is understood that the dosage regimen will be determined by the attending physician considering varioustactors which modify the action of the subject compounds of the ivention aglf ActRl polypeptides). The various factors include, but are not limited to, 30 amount of bone weight desired to be fbrned, the site of bone damage, the condition of the damaged bone, the size of a wound, type of damaged tissue, the patient's age, sex, and diet. the severity of any infection, time of administration and other clinical tators Optionally, - 52 the dosage miay vary with the type of matrix used in the reconstitution and the types of compounds in the composition The addition of other known growth factors to the final composition 4 may also effect the dosage. Progress can be monitored by periodic assessment of bone growth andaor repair, fr example, X-rays, histomorphometric determinations, and 5 tetracycline labeling, In certain embodiments of the invention, one or more ActR1l polypeptides can be adminiistered, togpetherora ifc t-) a admiistred ogeher(simultaneously) or at different times (sequentially Or overlapping). In addition, ActiRI polypeptides can be ad-ministered with another type of therapeutic agents. for example, a cartilage-inducing agent, a bone-inducing agent, a muscleinducing agent, a 10 fat-reducing, or a neuron-inducing agent; The two types of compounds may be administered siraulaneously or at different tiws It is expected that the ActRl polypeptides of the invention may act in concert with or perhaps synergiStiiily with another therapeutic agent, In a specific example, a variety of osteogenie, cartilage-inducing and bone-inducing factors have been described, particularly bisphosphonates, See e.g. European Patent 15 Application Nos 4,1 55 and 169,01 For example, other factors that can be combined with the subject AcR' polypeptides include various growth. factors such as epidermal growth factor (EGF) platelet derived growth factor (PDGF). transforming growth factors (T GFk-and UP I) and insulin-like growth factor (lGF). In certain embodiments/the present invention also provides gene therapy for the in 20 vivo production of AeRHl polypeptides Such therapy would achieve its therapeutic effect by introduction of the ActRI polynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of ActRII polynucleotide sequences can be achieved using a recombinant expression vector such as a chinmeric vimus or a colloidal dispersion system. Preferred for therapeutic delvery of ActRl polynucleotide sequences is the use of targeted 25 liposoies Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes vims, vacc inia, or preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivaive of a nurine or avian retrovirus Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited 30 to: Ialoney m urine leukemia virus (MoMu V), Harvey murine sarcoma virus (HaMuSV), murrine ramrmary tumor virs (MuN ) and Rous Sarcoma Virus (RSV), A number of - 53 additional retrovira vectors can incorporate multiple genes, All of these vectors can transfer or incorporate a gene for a selectablt niarke so that transduced cela can be identified and generated e Rtroviral vectors can be made targetsspeciwe by attaching, for example, a sugar, a glycolipid, or a protein, Preferred targeting is acconplished by using an anybody. Those 5 of skill in the art will ecognize that specific polynucleotide sequences can be inserted into the retroviral genomes or attached to a viral envelope to allew target specific delivery of the retroviral vector containing the ActRII polynucleotide fn ione preferred embodiment, the vector is targeted to bone, cartilage, nmscle or neuron cells/tissues Altemativel tissue culture cells can be directly transfcted with plasmids encoding 10 the retroviral structural genes gag, pol and env, by convention calcium phosphate transfection 'These cells are then transfected with the vector plasnid containing the genes of interest The resulting cells release the retroviral vector into the culture medium, Another targeted delivery systern for ActRIl polynuclekotides is a colloidal dispersion system Colloidal dispersion systems include macromolecule complexes, nanocapsules, 15 microspheres beads, and lipid-based systems including oiin-water emulsions, micelles, mixed micefles and liposomes, The preferred colloidal system of this invention is a liposoime. Liposones are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be dliered to ceds in a biologically active form (see e g Fraley, et al, Trends 20 Biochem Soi, 67, 1981), Methods for efficient gene transfer using a liposome vehicle, are known in the art, see e.g, Mannino, et al, Biotechniques, 6:682 1988, The coition of the liposome is usually a combination of phospholipids, usually in combination with steroids especially cholesterol Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on p1, ionic strength, and the presence of divalent 52 nations. Examples of lipids useful in liposomne production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylhol ine, phosphatidylse rine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides Illustrative phospholipids include egg phosphatidyicholine dipaliitoylphosphatdvrcholirne, and 30 distearoylphosphatidylchne. The targeting of liposomes is also possible based on, for example, organ-specicity cenapecificity. and organelle-specificity and is known in the art. - 54 - EXEMPLIFICATION The invention now being generally described, it will be more readily understood by reference to the tIlowing examples. which are included merly for purposes of illustration of certain embodinents and embodiments of the present invention, and are not intended to imit 5 the invention. Examile I. Generation of ActRIIB mutants: Applicants generated a series of mutations in the extracellular domain of ActRiEII and produced these mutant proteins as sohl fusion proteins bet ween extraelular ActR-B and an Fe domain A co, crystal stjeture of Activin and extraceiular ActRUB did not show any 10. role for the final (C-terminal) 15 anino acids (referred to as the "tail" herein) of the extracelluar domair in ligand binding. This sequence failed to resolve on the crystal stucture, suggesting that these residues are present in a flexible loop that did not pack unifornh in the crystal fTompson et al. EMBO J. 2003 Apr 1,22(7):1555-66- This sequence is also poorly conseved between ActRiUB and ActRIIA. Accordingly, these 15 residues were omitted inithe basic, or background, ActRUB~Fc fusion construct Additionaly, position 64 in the backgrmd frn is occupied by an aaime, which is generally considered the "wild type fonn, although a A64R allele occurs naturally. Thus, the background ActRiBFec fusion ias the sequence (Fe portion underined)(SEQ ID NO: 14); 20 S:RGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLBCYASWANSSGTIELVK KG3CWLEDPNCYDRQECVA-TE ENPQVYFCCCEGNF, WCNERPTHi-LPE ACGiiE C A.IYE.L GESV ELFEKP 112hua EEEE-_E'R'E QE Fn H N KTFKPREECOYNSTYRVVSVLT~ilD LGEKKSKLVIRIKKO REPOVYTLP SREEMT.OVSICLVKGFYPSDIAVFEN CFENN YKTTPPVLDS 25. ... . QQEL tiERQQysghE I MIKSSL SMIKE Surprisingly, as disVUssed below, the Genrina il was found to enhance activin and GDEI I binding, thus a preferred version of ActRUIB has a sequence (Fe portion underlined)(SEQ ID NO: 15), SCRGETRECIY YNANWELERTNQSGLERCEGEQDKRILNCYASWVAN$SGTIEI VK 30 KCWLDDFNCYDRQECVATEENEQVYFCCCEGNFCNERFTLPEAGGPEVTYEPPP TAPTO THTCPPCPAPEL OPSWVFLFP PKPKDTLMIS RTPE V TCV V VD VSHEDPEV - 55 - KFNWY'JD EVFHNAKTKPRBEQYN TRVVSVLTVLHQ WLNKEYKCKVSNKA LP PS OPEBNNYKTTPPViLDSDKSFFIXSKLTVDKSRWOOGl2NVFSCSVMHEALNUYlTQKSL SLSPGK 5 Various mutations were introduced into the background ActRIIBI, protein Mutations were generated in ActRIB extracelhuar doniain by PCRtmutagcnesis After PCR, fragments were purified thru Qgen column, digested with Sfol and Agel and gel purified, These fragments were ligated ho expression vector paid such that uponligation it created fusion chimera with human IGiO Upon transformation into E. coli DIIS alpha, colonies were 10 picked and DNAs were isolated Al mutants were sequence verified, All of the mutants were produced in HEK293T Ce by transient transfectionn summary, in a 500ml spinner. HEK293T cells were set up at ox I0 cells/mI in reestyle (Invirogen) media in 250rl volume and grown overnight 'Next daythese cells were treated with DNA:EI (1:1) complex at 0.5 ugml final DNA concentration After 4 hrs, 250 mil 15 media was added and eli were grown for 7 days, Conditioned media was harvested by spinning down the els and concentrated. All the mutants were purified over protein A column and eluded with low pH (3.0) glycine buffer. After neutralization, these were dialyzed against PBRS. Mutants were also produced in CHO cells by siar methodology; 20 Mutants were tested in binding assays and bioassays described below, Proteins expressed in CHO cells and HEK293 cells wem indistinguishable in the binng assays and bioassays, Exandie 2 CDF I and Acti" A Binding 25 Biding of ActRIIBFc proteins was tested in a BiaCorM assay. GiDF -or Activin A ("ActA.") were immobilized on a BiaCore CMS chip using standard amine coupling procedure The ActRIIB) -Fe mtant or wild-type protein was loaded onto the system, and biding was measured Results are surnuarized in Table I below. 30 Table 1 Soluble ActRiUB-Ft binding to GDFI I and Activin A (BiaCore assay) - 56 - ActRIB 3 AatAGD 1 WT} (64A7M WT (64R) naD 8,6e4M +15Stail KD 6S, e- 8M KI 1 Se.8M

--.-----------------------------

BE37A * A * R40A K55A R56A I * K74A KD4,35e-9 M KD) 53e9M K74F * Dgo * F82A + -- - -- ---- WT -inding ----- ---- - - - - - - -- -- -C $ <1/2 WT binding + w-r ++ - 2x increase-d binding -+-H+ ~- increased binding 10 +H 1--x increased binding ++- 40x increased binding As shown in able 1 stations had varyig effeas on ligand hinding he addition of the C-erminal 15 amino acids of the extracellular domain caused a substantial increase in 15 binding affinity for both Actaivin A and GDF-1, and it is expected that this effect will translate to essentially all of the other mutations Other mutations caused an overall increase in ligand binding affiMity, including the narally occurrinallllele A64R and K74A, The - 57 - R40A mutaton caused a moderate decrease in binding affini ty for both Activin A and ODF 1, Many mutations abolished detectable binding to Activin A and GDE- 1, including E37A. RS6A 78, 80, 80, D80A D800f' D80F. D)8GM and D)80N, Certain mutations caused a shift in selecivity The following matadons caused an increase in the 5 ratio of GDP-1 I to Activin A binding: K74Y K4F K741 and DSOL The following mutations caused a decrease in the ratio of GDF-I to Activin A binding DS4A, K5SA, I,9A and F2A man l ta~ gfrD-i I and Activinmediated sjgnaiinum 10 An A-204 Reporter Gene Assay was sed to evaluate the effects of ActRIB-e proteins on signaling by GDE-i - and Activin A. eU line: Human Rhabdomyosarcoma (derived from muscle), Reporter vector: pG3(CAGA)12 (Described in Dennier et al, 1998, EMBO 173091 310E) See Figure S. The CAGA1 naotif Is present in TGF-Beta responsive genes (PAl- I gene) , so tis vector is of general use for factors signaling through 15 Snnad2 and V Day I Sphit A-204 cells into 48-well plate. Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 or pGL3(CAA) I2(10 ug)+ pRLCMV (1 ug) and Fugene, Day : Add factors (dilated into median+ 0_1 % BSA), Inhibitors need to be 20 preincubated with Factors for 1 hr before adding to cells, 6 irs later, cells rinsed with PBS, and lvse cel's, This is followed by a Luciferase assay in the absence of any inhibitors, Actvin A showcd 10 fold stimulation of reporter gene expression and an ED5 -2 ng/mi. GDF-11: 16 fold stimulation, ED$0: 1 .5 ag/mil. 25 As shown in Figure 16, wild-type (background A64) ActRilIEc inhibits ODFI signaling in the A-204 Reporter Gene Assay, The background mA construct showed an inhibitory effect on GD-11 activity, The A64K mutation (also a naturally occurring frm) caused a substantial increase in GDE-I inhibition, and a combination of the A64K mutation with the addition of the 15 C-texnninal amino acids of the extracellular domain (the 15 amimo 30 acid tcai7) produced an even more potent inii- of DI activity. As shown in Figure - 58 - 17, the background A4 construct showed an inhibitory eMct on Activin A activity. The K74A mutation caused a substantial increase in Activin A inhibition; A control sample lacking AtivinA showed no atlivy. These data from the bioassay system correlate well with the binding assays shown in 5 Table 1 and demonstrate that the effects of the various mutations translate to a biological system. Example 4: ActRIAic Fusion Proteins As shown in Figure 14, ActRITA and ActRIIB are highly conserved 10 Accordingly, nost of the mutations tested in ActURB are expected to have similar effects in ActRKIA. Thus, a background ActRilA- fusion may be constructed with the following sequence (Fe porton undedined)(SEQ ID NO:6): A1LiRSETQECLFFM4-NW EKDRTNQOV IEPCYGDKDKRRHCFAT\VKN ISGSII2IVKQ GCWLDDINCYDRTDC\VEKKDSPVIYFCCCEGNMCN EKF2SYIPEMOI TC-PC PA 15 PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVINAK POVYTPPSREEMTKNOVSLTCLVKGFYPSDIAVEWESNG OPENNYKTPPVLDSDG SE\LY _V9RQONVFSCSVMHEAUNITQKSLSLSPGK As discussed beiow, the C-teminal tail was found to enhance activin and GDF-I 1 20 binding, thus a preferred version of ActRIA-Fk has a sequence (Fc portion underbned)(SEQ ID NO:17)Y AILGRSETQELFFNANWEKDRTNQTGVEPCYGDKKRRHCrATWKNISGSIVK.Q GCWL DDTNCY DRTDCVEKKDSPEVYPCCCEGNMCNEKFSYPPEMEVTQPTSNPVTP KPPGOGThTCPPCPAPEL LGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK 25 FNWYDQVEVHNAKTKPREC YQNTYRVV SVLIQD LNGKEXYCKVSNKAL -SP5K - 59 - Additional mutations, corresponding to those made in ActRUB, may be made in the background version of ActRHIA or the "taif" verAo of ActRJIA The corespondence between ActRITB and AcxRRA mutations s shown in Table 2 below. ActRilB Mutant mFutional Effect Corresponding ActRIA Mutant WT (64A) Backgrotnd. WT is.K65so K65A mutation is expected to decrease binding to all ligand\ WT (64R) nerease binding to all lgands K65 background 15tad Increase bindnig to al ligands, 1 5 tail EP7A L$indnate detectable binding to al ugnds. E38A R4 A1 Decrease binding to all ligands R41A - --- ------------------ D54A DDecease DFI /Activin b -ing ratio. D55A ---------------- ---- --- ------. . .. K55A Dcrease GDFI Activin binding ratio, K 6 R56A Elinina e detectable biding to all ugands R57A ----------_- -- --- .______ ..... ________________ K74A inrease binding to ail gand. K75A K74Y arease CF- /Activ in biding ratio K?5Y K74F Ncrease GDEI I/Activin binding ratio. K75F K741 increase GDF-1 /Activin binding ratio, K751 W78A lminate detectable binding to all ligand. \ W79A L79A Decrease ODF-II/Activin bi ending ratio L80A DS0R Eliminate detectable binding to all hgands. US!R D)80A Winirate detectable binding to all iganda D)81A D80F Eliminate detectable binding to all igands. 1 1Dh F D80Tiae detectable binding to all tgan D --- ----------- - ------------- 4------ ------ Eliminate detectable bind ngic a gandsl D8 1N - - -- -- ----- ---------- --.............-...t SOM. Eini ii atectL busding to) all liganda. I S IF DSOG E imninate detectable bi-nding to -all uigands- tDg N D80 rease 00 -l /Activin binding rao. D911 F82A Decrease GDE-I1/Activin binding ratio, h3A 5 - 60 - INCORPORATION BY REFERENCE All publicadmis and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent wasspeifically and individually indicated to be incorporated by reference. 5 While specific embodiments of the subject matter have been discussed, the above specification is ilustrative and not restrictive Many variations will become apparent to those skilled in the an upon review of this specification and the claims below. Ae M scope of the invention should be determined by reference to the claimsalong With their aill scope of equivalents, and the specification along with such vaiationas - 61 -

Claims (41)

1. A pharmaceutical preparation for rating an ActRi associated disorder, comprising; a) a soluble Actl(U polypeptide; and b) a pharnaceutially eptable carrier. 5 2 Tlhe pharmaceutical preparation of claim I wherein the solble ActRU polypeptide is selected from the group consisting of a) a polypeptide comprisng an amino acid sequence selected from SEQ ID NOs: 1-2 and 9~12; b) a polypeptide comprising an amino acid sequence at least 90% identical to an 10 amino acid sequence selectd from SEQ ID NOs: 1-2 and 9-12; and c) a polypeptide comparing at last 10 consecutive amino acids selected from SEQ D Os:1-2 and 9- 12, wherein said soluble ActRji polypepdde has one or more of the following characteristics: 15 i) binds to an ActRil ligand with a Kd of at least l M; and ii) inhibits ActRII signaling in a cell, 3 The pharmaceutical preparation of chim 2., wherein the ActRii ligand is selected from the group consisting of: activin, EM??, (IDs, GMG DFI, and Nodal

4. The pharmaceutical preparation of claim 1, wherein the solube AciRli polypeptide is 20 selected from the group counting of, a soluble ActR11A polypeptide and a soluble ActRUiB polyp epti de

5. The phanmacetical preparation of claim 1, wherein the soluble ActRR1 polypeptide comprises a mutation in SEQ ID NO: I or 2,

6. The pharmaceutical preparation of claim 5, wherein the mutation alters binding of an 25 ActRil ligand to the soluble ActRa polypeptide

7. The pharmaceutical preparation of claim 6. wherein the Asp amino acid residue at position 62 of SEQ ID N: 2 is mutated to an amino acid residue selected from the group consisting of: a uncharged amino acid, a negative amino acid, and a hydrophobic amino acid, - 62 -

8. The pharmaceutical preparation of claim 1, wherein said soluble AcRili polyepttide is a fusion protein including, in addition to an ActRil poiypeuptide domain, one or more polypeptide portions that enhance one or mre of nin stability in vivo half lite, uptak/administration, tissue localization or dstribution., formation of protein complexes, 5 and/or purification.

9. The pharmaceutical preparation of claim 8, wherein said fusion protein includes a poiypeptide portion selected front the grup consisting of an immnnoglobuin Fc domain and a serum albumin,

10. the pharmaceutical preparation of claim 8, wherein said fusion protein includes a 10 purification subsequence selected from: an epitope tag, a FLAG tag, a polyhistidine sequence, and a G$T fusion, II The phannaceutical preparation of claim 1,. wherein said soluble ActRl polypeptide includes one or more modified amino acid residues selected fron- a glycosylated amino acid a PEGylated amino acd a famesylated amino acid, an acetylated amio acid a biotinylated 15 amino acid, an amino acid conjdgated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent 12 The pharmaceutical preparation of claim .1 wherein said preparation is substantially pyrogen free.

13. The pharnaeuticaT preparation of claim I, wherein Che soluble ActRi polypeptide 20 binds to an Aciill ligand with a Kd of less than I micromolar,

14. A packaged phannaceuical comprising a pharmaceudial preparation of claim land labeled for usei prom oting gwth of a tissue or diminishing or preventing loss of a tissue in a haman, wherein the tissue is selected from the group consisting of bone, cartilage, muscle, fat, and neuron. 25 15 A packaged pharmaceutical comprising a pharmaceutical preparation of claim 1, and labeled for use in promoting growth of a tissue or diminishing or preventing loss of a tissue in a nonhuman, wherein the tissue is selected from the group consisting of: bone, cartilage, muscle, fat, and neuron.

16. A stabilized ActRi polypeptide comprising: a solible ActRil polypeptide and a 30 second portion comprising a stabilizig domain. - 63 - 17 The stabilized AcdR1 polypeptide of claim 16, wherein the second portion is a polypeptide covalently fused to the soluble ActR polypeptide. 18 The stabilized ActRil polypeptide of claim 17, wherein the second portion is a podypeptide fused to the carboxyl terininus of the soluble ActRU polypeptide, 5 19 The stabilized ActRIU polypeptide of claim 17, wherein the second portion is selected from the group consisting of: serum albumin and an lgG Fe domain.

20. The stabilized ActR polypeptide of claim 17, wherein the second portion is a non amino acid moiety 21 The stabilized ActRi1 polypeptide of claim 17, wherein the second portion comprises 10 polyethylene glycol,

22. An isolated polynucleotide comprising a coding sequence obr a soluble ActR1 polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from SEQ D NOs 1-2 and 9-12; 15 b) a polypeptide comprising an amino acid sequence at least 90% identical to an anino acid sequence selected from SEQ ID NOs: 12 and 9-12 and C) a polypeptide comprising at least' I consecutive amino acids selected from SEQ ID NOs: 1-2 and 9-12, wherein said soluble ActRul polypeptide has one or more of the following 20 characteristics: i) binds to an ActRE ligand with a Kd of at least 110's M; and ii) inhibits ActRil signaling in a cel.

23. An isolated polynucleotide, comprising: a) a sequence encoding an Actdl polypeptide; 25 b) a stop codon; and c) a sequence that is at least 90% identical to a sequence encoding an Actl polypeptidet - 64 - wherein the strip codon is positioned between the sequence of (a) and the sequence of (c) or within the sequence of (c).

24. An isolated polynucleotide comprising a polyrucleotide sequence selected from the group consisting of SEQ ID NOs: 7 and 8, said isolated polynucleotide further comprising a 5 non-natural transcription termination codon at least six hundred nucleotides before the 3' terminus,

25. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 22, 23 or 24,

26. A cell transformed with a recombinant polynucleotide of claim 25. 10 27. The cell of claimn 26, wherein the cell is a mammalian cell.

28. The cell of claim 27, wherein the cell is a human cell.

29. A method of making a soluble ActRII polypeptide, comprising: a) culturing a cell under conditions suitable for expression of the soluble ActRII polypeptide, wherein said cell is transformed with a recombinant polynucleotide of claim 25; 15 and b) recovering the soluble ActRII polypeptide so expressed,

30. A method for treating a subject having a disorder associated with muscle loss or insufficient muscle growth, comprising administering to the subject an effective amount of a soluble ActRIl polypeptide. 20 31. The method of claim 30, wherein the subject has muscle atrophy. 32, The method of claim 30, wherein the subject has a muscular dystrophy 33, The method of claim 30, wherein the subject has ALS 34, The method of claim 30, wherein the soluble ActRIl polypeptide is selected from the group consisting of: 25 a) a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 -2 and 9-42; b) a polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NoOs: 1-2 and 9-12; and - 65 - c) a polyeptide comprising at least 10 consecutive amino acids selected from SEQ ID NOs: P2 and 9-12, d) a stabilized ActRl polypeptide.

35. The method fdfaim 30,wherein the disorder is a muscle wasting disorder. 5 36, The method of claim 35wherein the disorder is selected from the group consisting of cachexia.anoxiaia DMD syndrome, BMD syndrome AIDS wasting syndromenmscular dystrophies neuwron-scular diseases, notor neuron diseases, diseases of the neurouscular jumtion. and inflammatory nyopathies. 37, The method of claim 35wherein the soluble ActRIlpolypeptide has one or more of 10 the following characteristics: i) binds to an ActRH ligand with a Kd of at least 10 i; and ii) inhibits ActRU signaling in a cell,

38. A method ior treating a subject having a disorder associated with neurodegeneration, comprising administering to the subject an effective amount of a soluble ActRII polypeptide. 15 39, The method of clam 38, wherein the disorder is selected from the group consisting of Alzheimer's Disease (AD);Parkinsons Disease (PD) Amyotrophic Lateral Scerosis (AIS), Huntington' s disease (HD).

40. The method of claim 38, wherein the soluble ActRil polypeptide is selected from the group consisting of: 20 a) a polypeptide comprising an rnino acid sequence selected from SEQ ID NOs: 12 and 9-12; b) a polypepfide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: -2 and 9-12; and c) a polypeptide comprising at least 10 consecutive amino adds selected from SEQ 2$ ID NOs 1-2 and 9 12, d) a stabilized ActRil polypeptide, - 66 - 41, A method for treating a subject having a disorder associated with abnormal cell growth and differentiation, conmprisig administering to the subject an effective amount of a solAble AcIRII polypeptide

42. The method of claim 41, wherein the disorder is selected from the group consisting of 5 inflammation, anergy. autoinuune diseases infectious diseases, and tumors,

43. The method of claim 41, wherein the soluble ActRil polypepfide is selected from the group consisting of, a) a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1-2 and 9-12; 10 b) a polypeptide comprising an amino acid sequence at last 90% identical to an amino acid sequence selected frorn SEQ ID NOs: 1-2 and 9-12; and c) a polypeptide comprising at least 10 consecutive amino acids selected from SEQ ID NIOs: 1-2 and 9-12, d) a stabilized ActII pohypeptide, 15 44, A method for increasing growth of a tissue or decreasing loss of' a tissue in a subje; comprising administering to the subject an effective amount of a soluble ActRiI praypeptide suficient to increase growth of the tissue or decrease loss of the tissue, wherein the tissue is selected from the group consisting of: bonecartilage muscle, fat, and neuron,

45. The method of claim44; wherein the soluble AciMi polypeptide is selected from the 20 group consisting of, a) a polypeptide comprising an amin acid sequence selected from SEQ ID NIOs: In2 and 9-12; b) a polypeptide comprising an amino acid sequence at least 90% identical to an aino acid sequence selected from SEQ ID NOs':1-2 and 942 and 25 c) a polypeptide comprising at least 10 consecutive amino acids selected from SEQ ID NOs: l-2 and 9-12, d) a stabilized ActRIl polypeptide. - 67 - 46, A method for decreasing the body fa content or reducing the rate of increase in body fat content in a subject, comprising administering to a subject in need thereof an effective amount of a sohble ActRi polypeptide

47. A method far treating a disorder associated with u desirable body weight gain in a 5 subject, comprising administering to a subject in need thereof an effetive amount of a sokable ActRIl polypeptide,

48. The method of claim 4 wherein said disorder is seleced from the group consisting of obesity, non-insulin dependent diabetes mellitus (,NDDM), cardiovascular disease, cancer hypertension, osteoarthritis, stroke, respiratory problems, and gall bladder disease. 10 494 The method of claim 46 or 47,whein the soluble ActRIl polypepride is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from SEQ ID NOs 1 2 and 912; b) a polypeptide comprising an amino acid sequence at least 90% identical to an 15 amino acid sequence selected from SEQ ID NOs: 1 2 and 9-12, and c) a polypeptide comprising at least 10 consecutive amino acids selected from SEQ ID NOs: 1-2 and 912, d) a stabilized ActRIl polypeptide.

50. A method of identifying an agent that stimulates growth of a tissue seleced from the 20 group consisting of: bone, cartilage, muscle, fat, and neuron, comprising a) identifying a test agent that binds to a ligand-binding domain of an ActRIl polypeptide competitively with a soluble ActRfl polypeptide; and b) evaluating the effect of the agent on growth of the tissue. 51 A method of antagonizing activity of an ActRIR polypeptide in a cell, comprising 25 contacting the cell with a sohuble AcRl polype tide.

52. A method of antagonizing activity of an ActRRl ligand in a cell, comprising contacting the cell with a solble ActRRl polypeptide, wherein the ActRi ligand is seIected from, the group consisting of: activin BMP7 iDES, .DFIl and Nodal. - 68 - 5$2 The method of claim 51 or 52,wherein the activity is mnmitored by a signaling transduction mediated by the ActRIWActRIl ligand complex.

54. The method of claim 51 or 52, wherein the activity is monitored by cell proliferation, 55 The method of claim 51 or 52, wherein the cell is selected from the group consisting 5 of an osteoblast, a chondrocyte, a myocyte an adipocyte, a muscle cell, and a neuronal cell, 56, Use of a soluble ActRII polypeptide for making a medicament for the treatment of a disorder associated with abnormal amount, development or metabolic activity of bone or cartilage tissue, comprising administering to a subject in need thereof an effective amount of a soluble ActRil polypeptide 10 57, Use of a soluble ActRI polypeptide for making a medicament for the treatment of a disorder associated with abnormal amount development or metabolic activity of muscle ssuec, omprising admtinisteng to a subject in need thereof an effective amount of a soluble ActRII polypeptide 58, Use of a soluble ActRIl polypeptide for making a mcdicament for the treatment of a 15 disorder associated with undesirable body fat content composing administering to a subject in need thereof an effective amount of a soluble ActRRI polypeptide

59. Use of a sohble ActRIl poiypeptide for making a medicament for the treatment of a disorder associated with neurodegeneration.comprising administering to a subject in need thereof an effective amount of a soluble ActRI polypeptide. 20 60. A GDF8 antagonist comprising an altered GDF8-binding domain of an ActRlU receptor that includes one or more mutations increasing the selectivity of the binding domain for GDF8 relative to a GDFS-binding domain of a wild-type receptor, 61 The GDFS antagonist of claim 60., wherein said one or more mutations increase the selectivity of the altered binding domain for GDF8 over activin 25 62. The GDFS antagonist of claim 61, wherein the altered binding domain has a ratio of 4 for activin bitiding to 4 for GDF8 binding that is at least 2 fold greater tbr the altered binding domain relative to the ratio for the GDF8-binding domain of a wild type receptor 63, The GDF8 antagonist of claim 61, wherein the altered binding domain has a ratio of - for activin binding to K4for GDFS binding that is at least $ fbld greater for the altered 30 binding domain relative to the ratio for the GDES-binding domain of a wild-type receptor: - 69 -

64. The GDF8 antagonist of claim 61 wherein the altered binding domain has a ratio of Ki for activin binding to K4 for GDFS binding that ia at least 1.0 fold greater for the altered binding domain relative to the ratio for the ODES-binding domain of a wild-type receptor, 65, The GDF antagonist of claim 61 wherin the altered binding domaIn has a ratio of 5 K for ac tivin binding to K for ODES binding that is at least 100 fold greater for the altered binding domain relative to the ratio for the GDESbinding domain of a wild-type receptor. 66 The GD8 antagonist of clain 6l, herein the altered binding domain has a ratio of ICso for inhibiting activin to It- for inhibiting i DF8 that is at least 2 fold greater for the altered binding domain than the ODFSinding domain of a wild-type receptor, 10 67. The GDF8 antagonist of claim 61, wherein the altered binding donain has a ratio of IC% for inhibiting activin to ICsj for inhibiting GDFS that is at least 5 fold greater for the altered binding domain than the ODE8-binding domain of a wildtype recptor. 65, The ODES antagonist of claim 61, wherein the alveed binding domain has a rati of lso for inhibiting activin to ICs for inhibiting ODF8 that is at least 10 fold greater for the 15 altered binding domain than the GDES-binding domain of a wild-type receptor,

69. The ODES antagonist of Claim 61, wherein the adered binding domain has a ratio of IC for inhibiting aetivin to so for inhibiting OF8 that sat least00 fold greater for the altered binding domain than the ODFS-binding doma in of a wild-type receptor

70. The GDF8 antagonist of Claim 60, which inhibits GDF8 with an 1050 at least 2 times 20 less than tie ICs, of the antagonist for inhibiting activin 71 The GDF8 antagonist of claim 60, which inhibits GDF8 with an ICsn at least 5 times less than the ICaO of the antagonist for inhibiting activin, 72, The ODF8 antagonist of claim 60, which inhibits GDF8 with an 1lf0 at least 10 times less than the ICs of the antagonist for inhibiting activin 25 73. The GDF8 antagonist of claim 60, which inhibits GDF8 with an ICao at least 100 times less than the IC- of the antagonist for inhibiting activin, 74, The ODES antagonist of any of claims 60, 61, 62, 66, and 70, wherein the altered GDF8 binding domain includes one or more stations in an ActRIIB polypeptide at residues selected from. the group consisting of E37,39, R40K 55 R56, Y60, A64, K.74, W78, 1,79, 30 DS0, F82 and F101, - 70 - 7$, The GDF8 antagonist of claim 60, wherein the GDF8 antagonist is a ftsion protein. 76, The GDFS antagonist of claim 75, wherein said altered GDES-binding domain of an ActRU receptor is fused to an IgG Fe domain,

77. The GDF8 antagonist of claim 76 wherein said IgG Fc domain comprises one or 5 more mutations, 78, The GDF8 antagonist of claim 77, wherein the Fe domain has reduced ability to bind to the Fey receptor relative to a wildtype FC domain.

79. The GDF8 anagonist of claims 77, wherein the Fc domain has incase ability to bind to the MC class i-related lk-receptor (FRN) elaive to a wild-type Fe domain. 10 80. The GDFS antagonist of claim 8flwherein the Fe domain has a mutation at residues selected from the group consisting of Asp-65, lysine 322, and Asn-434 SL The GDF8 antagonist of claim 77, wherein the mutation is shown in Figure 12, 82, The ODFS antagonist of claim 76, wherein the Ig Fe domain has an amino acid sequence as set forth in SEQ ID NO, 13, 15 83 A GDF8 antagonist comprising a GDFES-binding domain of an ActRII receptor fised to'an Fe domain, wherein the IgG Fe domain comprises one or more mutations,

84. The GDF8 antagonist of claim 83, wherein the .Fe domain has increase ability to bind to the MHC class I-related Fc-receptor (FcRN) relative to a wild-type Fc domain,

85. The GDF8 antagonist of claim 83, wherein the FEc domain has increase ability to bind 20 to the MUC class related Fr-receptor (FeRN) relative to a wild-tye Fe domain,

86. A method for treating a disorder associated with abnormal activity of GDE8 comprising administering to a subject in need thereof an eoietive amount of a souble A\ctRRl polypeptide, 87, The method of clai 36 wherein the disorder is selected from the group consisting 25 of; metabolic disorders such as type 2 diabetes, impaired glucose tolerance, metabolic syndrome (e g., syndrome X), and insun resistance induced by trauma (e.g, burns or nitrogen imbalance); adipose tissue disorders (e g, obesity) muscular dystrophy (including Duchenne 's muscular dystrophy) amyotrophic lateral sclerosis(ALS); muscle atrophy; organ atrophy; frailty; carpal tunnel syndrome; congestive obst-uctive pulmonary disease; - 71 - sarcopenia, cachexia and other muscle wasting syndromes; osteoporosis; glucocorticoid induced osteoporosis'; osteopenia; osteoarthritis; \steoporOsisrelated fractures; low bone mass due to chronic glucocorticoid therapy, premature gonadal failure., androgen suppression, vitania D deficiency, secondary hyperparathyroidisn ntzttonal deficiencies, and anorexia 5 nervous.

88. The GDF8 antagonist of any of claims 60, 61, 62, 66, and 70, wherein the altered GDFS binding domain includes one or more mutations in an ActRIJA polypeptide at residues selected from the group consisting of: E38 E4Q, R41 K56 (R57, Y61, K65. K75, W79, L80, D81, 183 and FP02 10 15 - 72 -