CA2290755A1 - The use of a bone morphogenetic protein (bmp) receptor complex for screening - Google Patents

Abstract

The present invention relates to the use of a type II receptor that is shared between activins and bone morphogenetic proteins, together with a bone morphogenetic protein type I receptor, for screening cellular differentiation actives. The invention further relates to cells comprising DNA coding for this receptor and to that DNA. The present invention relates to a method for determining whether a compound is capable of binding to a new BMP complex and whether a test compound produces a signal upon binding to this BMP receptor protein complex. The invention further relates to a method for determining the concentration of a BMP receptor ligand in a clinical sample using a new BMP complex. The invention further relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for components of this complex, or soluble fragments thereof.

Description

I
THE USE OF A BONE MORPHOGENETIC PROTEIN (BMP) RECEPTOR COMPLEX FOR SCREENING
TECHNICAL FIELD
The present invention relates to the field of bone formation and development and cellular differentiation Specifically, the present invention relates to the use of a type II receptor that is shared between activins and bone morphogenetic proteins, together with a bone morphogenetic protein type I receptor, for screening cellular differentiation actives. The invention further relates to cells co-transfected with DNA coding for this receptor and DNA coding for a type I bone morphogenetic protein receptor.
BACKGROUND
Humans and other warm-blooded animals can be afflicted by a number of bone-related disorders. Such disorders range from bone fractures, to debilitating diseases such as osteoporosis. While in healthy individuals bone growth generally proceeds normally and fractures heal without the need for pharmacological intervention, in certain instances bones may become weakened or may fail to heal properly. For example, healing may proceed slowly in the elderly and in patients undergoing treatment with corticosteroids (e.g., transplant patients).
Osteoporosis is a condition in which bone hard tissue is lost disproportionately to the development of new hard tissue. Osteoporosis can generally be defined as the reduction in the quantity of bone, or the atrophy of skeletal tissue; marrow and bone spaces become larger, fibrous binding decreases, and compact bone becomes fragile. Another bone related disorder is osteoarthritis, which is a disorder of the movable joints characterized by deterioration and abrasion of articular cartilage, as well as by formation of new bone at the joint surface.
While a variety of treatments are available for such bone-related disorders, none of the treatments provide optimum results. One of the difficulties facing individuals who treat bone-related disorders is a lack of complete understanding of bone metabolism and of the bone-related disorders. A key to such understanding is identifying and characterizing each of the components involved in bone growth.
Bone morphogenetic proteins (BMPs) have been demonstrated to play a role in bone formation and development (J. M. Wozney, Molec. Reproduct. and Develop., 32: 160-167 (1992); B.L.M Hogan, Genes c& Dev. 10: 1580-1594 (1996)).
Furthermore, the role of BMPs may not be limited to their role in bone. The finding that the BMPs are found at significant concentrations in other tissues such as brain, kidney, stratified squamous epithelia, and hair follicle (N.A. Wall, M.
Blessing, C.V.E. Wright, and B.L.M. Hogan, J. Cell Biol., 120: 493-502 (1993);
E.
Ozkaynak, P.N.J. Schnegelsberg, D.F. 3in, G.M. Clifford, F.D. Warren, E.A.
Drier, and H. Oppermann, J. Biol. Chem., 267: 25220-25227 (1992); K.M. Lyons, C.M.
Jones, and B.L.M. Hogan, Trends in Genetics, 7: 408-412 (1991); V. Drozdoff, N.A. Wall, and W.J. Pledger, Proceedings of the National. Academy of .Sciences.
U.SA., 91: 5528-5532 (1994)} suggests that they may play additional roles in development and differentiation. In support of this, BMPs have recently been found to promote nerve cell differentiation, to affect hair follicle formation, and have been implicated in cardiac and kidney development as well as the development of a variety of other organs (K. Basler, T. Edlund, T.M. 3essell, and T. Yamada, Cell, 73: 687-702 (1993); V.M. Paralkar, B.S. Weeks, Y.M. Yu, H.K. Kleinman, and A.H: Reddi, J. Cell Biol., 119: 1721-1728 (1992); M. Blessing, L.B. Nanney, L.E.
King, C.M. Jones, and B.L. Hogan, Genes Dev., 7: 204-215 (1993); A.T. Dudley, K.M. Lyons, and E.J. Robertson, Genes & Dev. 9:2795-2807(1995); G.C. Luo, A.L.J.J. Hofmann, M. Bronckers, A. Sohocki, A. Bradley, and G. Karsenty, Genes WO 98152038 PCT/fJS98/09519 & Dev. 9:2808-2820(1995); T.M. Schultheiss, J.B.E. Bunch, and A.B. Lasser, Genes & Dev. 11:451-462 (1997). B.L.M Hogan, Genes & Dev. 10: 1580-1594 ( 1996)).
A BMP initiates its biological effect on cells by binding to a specific BMP
receptor expressed on the plasma membrane of a BMP-responsive cell. A receptor is a protein, usually spanning the cell membrane, which binds to a ligand from outside the cell, and as a result of that binding sends a signal to the inside of the cell which alters cellular function. In this case, the ligand is the protein BMP, and the signal induces the cellular differentiation.
Because of the ability of a BMP receptor to specifically bind BMPs, purified BMP receptor compositions are useful in diagnostic assays for BMPs, as well as in raising antibodies to the BMP receptor for use in diagnosis and therapy. In addition, purified soluble BMP receptor compositions may be used directly in therapy to bind or scavenge BMPs, thereby providing a means for regulating the activities of BMPs in bone and other tissues. In order to study the structural and biological characteristics of BMP receptors and the role played by BMPs in the responses of various cell populations to BMPs during tissue growth/formation stimulation, or to use a BMP receptor effectively in therapy, diagnosis, or assay, purified compositions of BMP receptor are needed. Such compositions, however, are obtainable in practical yields only by cloning and expressing genes encoding the receptors using recombinant DNA technology. Efforts to purify BMP receptors for use in biochemical analysis or to clone and express mammalian genes encoding BMP receptors have been impeded by lack of a suitable source of receptor protein ' or mRNA. Prior to the present invention, few cell lines were known to express high levels of high affinity BMP receptors consisting of the receptor subunits described herein, which precluded purification of the receptor for protein sequencing or construction of genetic libraries for direct expression cloning. Availability of the BMP receptor sequence will make it possible to generate cell lines with high levels of recombinant BMP receptor for biochemical analysis and use in screening experiments.
The BMPs are members of the TGF-(3 superfamily. Other members of the TGF-(3 superfamily include TGF-Vii, activins, inhibins, Miillerian Inhibiting Substance, and the Growth and Differentiation Factors (GDFs) (B.L.M Hogan, Genes & Dev. 10: 1580-1594 (1996)). As expected, the receptors for various members of the TGF-(3 superfamily share similar structural features. Receptors of the TGF-(3 ligand superfamily are typically classified into one of twv sub-groups, designated as type I and type II. The type I and type II receptors are classified as such based on amino acid sequence characteristics. Both the type i and type II
receptors possess a relatively small extracellular ligand binding domain, a transmembrane region, and an intracellular protein kinase domain that is predicted to have serine/threonine kinase activity (Lin and Moustakas, Cellular and Molecular Biology, 40: 337-349 ( 1994); L.S. Mathews, Endocrine Reviews, 15:
310-325 (1994); L. Attisano, J.L. Wrana, F. Lopez-Casillas, and J. Massague, Biochimiccr et Biophysica Acta, 1222: 71-80 (1994); P. Ten Dijke, K. Miyazono, and C.-H. Heldin, Current Opinion in Cell Biology 8:139-145 {1996), H.
Yamashita, P. Ten Dijke, C.-H. Heldin, and K. Miyazono, Bone 19:569-574 (1996), J. Massague, F. Weis-Garcia, Cancer Surveys 27: 41-64 (1996)) The type I receptors cloned to date belong to a distinct family whose kinase domains are highly related and share > 85% sequence similarity (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)). The intracellular juxtamembrane region of the type I receptors is characterized by an SGSGSG
motif 35-40 amino acids from the transmembrane region, and the carboxy terminus of these receptors is extremely short (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994); L. Attisano, J.L. Wrana, F. Lopez-Casillas, and J.
Massague, Biochimica et Biophysica Acta, 1222: 71-80 (1994}). The extracellular domain of the type I receptors contains a characteristic cluster of cysteine residues, termed the "cysteine box", located within 25-30 amino acids of the transmembrane region, and another cluster of cysteine residues, termed the "upstream cysteine box", located after the putative signal sequence (B. B. Koenig, et al., Molecular and Cellular Biology, 14: 5961-5974 (/994); L. Attisano, et al., Biochimica et BiophysicaActa, 1222: 71-80 (1994); J. Massague, F. Weis-Garcia, Cancer Surveys 27: 41-64 (1996)).
Three distinct mammalian type I receptors have been reported for the BMPs:
Bone Morphogenetic Protein Receptor Kinase-1 (herein referred to as "BRK-1 ") (see U.S.S.N. 08/158,735, filed November 24, 1993 by J. S. Cook, et al.; and B.B.
Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)), ALK-2, and ALK-6. BRK-1 is the mouse homologue of human ALK-3, and is also known as BMPR-IA or TFR-1 I (see P. Ten Dijke, K. Miyazono, and C.-H. Heldin, Current Opinion in Cell Biology 8:139-145 (1996)); the rat homologue of BRK-1 has also been cloned {K. Takeda, S. Oida, H. Ichijo, T. Iimura, Y. Marnoka, T. Amagasa and S. Sasaki, Biochemical and Biophysical Research Communications, 204: 203-209 (1994}). BRK-I has been shown to bind both BMP-2 and BMP-4 more efficiently than it binds BMP-7, as measured by affinity labeling (J.M. Graff, R.S. Thies, J.J.
Song, A.J. Celeste, and D.A. Melton, Cell 79:169-179 {1994); B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994); A. Suzuki, R.S. Thies, N.
Yamaji, J.J. Song, J. M. Wozney K. Murakami, and N. Ueno, Proceedings of the National Academy of Sciences, U.S.A., 91:10255-10259 (1994); P. ten Dijke, H.
Yamashita, T.K. Sampath, A.H. Reddi, M. Estevez, D. L. Riddle, H. Ichijo, C.-H.
Heldin, and K. Miyazono, J. Biological Chemistry 269:16985-16988 (1994)), whereas it does not bind GDF-5 at all (H. Nishitoh, H. Ichijo, M. Kimura, T.
Matsumoto, F. Makishima, A. Yamaguchi, H. Yamashita, S. Enomoto, and K.
Miyazono, J. Biological Chemistry 271:21345-21352 (1996)). The binding properties of ALK-6, also known as BMPR-IB (see P. Ten Dijke, K. Miyazono, and C.-H. Heldin, Current Opinion in Cell Biology 8:139-145 (1996)) are similar to that of BRK-l, except that ALK-6 is also capable of binding GDF-5 (P. ten Dijke, H.
Yamashita, T.K. Sampath, A.H. Reddi, M. Estevez, D. L. Riddle, H. Ichijo, C.-H.
Heldin, and K. Miyazono, J. Biological Chemistry 269:16985-16988 (1994); H.
Nishitoh, H. Ichijo, M. Kimura, T. Matsumoto, F. Makishima, A. Yamaguchi, H.
Yamashita, S. Enomoto, and K. Miyazono, J. Biological Chemistry 271:21345-21352 (1996)). It is also postulated that ALK-6 is the mouse homologue of the chicken receptor Bone Morphogenetic Protein Receptor Kinase-2 (herein referred to as "BRK-2") (also referred to as RPK-1) (S. Sumitomo, T. Saito, and T. Nohno, DNA Sequence, 3: 297-302 (1993)). ALK-2, also known as ActRI, Tsk7L, or SKR1 (see P. Ten Dijke, K. Miyazono, and C.-H. Heldin, Current Opinion in Cell Biology 8:139-145 (1996)); K. Matsuzaki, J. Xu, F. Wang, W.L. McKeehan, L.
Krummen, and M. Kan, J. Biological Chemistry 268:12719-12723 (1993)) binds BMP-7 but does not bind BMP-4 or GDF-5 (K. Matsuzaki, J. Xu, F. Wang, W.L.
MeKeehan, L. Krummen, and M. Kan, J. Biological Chemistry 268:12719-12723 (1993); H. Nishitoh, H. Ichijo, M. Kimura, T. Matsumoto, F. Makishima, A.
Yamaguchi, H. Yamashita, S. Enomoto, and K. Miyazono, J. Biological Chemistry 271:21345-21352 (1996); P. ten Dijke, I-i. Yamashita, T.K. Sampath, A.H.
Reddi, M. Estevez, D. L. Riddle, H. Ichijo, C.-H. Heldin, and K. Miyazono, J.
Biological Chemistry 269:16985-16988 (1994)).
In contrast to the type I receptors, the kinase domains of the type II
receptors are only distantly related to one another. The SGSGSG motif found in type I
receptors is not found in type II receptors. Also, the "upstream cysteine box"
of type I receptors is not present in type II receptors. Furthermore, while all of the activin type II receptors contain a proline-rich sequence motif in the intracellular juxtamembrane region, there is no characteristic sequence motif that is common to all type II receptors (L.S. Mathews, Endocrine Reviews, 15: 310-325 (1994)).
The length of the carboxy terminus of the type II receptors is considerably variable, with the longest known carboxy terminus being found in the nematode BMP type II

receptor DAF-4 (M. Estevez, L. Attisano, J.L. Wrana, P.S. Albert, J. Massague, and D.L. Riddle, Nature, 365: 644-49 (1993)) that was cloned from C. elegans, and the mammalian BMP-specific type II receptor BRK-3 described in U. S. Patent application serial number 081334,179 by Rosenbaum and Nohno, incorporated herein by reference, also known as BMP-RII (B.L. Rosenzweig, T. Imamura, T.
Okadome, G.N. Cox, H. Yamashita, P. Ten Dijke, C.-H. Heldin, and K. Miyazono, Proceedings of the National Academy of Sciences, U.SA., 92: 7632-7636 (1995);
T.
Nohno, T. Ishikawa, T. Saito, K. Hosokawa, S. Noji, D.H. Wolsing, and J. S.
Rosenbaum, J. Biological Chemistry 270:22522-22526 (1995), F. Liu, F. Ventura, J. Doody, and J. Massague, Molecular and Cellular Biology, 15: 3479-3486 (1995}). The extracellular domain of the type II receptors contains a single cysteine box located near the transmembrane region. Aside from the presence of the cysteine box, there is little sequence similarity amongst the extracellular domains of the type II receptors for TGF-~3, activin, and BMPs.
Signaling by members of the TGF-~3 ligand superfamily requires the presence of both type I and type II receptors on the surface of the same cell (L.S.
Mathews, Endocrine Reviews, I 5: 310-325 ( 1994}; L. Attisano, J.L. Wrana, F.
Lopez-Casillas, and J. Massague, Biochimica et Biophysica Actrr, 1222: 71-80 (1994); P. Ten Dijke, K. Miyazono, and C.-H. Heldin, Current Opinion in Cell Biology 8:139-145 (1996), H. Yamashita, P. Ten Dijke, C.-H. Heldin, and K.
Miyazono, Bone I 9:569-574 ( 1996), J. Massague, F. Weis-Garcia, Cancer Surveys 27: 41-64 (1996)). Similar to what has been demonstrated for the TGF-(3 and activin receptor systems (for reviews see (T. Brand and M.D. Schneider, Circulation Research 78:173-179 (1996); P. Ten Dijke, K. Miyazono, and C.-H. Heldin, Current Opinion in Cell Biology 8:139-145 (1996)), BMPs bind to a heteromeric receptor complex consisting of a type I (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994); P. ten Dijke, H. Yamashita, T.K. Sampath, A.H.
Reddi, M. Estevez, D. L. Riddle, H. Ichijo, C.-H. Heldin, and K. Miyazono, J.

Biological Chemistry 269:16985-16988 {1994)) and a type II (B.L. Rosenzweig, T.
Imamura, T. Okadome, G.N. Cox, H. Yamashita, P. Ten Dijke, C.-H. Heldin, and K. Miyazono, Proceedings of the National Academy of Sciences, U.S.A., 92: 7632-7636 (1995); T. Nohno, T. Ishikawa, T. Saito, K. Hosokawa, S. Noji, D.H.
Wolsing, and J. S. Rosenbaum, J. Biological Chemistry 270:22522-22526 (1995), F.
Liu, F. Ventura, J. Doody, and J. Massague, Molecular and Cellular Biology, 15:
3479-3486 (1995); A. Letsou, K. Arora, J.L. Wrana, K. Simin, V. Twombly, J.
Jamal, K. Staehling-Hampton, F.M. Hoffmann, W.M. Gelbart, J. Massague, and M.B. O'Connor, Cell 80:899-908 (1995); E. Ruberte, T. Marty, D. Nellen, M.
Affolter, and K. Basler, Cell 80:889-897(1995); H. Yamashita, P. ten Dijke, D.
Huylebroeck, T.K. Sampath, M. Andries, J.C. Smith, C.-H. Heldin, and K.
Miyazono, J. Cell Biology 130:217-226 ( 1995)) receptor, and BMP-mediated signaling requires the presence of both the type I and type II receptors (F.
Liu, F.
Ventura, J. Doody, and J. Massague, Molecular and Cellular Biology, 15: 3479-3486 {1995); B.L. Rosenzweig, T. Imamura, T. Okadome, G.N. Cox, H. Yamashita, P. Ten Dijke, C.-H. Heldin, and K. Miyazono, Proceedings of the National Academy of Sciences, U.S.A., 92: 7632-7636 (1995); E. Ruberte, T. Marty, D.
Nellen, M. Affolter, and K. Basler, Cell 80:889-897(1995); H. Yamashita, P.
ten Dijke, D. Huylebroeck, T.K. Sampath, M. Andries, J.C. Smith, C.-H. Heldin, and K.
Miyazono, J. Cell Biology 130:217-226 (1995) P.A. Hoodless, T. Haerry, S.
Abdollah, M. Stapleton, M.B. O'Connor, L. Attisano, and J.L. Wrana, Cell 85:

500 ( 1996)}.
Unlike the TGF-(3 (L. Attisano, J. Carcamo, F. Ventura, F.M.B. Weis, J.
Massague, and J.L. Wrana, Cell 75:671-680 (1993); R. Ebner, R.-H. Chen, S.
Lawler, T. Zioncheck, and R. Derynck, Science 262:900-902 (1993); P. Franzen, P. ten Dijke, H. Ichijo, H. Yamashita, P. Schulz, C.-H. Heldin, and K.
Miyazono, Cell 75:681-692 (1993)) and activin (J. Carcamo, F.M.B. Weis, F. Ventura, R.
Wieser, J.L. Wrana, L. Attisano, and J. Massague, Molecular and Cellular Biology 14:3810-3821 (1994); R. Ebner, R.-H. Chen, S. Lawler, T. Zioncheck, and R.
Derynck, Science 262:900-902 (1993)) type I receptors, the type I receptors for BMPs are capable of binding ligand on their own when expressed in COS cells (J.M. Graff, R.S. Thies, J.J. Song, A.J. Celeste, and D.A. Melton, Cell 79:169-(1994); (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994);
A. Suzuki, R.S. Thies, N. Yamaji, J.J. Song, J. M. Wozney K. Murakami, and N.
Ueno, Proceedings of the National Academy of Sciences, U.S.A., 91:10255-10259 (1994); P. ten Dijke, H. Yamashita, T.K. Sampath, A.H. Reddi, M. Estevez, D.
L.
Riddle, H. Ichijo, C.-H. Heldin, and K. Miyazono, J. Biological Chemistry 269:16985-16988 (1994)), although binding affinity and/or crosslinking efficiency to the type I receptor is enhanced in the presence of the type II receptor (A.
Letsou, K. Arora, J.L. Wrana, K. Simin, V. Twombly, J. Jamal, K. Staehling-Hampton, F.M. Hoffmann, W.M. Gelbart, J. Massague, and M.B. O'Connor, Cell 80:899-908 (1995); F. Liu, F. Ventura, J. Doody, and J. Massague, Molecular and Cellular Biology, 15: 3479-3486 (1995); T. Nohno, T. Ishikawa, T. Saito, K. Hosokawa, S.
Noji, D.H. Wolsing, and J. S. Rosenbaum, J. Biological Chemistry 270:22522-22526 (1995); B.L. Rosenzweig, T. Imamura, T. Okadome, G.N. Cox, H.
Yamashita, P. Ten Dijke, C.-H. Heldin, and K. Miyazono, Proceedings of the National Academy of Sciences, U.S.A., 92: 7632-7636 (1995); H. Yamashita, P.
ten Dijke, D. Huylebroeck, T.K. Sampath, M. Andries, J.C. Smith, C.-H. Heldin, and K.
Miyazono, J. Cell Biology 130:217-226 (1995)). In further contrast to the TGF-~3 and activin receptor systems, crosslinking of BMP-2 or BMP-4 to the mammalian BMP type II receptor BRK-3 is not detectable in the absence of the type I
receptor, and only a very low level of binding is detectable at the whole cell level in cells transfected with the type II receptor alone (T. Nohno, T. Ishikawa, T. Saito, K.
Hosokawa, S. Noji, D.H. Wolsing, and J. S. Rosenbaum, J. Biological Chemistry 270:22522-22526 (1995)). Together, these data imply that, while BMPs might possibly bind to either subunit alone, high affinity binding and signaling are only WO 98!52038 PCT/US98/09519 i0 obtained when the appropriate heteromeric receptor complex is formed, and the differences in sequence of the various type II receptor cytoplasmic domains suggests that unique signaling receptor complexes will be produced by ligand binding to different type I: type II receptor subunit combinations. Hence, there is a need for identification of high affinity mammalian type I: type II BMP
receptor complexes in addition to the type I receptor and type II receptor complexes that have already been identified, if one is to use these receptor complexes in screening assays for the identification of novel cellular differentiation agents that act through an interaction with high affinity BMP receptor complexes.
We have previously reported the use of the type II BMP receptor kinase protein BRK-3 which enables the formation of a signaling complex with a BMP
type I receptor, as described in U. S. Patent application serial number 08/462,467 by Rosenbaum, incorporated herein by reference, and we have previously reported that the BRK-2 type I receptor forms a high affinity complex with BRK-3, whereas this is not the case when BRK-1 is the type I receptor {T. Nohno, T. Ishikawa, T.
Saito, K. I-Iosokawa, S. Noji, D.H. Woking, and J. S. Rosenbaum, J. Biological Chemistry 270:22522-22526 (1995)). This implies that additional mammalian high affinity BMP receptor complexes exist in which a type II receptor other than forms a complex with BRK-1 in response to BMP-2 and BMP-4 ligands. In the fruit fly Drosophila melanogaster the product of the 25DlTkv locus is the fly homologue of mammalian BRK-1 and BRK-2 that is capable of binding the Drosophila homologue of BMP-2 and BMP-4, the product of the decapentaplegic (Dpp) gene, as well as the mammalian BMP-2 (H. Okano, S. Yoshikawa, A. Suzuki, N. Ueno, M. Kaizu, M. Okabe, T. Takahashi, M. Matsumoto, K. Sawamoto, and K.
Mikoshiba, Gene 148: 203-209 ( 1994); A. Pelton, Y. Chen, K. Staehling-Hampton, J.L. Wrana, L. Attisano, J. Szidonya, J. A. Cassill, J. Massague, and F.M.
Hoffmann, Cell 78: 239-250 (1994)). The product of the Drosophila punt gene product, which was originally identified as an activin type II/type IIB
receptor homologue (S.R. Childs, J.L. Wrana, K.Arora, L. Attisano, M.B. O'Connor, and J.
Massague, Proceedings of the National Academy of Sciences, U.S.A., 90: 9475-(1993)), is required in concert with the Tkv receptor in order for Dpp to signal;
implying that Tkv and Punt form a signaling receptor complex in the presence of Dpp ligand (D. Nellen, R. Burke, G. Struhl, and K. Basler, Cell 85: 357-368 (1996);
E. Ruberte, T. Marty, D. Nellen, M. Affolter, and K. Basler, Cell 80:889-897(1995); A. Letsou, K. Arora, J.L. Wrana, K. Simin, V. Twombly, J. Jamal, K.
Staehling-Hampton, F.M. Hoffmann, W.M. Gelbart, J. Massague, and M.B.
O'Connor, Cell 80:899-908 (1995)). The mammalian activin type II receptor has been reported to bind BMP-7 with high affinity and to signal in concert with the ALK-2 or BRK-2 type I receptors, but a signal was not produced when BRK-1 was the type I receptor, suggesting that mammalian ActRII and BRK-1 do not form a signaling receptor complex (H. Yamashita, P. ten Dijke, D. Huylebroeck, T.K.
Sampath, M. Andries, J.C. Smith, C.-H. Heldin, and K. Miyazono, J. Cell Biology 130:217-226 ( / 995)).
The ActRIIB receptor exists in four distinct splice variants, described as ActRIIB~, AciRIIB2, ActRIIB3, and ActRIIB4, each of which is capable of binding activin ligand (L. Attisano, J.L. Wrana, S. Cheifetz, and J. Massague, Cell 68: 97-108 ( 1992)). We demonstrate here that there is an absolute requirement for the eight amino acids only present in the extraceilular juxtamembrane region of ActRIIB~
and ActRIIB2 in order for this type II receptor to bind BMP-2 and BMP-4 ligands and to form a complex with BMP type I receptors BRK-I and BRK-2 in the presence of BMP-4 or BMP-2 ligands. We further demonstrate that the BRK-1 + ActRIIB, complex binds BMP-4 Iigand with higher affinity than does the BRK-I type I
receptor alone, indicating that the BRK-1 + ActRIIB2 complex represents a high affinity complex for BMP-4, analagous to what is observed with the BRK-2 +
BRK-3 BMP receptor complex {T. Nohno, T. Ishikawa, T. Saito, K. Hosokawa, S.
Noji, D.H. Wolsing, and J. S. Rosenbaum, J. Biological Chemistry 270:22522-22526 {1995)), and that the BRK-I + ActRIIB2 complex therefore represents a distinct high affinity BMP receptor complex. Finally, we demonstrate that the BRK-1 + ActRIIB2 complex, described in detail below, is competent for signaling a response to BMPs. ActRIIB2 and ActRIIB, therefore can be used as BMP type II
receptors in concert with mammalian BMP type I receptors in order to identify novel compounds which interact with this BMP receptor complex and to determine if these novel compounds behave as BMP receptor agonists or antagonists which will be useful as therapeutic agents in humans and other mammals.
QBJECTS OF THE PRESENT INVENTION
It is an object of the present invention to provide a method for identifying compounds capable of binding to a BMP receptor kinase protein complex.
It is also an object of the present invention to provide a method for determining the amount of a compound capable of binding a BMP receptor kinase protein complex in a sample.
It is also an object of the present invention to provide a host cell comprising a recombinant expression vector encoding a BMP type II receptor kinase protein and a recombinant expression vector encoding a BMP type I receptor kinase protein comprising said BMP receptor kinase protein complex.
It is also an object of the present invention to provide a method for determining whether a test compound produces a signal upon binding to a BMP
receptor protein complex.
SUMMARY
The present invention relates to a method for determining whether a compound is capable of binding to a BMP receptor kinase protein complex, the method comprising introducing a sample comprising the compound to the BMP
receptor kinase protein complex and allowing the compound to bind to the BMP
receptor kinase protein complex, wherein the BMP receptor kinase protein complex is comprised of a BMP type I receptor kinase protein and the BMP/Activin type II

receptor kinase protein, comprising the eight amino acid juxtamembrane region, which is characteristic of ActRIIB~ or ActRIIB2.
The invention further relates to a method for determining the concentration of a BMP receptor ligand in a clinical sample, the method comprising introducing the sample comprising the ligand to a BMP receptor kinase protein complex and allowing the ligand to bind to the BMP receptor kinase protein complex, wherein the BMP receptor kinase protein complex is comprised of a BMP type I receptor kinase protein and BMP/Activin type II receptor kinase protein, ActRIIB~ or ActRIIB2.
The invention further relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for the BMP/Activin type II
receptor kinase protein ActRIIB2 and an expression vector comprising a DNA sequence that codes for a BMP type I receptor kinase protein.
The invention further relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for a soluble BMP type I receptor kinase protein and a soluble BMPIActivin type II receptor kinase protein ActRIIB2.
The invention further relates to a method for determining a test compound produces a signal upon binding to a BMP receptor protein complex, the method comprising: (a) transfecting BMP receptor protein complex expressing cells with a 3TP-Lux luciferase reporter gene (J.L. Wrana, L. Attisano, J. Carcamo, A.
Zentella, J. Doody, M. Laiho, X.-F. Wang, and J. Massague, Ce1171:1003-1014 (1992)) in conjunction with a beta-galactosidase gene, wherein the cells have been transfected with a DNA sequence coding for BMP receptor kinase protein ActRIIB, or ActRIIB2 and a DNA sequence coding for a BMP type I receptor kinase protein;
(b) culturing (i) a first set of the cells in the presence of the test compound, and (ii) a second set of the cells in the absence of the test compound; (c) quantitating via the arbitrary light units the level of luciferase activity produced by activation of the WO 98/52038 PC'T/US98/09519 luciferase enzyme that results from stimulation of the reporter construct produced from step (b); and (d) comparing the amount of arbitrary light units quantitated in step (c) from the first set of cells to the amount of arbitrary light units quantitated in step (c) for the second set of cells.
nF~~RIPTION
The present invention answers the need for a method for determining whether a compound has BMP receptor affinity. The method comprises introducing a sample comprising a test compound to a BMP receptor kinase protein complex and allowing the compound to bind to the BMP receptor kinase protein complex, wherein the receptor complex comprises a BMP type I receptor kinase protein and the BMP/Activin type II receptor kinase protein, generally refered to as ActRIIB, with anspecific eight amino acid juxtamembrane region, characteristic of a protein designated herein as "ActRIIB2". The invention also answers the need for a host cell that is co-transfected with an expression vector comprising a DNA
sequence that codes for BMP/Activin type II receptor kinase protein ActRIIB2 and an expression vector comprising a DNA sequence that codes for a BMP type I
receptor kinase protein. Also provided is a method for determining the concentration of a BMP receptor ligand in a clinical sample, the method comprising introducing the sample comprising the ligand to a BMP receptor kinase protein complex and allowing the ligand to bind to the receptor complex, wherein the receptor complex is comprised of a BMP type I receptor kinase protein and BMP receptor kinase protein ActRIIB2. The invention also answers the need for a host cell that is co-transfected with an expression vector comprising a DNA sequence that codes for a soluble BMP/Activin type II receptor kinase protein ActRIIB2 and an expression vector comprising a DNA sequence that codes for a soluble BMP type I receptor kinase protein.
As used herein, "mouse ActRIIB2" means a protein having the amino acid sequence SEQ ID N0:4, as well as proteins having amino acid sequences substantially similar to SEQ ID N0:4, and which are biologically active in that they are capable of binding a BMP molecule (including, but not limited to BMP-2, BMP-4, and/or BMP-7), or transducing a biological signal initiated by a BMP
molecule binding to a cell, or crossreacting with antibodies raised against ActRIIB2 protein, or peptides derived from the protein sequence of ActRIIB2, or forming a complex with a BMP type I receptor, or co-immunoprecipitating with a BMP type I receptor when antibodies specific for either ActRIIB2 or a BMP type I receptor are used.
As used herein, "mouse BMP receptor kinase protein" or "m-BRK-3" means a protein having amino acid sequence SEQ ID N0:12 or a sequence substantially similar to that sequence. Also included in this definition are proteins of this ilk which are biologically active in that they are capable of binding a BMP
molecule {including, but not limited to BMP-2, BMP-4, and/or BMP-7), or transducing a biological signal initiated by a BMP molecule binding to a cell, or crossreacting with antibodies raised against this protein, or peptides derived from the protein sequence of this protein, or forming a complex with a BMP type I receptor, or co-immunoprecipitating with a BMP type 1 receptor when antibodies specific for either this protein or a BMP type I receptor are used.
As used herein, "BMP receptor kinase protein BRK-3" or "BRK-3" refers individually and collectively to the receptor proteins h-BRK-3 (SEQ ID NO:10), and m-BRK-3 (and soluble and incomplete fragments of any of these). Also included in this definition are proteins of this ilk which are biologically active in that they are capable of binding a BMP molecule (including, but not limited to BMP-2, BMP-4, and/or BMP-7), or transducing a biological signal initiated by a BMP molecule binding to a cell, or crossreacting with antibodies raised against this protein, or peptides derived from the protein sequence of this protein, or forming a complex with a BMP type I receptor, or co-immunoprecipitating with a BMP type I
receptor when antibodies specific for either this protein or a BMP type I
receptor are used. Also included are BMP receptor kinase proteins substantially similar to h-BRK-3 and m-BRK-3 (and soluble and incomplete fragments as above). Such receptor proteins, DNA sequences coding for the proteins, and recombinant expression vectors comprising said DNA are described and claimed in U. S.
Patent application serial number 08/462,467 by Rosenbaum, incorporated herein by reference.
As used herein, a "BMP Type I Receptor Kinase" is a protein capable of binding BMP-2, BMP-4 and/or other known BMPs, and bears sequence characteristics of a type I receptor including, but not limited to, an extracellular ligand binding domain containing a cysteine box and an upstream cysteine box, an SGSGSG motif, designated the GS domain, in the intracellular juxtamembrane region, an intracellular kinase domain that is greater than about 85% similar to other type I receptors for other ligands in the TGF-(3 superfamily, and/or a relatively shorE
carboxy terminus. As used herein, "BMP Type I Receptor Kinase" also includes receptor proteins having the characteristics of a BMP type I receptor as described in the literature, such as in: B.B. Koenig et al., Molecular and Cellular Biology, 14:
5961-5974 (1994); L. Attisano, et al., Biochimica et Biophysica Acta, 1222: 71-{1994); J. Massague, L. Attisano, and J. L. Wrana, Trends in Cell Biology, 4:

178 (1994); and ten Dijke, et al., J. Biological Chemistry, 269: 16985-16988 (1994).
Examples of BMP type I receptors include, but are not limited to: BRK-1 (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994), the rat homologue of which is BMPR-Ia (K. Takeda, S. Oida, H. Ichijo, T. Iimura, Y.
Maruoka, T. Amagasa, and S. Sasaki, Biochem. Biophys. Res. Communicu., 204:
203-209 (1994)}; BRK-2, also referred to as RPK-1 (S. Sumitomo, T. Saito, and T.
Nohno, DNA Sequence, 3: 297-302 (1993), and postulated to be the chicken homologue of ALK-6 (P. ten Dijke, H. Yamashita, H. Ichijo, P. Franzen, M.
Laiho, K. Miyazono, and C.-H. Heldin, Science, 264: 101-104 (1994)); ALK-2, which has been shown to be a receptor for BMP-7 (ten Dijke et al., J. Biological Chemistry, 269: 16985-16988 (1994)}; the Xenopus BMP type I receptor that binds BMP-2 and BMP-4 and which is involved in mesoderm induction (J.M. Graff, R.S. Thies, J.J.
Song, A.J. Celeste, and D.A. Melton, Cell, 79: 169-179 (1994)); and type I
receptors from Drosophila that bind the decapentaplegic peptide, which is the Drosophila homologue of BMP-? and BMP-4. These Drosophila receptors are designated 25D1, 25D2, and 43E (T. Xie, A.L. Finelli, and R.W. Padgett, Science, 263: 1756-1759 (1994); A. Penton, Y. Chen, K. Staehling-Hampton, J. L. Wrana, L.
Attisano, J. Szidonya, J. A. Cassill, J. Massague, and F.M. Hoffmann, Cell, 78: 239-250 (1994); and T. J. Brummel, V. Twombly, G. Marques, J. L. Wrana, S. 3.
Newfeld, L. Attisano, J. Massague, M. B. O'Connor, and W. M. Gelbart, Cell, 78:
251-261 (1994)). Preferred BMP type I receptors useful in the present invention include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID N0:14 (BRK-1), SEQ ID N0:16 (BRK-2).
As used herein, "soluble fragment" refers to an amino acid sequence corresponding to the extracellular region of BRK-1, BRK-2, or the ActRIIB, preferably Act RIIB1 or ActRIIB2, which is capable of binding BMPs. Soluble fragments include the complete extracellular domain of the receptor protein, prior to the start of the predicted transmembrane region.
Examples of such soluble fragments for ActRIIB2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID N0:4, wherein amino acids 1-134 are present. Examples of such soluble fragments for ActRIIB2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID N0:4, wherein amino acids 1-134 are present. It is understood from these examples that by homology one can determine the essential and non-essential region, given these examples.
Examples of soluble fragments for BRK-1 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to amino acid residues 1-I53 in SEQ ID N0:14.

Examples of soluble fragments for BRK-2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to amino acid residues 1-126 in SEQ ID N0:16.
As used herein, "incomplete receptor kinase fragment" refers to an amino acid sequence corresponding to the extracellular, transmembrane, and intracellular juxtamembrane region of BRK-l, BRK-2, or the ActRIIB, preferably ActRIIBI or ActRIIB2, which is capable of binding BMPs in a manner similar to the full-length receptor, but which is incapable of signaling due to deletion of the intracellular kinase domain (otherwise known as a dominant negative receptor construct).
Examples of such incomplete receptor fragments for ActRIIB2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID N0:4; wherein amino acids I-161 are present and amino acids 162-191 are optionally present, for ActRIIBI, with amino acid sequences substantially similar to SEQ ID N0:2, amino acids 1-161 are present and amino acids 162-215 are optionally present.
Examples of incomplete receptor fragments for BRK-1 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID N0:14; wherein amino acids I-177 are present and amino acids 178-229 are optionally present.
Examples of incomplete receptor fragments for BRK-2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID N0:16; wherein amino acids I-149 are present and amino acids I50-199 are optionally present As used herein, a "BMP receptor kinase protein complex" is the combination of a BMP type I receptor and BMP receptor kinase protein ActRIIB2. The combination of the type I and ActRIIB2 receptors includes, but is not limited to, a combination of the type I and ActRIIB2 receptors in solution (e.g., as soluble fragments); a combination of the receptors (e.g., as soluble fragments) attached to a solid support; or a combination of the receptors {e.g., as full-length or incomplete fragments) within a cell membrane of transfected cells.
As used herein, "substantially similar" when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, a sequence altered by mutagenesis, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is to retain biological activity of the ActRIIB2 protein. Alternatively, nucleic acid sequences and analogs are "substantially similar" to the specific DNA sequence disclosed herein if the DNA sequences, as a result of degeneracy in the genetic code, encode an amino acid sequence substantially similar to the reference amino acid sequence. In addition, "substantially similar" means a receptor protein that will react with antibodies generated against the ActRIIB2 protein or peptides derived from the protein sequence of ActRIIB2.
"Homologues" are proteins that maintain the similar function and have substantially the same amino acid chain as those proteins listed in the sequence listings, however there may be innocuous substitutions in the chain that would not alter structure or function, for example, one hydrophobic amino acid for another, e.g., leucine for isoleucine; or an acidic amino acid for another, such as glutamic acid for aspartic acid, and the like. As such these proteins include whole proteins with at least 90% homology as understood by the art, with deletions and/or insertions or fragments thereof. For example, a rat protein which is 95%
homologous to that of a human based on the peptide sequence derived from the DNA or cDNA sequence, and a similarly derived bovine protein with the same function and similar homology, are both considered homologues. Thus homologous cDNAs cloned from other organisms give rise to homologous proteins.
Likewise proteins may be considered homologues based on the amino acid sequence alone. Practical limitations of amino acid sequencing would allow one to determine that a protein is homologous to another using for example comparison of the first SO amino acids of the protein. Hence 90% homology in would allow for differing amino acids in the chain of the first 50 amino acids of the homologous protein.
In addition, it is acknowledged that certain proteins have regions that are important to their function that are evolutionarily conserved. Such areas, may render the protein homologous. Thus the term "homologous" can be defined in terms of function or structure. Evidence of this evolutionary conservation is found in the group of proteins making up the complex. For example, type II receptors for members of the TGF-(3 superfamily. When expressed in COS cells, this human type II receptor is capable of forming differential heteromeric complexes with either the murine BRK-1 or the chicken BRK-2 type I receptors in the presence of BMP-4.
It has been demonstrated previously that murine and human BMP type I receptors form a complex with the nematode DAF-4 type II receptor (see Koenig, B. B., Cook, J.S., Wolsing, D.H., Ting, J., Tiesman, J.P., Correa, P.E., Olson, C.A., Pecquet, A.L., Ventura, F., Grant, R.A., Chen, G.-X., Wrana, J.L., Massague, J., and Rosenbaum, J.S., Mol. Cell. Biol., Vol. 14, pp. 5961-5974 (1994); and ten Dijke, P., Yamashita, H., Sampath, T.K., Reddi, A.H., Estevez, M., Riddle, D.L., Ichijo, H., Heldin, C.-H., and Miyazono, K., J. Biol. Chem., Vol. 269, pp. 16985-16988 (1994)), that a dominant negative construct of the murine BMP type I receptor BMPR-IA/TFR11/BRK-1 alters dorsal-ventral patterning when expressed in ventral blastomeres of Xenopus embryos (see Suzuki, A., Thies, R.S., Yamaji, N., Song, J.J., Wozney, J.M., Murakami, K., and Ueno, N., Proc. Natl. Acad. Sci. U.S.A., Vol.
91, pp. 10255-10259 (1994)), that the chicken BMP-related ligand dorsalin-1 induces alkaline phosphatase activity in murine bone marrow stromal cells (see Basler, K., Edlund, T., Jessell, T.M., and Yamada, T. Cell, Vol. 73, pp. 687-(1993)), and that the Drosophila BMP-2 and BMP-7 homologues, Dpp and 60A, induce ectopic bone formation in rats {see Sampath, T.K., Rashka, K.E., Doctor, J.S., Tucker, R.F., and Hoffmann, F.M., Proc. Natl. Acad. Sci. U.S.A., Vol.
90, pp.

6004-6008 (1993)). Given the high degree of sequence conservation among the proteins of this family, it is unlikely that the observed differential binding properties for the type I-type II receptor complexes previously described herein (T.
Nohno, T.
Ishikawa, T. Saito, K. Hosokawa, S. Noji, D.H. Wolsing, and J. S. Rosenbaum, J.
Biological Chemistry 270:22522-22526 (1995)) are due to the species differences of the type I receptors but are, rather, due to the unique nature of individual type Iaype II receptor complexes. These homologous proteins, whether from nematode, chicken, frog, or mammal are all contemplated in this invention. Mere allelic or interspecies variations do not appear to be significant enough to distinguish between such variations. Without being bound by theory, the differential binding properties described below most likely reflect the different signaling potential of the receptor complexes upon ligand binding. (see Carcamo, J., Weis, F.M.B., Ventura, F., Wieser, R., Wrana, J.L., Attisano, L., and Massague, J., Mol. Cell. Biol., Vol. 14, pp.
3 810-3 82 I ( 1994)).
The skilled artisan will appreciate that the degeneracy of the genetic code provides for differing DNA sequences to provide the same transcript, and thus the same peptide. In certain cases preparing the DNA sequence, which encodes for the same peptide, but differs from the native DNA include;
--- ease of sequencing or synthesis;
--- increased expression of the peptide; and --- preference of certain heterologous hosts for certain codons over others.
These practical considerations are widely known and provide embodiments that may be advantageous to the user of the invention. Thus it is clearly contemplated that the native DNA, or DNA listed in the SEQ ID listed here, or incorporated by reference, are not the only embodiment or the DNA envisioned in this invention.
As used herein, "biologically active" means that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the present invention disclosed herein to be capable of binding detectable quantities of or BMP-4, or transmitting a BMP-2 or BMP-4 stimulus to a cell, for example, as a component of a hybrid receptor construct. Preferably, a biologically active BMP
type I receptor: ActRIIB2 receptor complex within the scope of the present invention means the receptor protein kinase complex is capable of binding (1251_ BMP-4 with nanomolar or subnanomolar affinity (Kd approximately equal to 10-9M). Preferably, the affinity is from about 1x10-1OM to 1x10-9M, with a proportion of binding sites exhibiting a Kd less than 10-9M.
As used herein, "signal" or "signaling" refers to a biological response caused by some external stimulus, preferably related to binding of a molecule, including a small molecule, peptide or the like, that may be detected if instrumentation is sensitive enough and the correct parameter is measured. Examples of signaling include, the agonism or antagonism of an enzyme, the triggering or inhibition of a biochemical cascade, or the like.
As used herein, "operably linked" refers to a condition in which portions of a linear DNA sequence are capable of influencing the activity of other portions of the same linear DNA sequence. For example, DNA for a signal peptide (secretory leader) is operabiy linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, contiguous in reading frame. The skilled artisan will appreciate that the particular use of the DNA, whether it be to transfect a cell, perform site directed mutagenesis or some other use will determine how DNA sequences are used and how they are operably linked. This is well within the scope of the skilled artisan to determine.

As used herein, "ATCC" means American Type Culture Collection, Rockville, Maryland.
As used herein, "bone morphogenetic protein 2" or "BMP-2" means a peptide encoded by a DNA sequence contained in ATCC No. 40345 (see ATCC/NIH REPOSITORY CATALOGUE OF HUMAN AND MOUSE DNA PROBES AND
LIBRARIES, sixth Edition, 1992, p. 57, hereinafter "ATCC/NIH REPOSITORY
CATALOGUE"). Isolation of BMP-2 is disclosed in U.S. Patent No. 5,013,649, Wang, Wozney and Rosen, issued May 7, 1991; U.S. Patent No. 5,166,058, Wang, Wozney and Rosen, issued November 24, 1992; and U.S. Patent No. 5,168,050, Hammonds and Mason, issued December 1, 1992; each of which is incorporated herein by reference.
As used herein, "bone morphogenetic protein 4" or "BMP-4" means a peptide encoded by a DNA sequence contained in ATCC No. 40342 (see ATCC/NIH REPOSITORY CATALOGUE). Isolation of BMP-4 is disclosed in U.S.
Patent No. 5,013,649, Wang, Wozney and Rosen, issued May 7, 1991, incorporated herein by reference.
As used herein, "bone morphogenetic protein 7" or "BMP-7" means a peptide encoded by a DNA sequence contained in ATCC No. 68020 and ATT
68182 (see ATCC/NIH Repository Catalogue), where the cDNA in ATCC 68182 is claimed to contain all of the nucleotide sequences necessary to encode BMP-7 proteins. Isolation of BMP-7 is disclosed in U S. Patent 5,141,905, issued August 25, 1992, to Rosen, et al., which is incorporated herein by reference.
As used herein, "DNA sequence" refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such sequences are preferably provided in the form of an open reading frame uninterrupted by internal nontranslated sequences {introns) which are typically present in eukaryotic genes. Genomic DNA containing the relevant sequences could also be used. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions. DNA sequences encoding the proteins provided by this invention can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.
As used herein, "recombinant" means that a protein is derived from a DNA
sequence which has been manipulated in vitro and introduced into a host organism.
Such an organism may produce the protein naturally, or may be devoid of any mechanism for making the protein initially: preferably the host organism does not produce the protein in its normal state and hence is a "heterologous host."
Recombinant proteins can be made using bacterial, fungal (e.g., yeast), or insect expression systems.
As used herein, "recombinant expression vector" refers to a DNA construct used to express DNA which encodes a desired protein (for example, ActRIIBZ ) and which includes a transcriptional subunit comprising an assembly of 1 } genetic elements having a regulatory role in gene expression, for example, promoters and enhancers, 2) a structural or coding sequence which is transcribed into mRNA
and translated into protein, and 3) appropriate transcription and translation initiation and termination sequences. Using methodology well known in the art, recombinant expression vectors of the present invention can be constructed. Possible vectors for use in the present invention include, but are not limited to: for mammalian cells, pJT4 or pJT6 (discussed further below), pcDNA-1 (Invitrogen, San Diego, Ca) and pSV-SPORT 1 (Gibco-BRL, Gaithersburg, MD); for insect cells, pBlueBac III or pBlueBacHis baculovirus vectors (Invitrogen, San Diego, CA); and for bacterial cells, pET-3 (Novagen, Madison, WI). The DNA sequence coding for a ActRIIB2 protein receptor kinase of the present invention can be present in the vector operably linked to regulatory elements.
The present invention relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for BMP receptor kinase protein ActRIIB2 and an expression vector comprising a DNA sequence that codes for a BMP type I receptor kinase protein. In one embodiment, the expression vector for the mouse ActRIIB2 protein comprises a DNA sequence coding for the mouse ActRIIB2 receptor protein, or a soluble or incomplete fragment thereof. (The DNA
can be genomic or cDNA.) Preferably the mouse ActRIIB2 protein is coded for by the nucleic acid sequence SEQ ID N0:3.
In a preferred embodiment of the present invention, the host cells of the present invention are co-transfected with the plasmid construct pJT6-mActRIIB2 and the plasmid construct pJT4-J 159F (for BRK-1 ) or plasmid construct pJT3-BRK-2 (for BRK-2), thereby resulting in co-expression of mActRIIB2 and BRK-1, or mActRIIB2 and BRK-2, respectively. Transfection with the recombinant molecules can be effected using methods well known in the art.
As used herein, "host cell" means a cell comprising a recombinant expression vector described herein. Host cells may be stably transfected or transiently transfected within a recombinant expression plasmid or infected by a recombinant virus vector. The host cells include prokaryotic cells, such as Escherichia coli, fungal systems such as Saccharomyces cerevisiae, permanent cell lines derived from insects such as Sf 9 and Sf 21, and permanent mammalian cell lines such as Chinese hamster ovary (CHO) and SV40-transformed African green monkey kidney cells (COS).
In one embodiment, the present invention relates to a method that is useful for identifying compounds capable of binding to a BMP receptor kinase protein.
In WO 98!52038 PCT/US98149519 another embodiment, the invention relates to a method that is useful for determining the concentration of a BMP receptor ligand (e.g., BMP-2, BMP-4, or BMP-7, or another as-yet identified BMP receptor ligand) in a clinical sample. In each of these methods, a sample comprising a putative ligand or a known ligand is introduced to a BMP receptor kinase protein complex, wherein the receptor complex is comprised of a BMP type I receptor kinase protein and BMP receptor kinase protein ActRIIB2.
Preferably, the ActRIIB2 receptor kinase protein is m-ActRIIB2, having an amino acid sequence SEQ ID N0:4, or the soluble fragment thereof or the incomplete fragment thereof. For example, BMP concentration in a sample can be determined by radioreceptor assay, in which unlabeled BMP in the sample competes with labeled tracer BMP for binding to the ActRIIB2 + BMP type I receptor complex.
As the amount of BMP in the sample increases, it reduces the amount of labeled BMP
which is able to bind to the receptor protein complex comprising ActRIIB2 and the type I receptor. Comparison with a standard curve prepared with known concentrations of unlabeled BMP allows accurate quantitation of BMP
concentration in the sample. Labeling of tracer BMP is preferably done by iodination with [1251]NaI.
ActRIIB2 can be expressed in the outer membrane of a stable cell line which also expresses the BMP type I receptor kinase, or supplied as a soluble fragment in solution with a soluble type I receptor fragment, or as a soluble fragment covalently attached to a solid support in conjunction with a type I receptor covalently attached to a solid support. To perform the assay, unlabeled BMP from the sample and labeled tracer BMP compete for binding to the receptor until equilibrium is reached.
The receptor-BMP complex is then isolated from free ligand, for example by washing (in the case of an adherent cell line), rapid filtration or centrifugation (in the case of a nonadherent cell line or receptor bound to a solid support), or precipitation of the receptor-ligand complex with antibodies, polyethylene glycol, or other precipitating agent followed by filtration or centrifugation (in the case of a soluble receptor}. The amount of labeled BMP in the complex is then quantitated, typically by gamma counting, and compared to known standards. These methods have been described in the literature using other receptors (M. Williams, Med.
Res.
Rev., I 1: 147-184 (1991); M. Higuchi and B.B. Aggarwal, Anal. Biochem., 204:

58 (1992); M.J. Cain, R.K. Garlick and P.M. Sweetman, J. Cardiovasc. Pharm., 17:
S I 50-S 151 ( I 991 ); each of which are incorporated herein by reference), and are readily adapted to the present BMP type I receptor: ActRIIB2 receptor/BMP
system. Such a radioreceptor assay can be used for diagnostic purposes for quantitation of BMP in clinical samples, where such quantitation is necessary.
The methods of the present invention is also useful in high-throughput screens to identify compounds capable of binding to ActRIIB2, or a homologous receptor protein, that is complexed to a BMP type I receptor kinase protein.
In such a method, the higher the affinity of the compound for the ActRIIB2/type I
complex, the more efficiently it will compete with the tracer for binding to the complex, and the lower the counts in the receptor-ligand complex. In this case, one compares a series of compounds within the same concentration range to see which competed for receptor binding with the highest affinity.
This invention is useful for determining whether a ligand, such as a known or putative drug, is capable of binding to and/or activating the receptors encoded by the DNA molecules of the present invention. Transfection of said DNA sequence into the cell systems described herein provides an assay system for the ability of ligands to bind to and/or activate the receptor complex encoded by the isolated DNA molecules. Recombinant cell lines, such as those described herein, are useful as living cell cultures for competitive binding assays between known or candidate drugs and ligands which bind to the receptor and which are labeled by radioactive, spectroscopic or other reagents. Membrane preparations containing the receptor isolated from transfected cells are also useful for competitive binding assays.
Soluble receptors derived from the ligand binding domain of the receptor can also be employed in high-throughput screening of drug candidates. Functional assays of intracellular signaling can act as assays for binding affinity and efficacy in the activation of receptor function. In addition, the recombinant cell lines may be modified to include a reporter gene operably linked to a response element such that a signal sent by the receptor turns on the reporter gene. Such a system is especially useful in high throughput screens directed at identification of receptor agonists.
These recombinant cell lines constitute "drug discovery systems", useful for the identification of natural or synthetic compounds with potential for drug development. Such identified compounds could be further modified or used directly as therapeutic compounds to activate or inhibit the natural functions of the receptor encoded by the isolated DNA molecule.
The soluble receptor protein complex of the present invention can be administered in a clinical setting using methods such as by intraperitoneal, intramuscular, intravenous, or subcutaneous injection, implant or transdermal modes of administration, and the like. Such administration can be expected to provide therapeutic alteration of the activity of the BMPs.
SEQ ID N0:3 and SEQ ID N0:7 represent the DNA sequences coding for m-ActRIIB2 and mActRIIB4 receptor proteins, respectively, that were isolated from mouse hair/skin samples. These sequences could be readily used to obtain the cDNA for ActRIIB2 or ActRIIB4 from other species, including, but not limited to, human, rat, rabbit, Drosophila, and Xenopus.
The present invention further relates to a method for determining whether a test compound produces a signal upon binding to a BMP receptor protein complex.
Such a method comprises employing the BMP receptor protein complex in a transcriptional reporter assay. The method for determining whether a test compound produces a signal upon binding to the BMP receptor protein complex comprises (a) transfecting BMP receptor protein complex expressing cells with a 3TP-Lux luciferase reporter gene (J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J.
Doody, M. Laiho, X.-F. Wang, and J. Massague, Cell 71:1003-1414 (1992)) in conjunction with a beta-galactosidase gene, wherein the cells have been transfected with a DNA
sequence coding for BMP receptor kinase protein ActRIIB, or ActRIIB2 and a DNA sequence coding for a BMP type I receptor kmase protein; (b) culturing (i) a first set of the cells in the presence of the test compound, and (ii) a second set of the cells in the absence of the test compound; (c) quantitating via the arbitrary light units the level of luciferase activity produced by activation of the luciferase enzyme that results from stimulation of the reporter construct produced from step (b); and (d) comparing the amount of arbitrary light units quantitated in step (c) from the first set of cells to the amount of arbitrary light units quantitated in step (c) for the second set of cells.
In addition, this same type of assay may be done using autoradiography. It will be apparent to the skilled artisan that a method for determining whether a test compound produces a signal upon binding to the BMP receptor portion complex comprises (a) labeling BMP receptor protein complex expressing cells with 3~P, wherein the cells have been transfected with a DNA sequence coding for BMP receptor kinase protein ActIZIIB, or ActRIIB~ and a DNA sequence coding for a BMP type I receptor kinase protein; (b) culturing (i) a first set of the cells in the presence of the test compound, and (ii) a second set of the cells in the absence of the test compound; (c) quantitating via autoradiography any phosphorylated proteins produced from step (b); and (d) comparing the amount of phosphorylated proteins quantitated in step (c) from the first set of cells to the amount of phosphorylated proteins quantitated in step (c) for the second set of cells.
For purposes of illustrating a preferred embodiment of the present invention, the following non-limiting examples are discussed in detail.
A PCR probe is generated from degenerate primers JT65 (SEQ ID N0:17) and JT69 (SEQ ID N0:18) from conserved protein sequences in kinase domain II
(JT65) and in kinase domain VIII (JT69) of the known receptor serinelthreonine kinases. This probe was subcloned into pBLUESCRIPT vector (Stratagene, La Jolla, CA) to give pBS2-54 and used to screen against a NIH3T3 library, which has been previously described in U. S. Patent application serial number 08/462,467 by Rosenbaum, incorporated herein by reference. A partial ActRIIB clone (38-4-2) which contains approximately 2.3 kb of the ActRIIB sequence is isolated.
In order to isolate the full-length mouse homologue of ActRIIB, a cDNA library is constructed from mouse hairlskin tissues (E82 strain, 2 days after hair shaving).
Total RNA (1.8 mg) is isolated from the cells using a Total RNA Separator Kit (Clontech, Palo Alto, CA). Messenger RNA (6.3 ~cg) is isolated from this total RNA
(1 mg) using the mRNA Separator Kit (Clontech, Palo Alto, CA). An aliquot of the mRNA (2 fig) is used to make cDNA library using the SUPER SCRIPT Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. The resulting library contains approximately 500,000 primary colonies, and is divided into 72 pools, each containing 7,000 colonies.
The initial screen of the library is accomplished by PCR. Piasmids used as templete are purified from each of the 72 pools, using QIAGEN columns (Qiagen, Chatsworth, CA). Positive pools are identified by PCR with primers designed to amplify all ActRIIB isoforms as described previously (Tsung-Chieh, J.Wu, M.H.
Jih, L. Wang, and Y.-J. Y. Wan, Molecular Reproduction and Development 38: 9-15 (1994)); these primers are provided herein and referenced as ActRIIBTF (SEQ ID
N0:19) and ActRIIBTR (SEQ ID N0:20). The PCR reaction was performed using the GENE-AMP PCR Kit with AMPLITAQ DNA Polymerase (Perkin Elmer, Applied Biosystems, Foster City, CA). An initial melting period at 94°C for 5 min was followed by 30 cycles of the following program: melting at 94°C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min.

For secondary screening, plates are streaked with the E. coli stocks from three positive pools (2,000 colonies/plate). A HYBOND nylon membrane is placed on top of the plate so that the bacterial colonies are transferred to the filter. The colonies are then allowed to recover at 37 °C for 2-3 hr. The filter is soaked in 10 % SDS for 3 min, then transferred to 1.5 M NaCI, 0.5 M NaOH for 5 min, neutralized in 1.5 M
NaCI, 0.5 M Tris, pH 7.5 for 5 min, and washed in 3X SSC. To remove proteins, the blots are then shaken with 50 fcg/ml of proteinase K (Boehringer Mannheim, Indianapolis, IN) in 0.1 M Tris, pH 7.6, 10 mM EDTA, 0.15 M NaCI, 0.02 % SDS
at SS°C for 1 hr. The mouse partial ActRIIB cDNA isolated from NIH3T3 library (clone 38-4-2) is cut with Mlu I and to give a 2.3 kb fragment. The fragment is randomly labeled with a-(32P]-dCTP having a specific activity of 3000 Cilmmol (NEN
Research Products, Boston, MA), using a PRIME-IT II Random Primer Labeling Kit {Stratagene, La Jolla, CA; a kit for random primer labeling of DNA, including Klenow DNA polymerase, primers, and buffers). The labeled probe is allowed to hybridize to the filters for 18 hr at 42°C in hybridization buffer (Sigma, St.
Louis, MO) consisting of 50% deionized formamide, 5 X SSPE (lx SSPE = 0.14 M NaCI, 8 mM sodium phosphate, 0.08 mM EDTA, pH 7.7), IX Denhardt's solutions, and 100 ~,g/ml of denatured salmon testis DNA. The blot is then washed in 0.25X SSPE, 0.5 %
sodium dodecyl sulfate (SDS), two times at 25°C for IS min each, then two times at 65°C for 30 min each. The blot is then exposed to Kodak X-OMAT AR autoradiography film for 18 hr at -80°C.
Colonies which corresponded to labeled spots on the autoradiograph are streaked on plates for tertiary screening, which is performed exactly as described above for secondary screening. Three positive clones are isolated.
The inserts from the 3 positive clones are sequenced using the TAQ DYE
DEOXY Terminator Cycle Sequencing Kit and an Applied Biosystems Model 373A
Automated DNA Sequencer. Comparison of the sequences shows that clone A46-3/pSPORT contains the complete coding of the ActRIIB4 variant whereas clone 8IpSPORT aligns with the ActRIIB2 variant approximately 50 base pairs from the beginning of the coding region (see Example 2 below).
The DNA sequence of this ActRIIB2 clone A49-8lpSPORT with the missing -- 50 by of coding region (assembled within the pJT6 expression vector as described below in Example 3) is shown in SEQ ID N0:3, and the deduced protein sequence of ActRIIB2 in SEQ ID N0:4. The sequence is identical to the marine ActRIIB2 sequence listed in GENBANK as accession number M84120 and described in Cell 68: 97-108 (1992), beginning at the ATG (base 44) of the published ActRIIB sequence through -900 bases, as well as the 3' end sequence (1460-1708 bases). For ActRIIB2, we have data confirming the sequence at ATG (base 44) through 900 bases, as well as the 3' end sequence (1460-1708). The sequencing data verifies this is the ActRIIB2 variant as it contains the first insert (bp 413-436).
The ActRIIB4 variant does not contain either of the inserts, as indicated by the DNA sequence of the A46-3lpSPORT clone shown in SEQID NO: 7 and the protein sequence shown in SEQ ID NO: 8. For ActRIIB4, the clone isolated was full length and in addition contained - 300 bases of unknown sequence 5' to the ATG at by 44.
('onstruction of expression vectors for m-BRK-3.
~~-1 RRK-2 ActRIIB~ and ActRIIB3 The pJT4 expression vector has been previously described in U. S. Patent application serial number 08/462,467 by Rosenbaum, incorporated herein by reference.
This expression vector has been optimized for transient expression in COS
cells, and includes the cytomegalovirus early promoter and enhancer, which gives very efficient transcription of message; an "R" element from the long terminal repeat of the human T-cell leukemia virus-1, which has been shown to increase expression levels further; an intron splice site from SV40, which is believed to enhance message stability;
a multiple cloning site; a polyadenylation signal derived from SV40, which directs the addition of a poly A tail to the message, as is required for most eukaryotic mRNA; and the origin of replication, which permits the replication of the plasmid to extremely high copy number in cells which contain the SV40 large T antigen, such as COS
cells. In addition, for manipulation and amplification of the vector in bacteria, the vector contains an E. coli origin of replication and an ampicillin resistance gene.
The expression vectors described herein are derivatives of the pJT4 expression vector, and are described below.
For mammalian expression, m-ActRIIB4 is subcloned into the mammalian expression vector pJT6. This vector is a derivative of pJT3, described in in U. S.
Patent application serial number 081462,467 by Rosenbaum, incorporated herein by reference, (see Example 4) in which the Not I site at the 5' end of the multiple cloning site has been deleted, and a spacer inserted between the Pst I and BamIiI
restriction sites in the multiple cloning site. To accomplish the subcloning, m-ActRIIB4 cDNA is excised from A46-3/pSPORT using Not I and Sal I, then subcloned into pJT6 at the Not I and Sal I sites to generate pJT6-A46L.
In order to assemble a full length clone of mouse ActRIIB2 variant in the pJT6 expression vector, a Sal I site is first placed at the 5' end of clone A46-3/pSPORT as follows. A primer is synthesized which contains a Sal I site followed by nucleotides 1-15 of the coding sequence of A46-3/pSPORT; the sequence of the primer is 5' ATC
GTC GAC CAT GAC GGC GCC CTG G 3' (SEQ ID N0:21). This is used together with the reverse primer, 5' GGG CGG AGG CCC CGG GTC 3' (SEQ ID N0:22), in order to amplify a DNA fragment using plasmid DNA from clone A46-3/pSPORT as the template. PCR is performed using the GENE-AMP PCR Kit with AMPLITAQ
DNA Polymerise Polymerise (Perkin Elmer, Applied Biosystems, Foster City, CA).
An initial melting period at 94°C for 5 min was followed by 30 cycles of the following program: melting at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. After the last cycle, the reaction was held at 72°C for 5 min to WO 98!52038 PCT/US98/09519 complete extension. The fragment amplified from A46-3/pSPORT is inserted into pJT6 vector as follows. The amplified fragment from A46-3lpSPORT is digested with Sal I
and Apa I. The insert from A49lpSPORT is digested with Apa I and Not I. The vector pJT6 is digested with Sal I and Not I. The three fragments are combined in a three-way ligation using T4 DNA ligase (3 hr, 25 °C) and used to transform electrocompetent E. coli, strain DHS-a, using a BIO-RAD Gene PULSER (BIO-RAD, Hercules, CA) according to the manufacturer's instructions. A positive colony is selected and is designated pJT6-A49. Sequencing of the S' portion of the insert that was amplified by PCR shows a sequence identical to that of clone A46-3lpSPORT, indicating that no mutations are introduced during the amplification.
The construction of the expression plasmids for the mBRK-3, mBRK-1, and chicken BRK-2 clones has been previously described in detail in U. S. Patent application serial number 081462,467 by Rosenbaum, incorporated herein by reference.
To determine the effects of co- _expression of mActRIIB, or mActRIIB4 with type I BMP receptors, it is necessary to co-express the cDNA for mActRIIB2 or mActRIIB4 with the cDNA for BRK-1 or the cDNA for BRK-2. The DNA sequence for mouse BRK-1 is shown in SEQ ID NO: 13, and the deduced amino acid sequence for mouse BRK-1 is shown in SEQ ID N0:14. The DNA sequence for chicken BRK-2 is shown in SEQ ID NO: 15, and the deduced protein sequence shown for chicken BRK-2 is shown in SEQ ID NO: 16. To compare the binding and signaling properties of mActRIIB2 with m-BRK-3 previously described in detail in U. S. Patent application serial number 08/462,467 by Rosenbaum, incorporated herein by reference, data obtained after co-expression of the type I receptors with BRK-3 are included in the following examples where indicated, for comparison purposes. The DNA sequence for mouse BRK-3 is shown in SEQ ID NO: I 1, and the deduced amino acid sequence for mouse BRK-3 is shown in SEQ ID N0:12.
Fxa'~mnle 4.
L~~~~~~~iian eynrPCSi~n of mActRIIB~ mArtRIIBa, m-RRi~-3. BR_K-1. and BWK-2 Transient expression of the receptors in mammalian cells using the expression plasmids described above is carried out in COS-1 cells (ATCC CRL 1650) or cells (J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J. Doody, M. Laiho, X.-F.
Wang, and J. Massague, Cell 71:1003-1014 (1992)) for the binding and immunoprecipitation studies (Examples 6-8 below) or RIBIL-17 cells (J.L.
Wrana, L.
Attisano, J. Carcamo, A. Zentella, J. Doody, M. Laiho, X.-F. Wang, and J.
Massague, Cell 71:1003-1014 (1992)) for the signaling assays (Example 9), using DEAE Dextran (Pharmacia Biotech, Piscataway, NJ).
For transient expression of BMP receptors in COS-1 cells, the cells are grown to approximately 70%-90% confluencey in DME high glucose media (Life Technologies, Gaithersburg, MD) supplemented with 10 % fetal bovine serum (HyClone, Logan, Utah), nonessential amino acids, and glutamine in T-175 flasks (Corning, San Diego, CA). The cells are washed twice with 37~C serum-free DME
media, after which 10 ml of DNA mixture is added to each T-175 flask. The DNA
mixture contains DME, 10% Nu-Serum (Collaborative Biomedical Products, Bedford, MA), 400 ~.g/ml DEAE-Dextran (Pharmacia, Piscataway, NJ), 0.1 mM chloroquine (Sigma, St. Louis, MO), and the cDNAs of interest: for mActRIIB2, 40 pg of pJT6-mActRIIB,, for mActRIIB4, 40 pg of pJT6-mActRIIB4, for mBRK-3S, 40 pg of pJT6-mBRK-3S, for BRK-1, 20 ~.g pJT4-J159F; for BRK-2, 20 ~,g pJT3-BRK2. When the experimental design requires transfection of a single receptor only, the empty expression vector pJT6 or pJT4 (described in detail in U.S. Patent Application Serial Number 081462,467 by Rosenbaum, incorporated herein by reference) is substituted for the corresponding receptor cDNA. The cells are then incubated at 37~C with the DNA
mixture for 3 hr. The solution is aspirated and the cells are incubated with 10 ml of a solution containing 10% dimethylsulfoxide (DMSO) in Dulbecco's phosphate buffered saline without calcium or magnesium (PBS; Life Technologies, Gaithersburg, MD).
After 2 min, the DMSO solution is aspirated, the cells are washed with the growth media described above, and fresh media is returned to the plates. The transfected cells are split into 12 well plates 24 hr post transfection for whole cell binding (Example 6) or 100 mm plates for affinity labeling and immunoprecipitation (Example 7). 36 to 72 hours after transfection the cells are suitable for binding analysis.
For transient expression of BMP receptors in RIBIL-17 cells for the binding and affinity labeling/immunnoprecipitation studies, the conditions are identical to that described above for the COS-1 cells except that MEM media (Life Technologies, Gaithersburg, MD) is used in place of DME-high glucose media, and R1B/L-17 cells were transfected 24 hours after seeding in a T-175 flask (Corning, San Diego, CA) at S-10 x 106 cells per flask. For transient expression of BMP receptors in RIBIL-cells for the 3TP-Lux assay, the conditions are identical to that described immediately above, except that 30 pg of the 3TP-Lux reporter plasmid (J.L. Wrana, L.
Attisano, J.
Carcamo, A. Zentella, J. Doody, M. Laiho, X.-F. Wang, and J. Massague, Cell 71:1003-1014 (1992)) and 15 ~g of the pCMV~i ~i-galactosidase expression plasmid (Clontech, Palo Alto, CA) are added in addition to the receptor plasmids. When the experimental design requires transfection of a single receptor only, the empty expression vector pJT6 or pJT4 (described in detail in U.S. Patent Application 08/462,467 by Rosenbaum and incorporated herein by reference) is substituted for the corresponding receptor cDNA.
hxa~nle 5 C'=Pn ration of the Radiolabeled BMP-4 Li~and Recombinant human BMP-2 and BMP-4 dimers are produced and purified from CHO cells as previously described (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)). (125I~-BMP-4 is prepared using IODOBEADS
(Pierce, Rockford, IL; immobilized chloramine-T on nonporous polystyrene beads).
Lyophilized BMP-4 (2 ~.g) is taken up in 50 ~.l of 10 mM acetic acid and added to 450 ~,1 of phosphate-buffered saline (PBS) (Sigma, St. Louis, MO) on ice. To the tube is added S00 Curie of 1251 (Amersham, Arlington Heights, IL) (2200Ci/mmol) in 5 ~1, and one IODOBEAD. The reaction is incubated on ice for 10 min with occasional shaking. The reaction is then terminated by removal of the reaction from the IODOBEAD. To remove unreacted 1251, ~e mixture is applied to a PD-10 gel filtration column (Pharmacia, Piscataway, NJ) previously equilibrated in 10 mM
acetic acid, 0.1 M NaCI, 0.25 % gelatin. The resulting labeled protein is > 95 %
precipitable by trichloroacetic acid, indicating that all 1251 is protein bound, and has a typical specific activity of 4000 to 9000 Ci/mmol.
Alternatively, BMP-4 is labeled with 1251 by the chloramine-T method (C.A.
Frolik, L.M. Wakefield, D.M. Smith, and M.B. Sporn, J. Biol. Chem., 259: 10995-11000 (1984)). BMP-4 (2 ~cg) is taken up in 5 p.l of 30% acetonitrile, 0.1 trifluoracetic acid (TFA) plus an additional S ~.1 of 1.5 M sodium phosphate, pH 7.4.
Carrier free 1251 (I mCi, 9 ~.1) is added, together with 2 ~,l of a chloramine T solution (100 ~cg/ml). An additional 2 ~,1 of the chloramine T solution is added at 2.0 min and at 3.5 min. After 4.5 minutes, the reaction is stopped by the addition of 10 ~l of 50 mM N-acetyl tyrosine, 100 ~,1 of 60 mM potassium iodide, and 100 ~,1 of 11M
urea in 1 M acetic acid. After a 3.5 minute incubation, unreacted iodine is removed on a gel filtration column (Pharmacia, Piscataway, NJ) run in 10 mM acetic acid, 0.1 M
NaCI, 0.25 % gelatin. The resulting labeled protein is > 95 % precipitable by trichloroacetic acid, indicating that all 1251 is protein bound, and has a typical specific activity of 3000-8000 Ci/mmol.
('haracterization of BMP-4 Binding; Affinit~to mActRIIB~in the re~sence of Binding of BMP-4 to mActRIIB2 in the presence of the BMP type I receptors can be demonstrated by whole cell binding of radiolabeled BMP-4, and by covalent crosslinking (affinity labeling) and immunoprecipitation of radiolabeled BMP-4 to the receptor. These two methods are described in detail in this Example and in Example 7 below.
Whole Cell Binding Competition Analysis:

COS-1 cells are transfected with pJT6-mActRIIB2 for mActRIIB2 expression, or pJT6-mActRIIB4 for mActRIIB4 expression; in the presence of pJT4-J159F for' expression, or pJT3-BRK2 for BRK-2 expression, as described in Example 4.
After transfection, cells are seeded into 12 well plates and the binding experiments are carried out at 24 to 36 hr. after plating. At that time, cells are washed once with binding buffer (SO mM HEPES, pH 7.4, 128 mM NaCI, 5 mM KCI, 5 mM MgS04, 1.2 mM CaCl2, 2 mg/ml BSA), then equilibrated in the same buffer at 4°C
for 30 - 60 min with gentle shaking. The buffer is then aspirated, and to each well is added 500 p,l of binding buffer (4° C), containing (125I~_gMP-4 tracer (100 - 400 pM), as well as varying concentrations of unlabeled BMP-2, BMP-4, or other unlabeled ligand, depending on the assay. For determination of nonspecific binding, BMP-4 is added to the binding buffer at a final concentration of 10 to 50 nM. To prevent degradation of ligand during the incubation, a protease inhibitor cocktail is also added, to give a final concentration of 10 ~glml leupeptin, 10 ~.glml antipain, 50 ~cglml aprotinin, 100 ~cglml benzamidine, 100 ~,g/mi soybean trypsin inhibitor, 10 ~g/ml bestatin, 10 ~cg/ml pepstatin, and 300 uM phenylmethylsulfonyl fluoride (PMSF}. The cells are incubated for 4 hr at 4°C with gentle shaking. At the end of the incubation period, the buffer is aspirated, and the cells are rinsed 4 times with 1 ml washing buffer (50 mM
HEPES, pH 7.4, 128 mM NaCI, 5 mM KCI, 5 mM MgS04, 1.2 mM CaCl2, 0.5 mg/ml BSA).
After the final wash is aspirated, 200 wl of RIPS buffer (20 mM Tris Base, 100mM
NaCI, 1 mNi EDTA, 0.5 % NP-40, 0.5 % Deoxycholic Acid, 0.1 % SDS, 10 mM NaI, 1 % BSA, pH 8.0) is added to each well and incubated at room temperature for min. The solubilized cells are then transferred to fresh tubes and counted in a Packard Model 5005 COBRA Gamma Counter (Packard Instruments, Meriden, CT).
The results of a [lzsl]BMP-4 competition experiment that compares the binding affinity of unlabeled BMP-4 in cells expressing BRK-1 alone vs. cells expressing mBRK-1 in the presence of mActRIIB~ indicates that the affinity of BMP-4 to the BRK-1 + ActRIIBz complex is higher (iCs° < 5 x 10-x° M) than it is to the BRK-1 type I

receptor alone (ICso > 10-9 M). This is evidenced as a shift in the competition curve to the left in cells co-expressing BRK-1 + ActRIIB2, compared to the position of the competition curve in cells expressing BRK-1 alone. A similar leftward shift of the competition curve was obtained in cells co-expressing BRK-1 + ActRIIB2 vs.
cells expressing only BRK-1 in three additional experiments. The data average to an ICso =
2.59 x 10-1° M (Log ICSO = -9.586 ~ 0.062; N = 4) in cells co-expressing BRK-1 +
ActRIIB2 and 2.33 x 10 -9 M (Log ICSO = -8.633 ~ 0.044; N = 4) in cells expresing BRK-1 only. As an increase in binding affinity has been observed in cells co-expressing the BRK-2 type I receptor with the BRK-3 type II receptor, but not in cells co-expressing BRK-1 + BRK-3 receptors (T. Nohno, T. Ishikawa, T. Saito, K.
Hosokawa, S. Noji, D.H. Wolsing, and J. S. Rosenbaum, J. Biological Chemistry 270:22522-22526 (1995)), these data indicate that the ActRIIBz type II
receptor represents a type II receptor that is capable of forming a high affinity binding complex for BMP-4 in the presence of the BRK-1 receptor; and as such BRK-1 + ActRIIB2 represents a novel high affinity BMP receptor complex.
Fx_amnl~.1 pPmonstration of Comp]Px Formation of mActRIIB~ but not mActRIIB~
v= i h T~~De I BMP Receptors Receptors of the TGF-13 receptor family have been shown to form complexes involving a type I and a type II receptor (L. Attisano, J.L. Wrana, F. Lopez-Casillas, and J. Massague, J. Biochim Biophys. Acta, 1222: 71-80 (1994)). In order to demonstrate that the eight amino acids in the extracellular juxtamembrane region of ActRIIB~ but not in ActRIIB4 (L. Attisano, J.L. Wrana, S. Cheifetz, and J.
Massague, Cell 68: 97-108 (1992)) are required for [~25IjBMP-4 binding and complex formation with the BMP type I receptors BRK-1 or BRK-2, COS-1 or RIBIL-17 cells are co-transfected with the cDNA for either mActRIIB2 or mActRIIB4 as the type II receptor in combination with either BRK-I or BRK-2 as the type I
receptor, as described in Example 4, and plated at a density of 3 x 10~
cells/dish into 100 mm dishes as described previously (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)). The receptors are crosslinked to (I25I~-BMP-4, then subjected to immunoprecipitation with antibodies specific for the type I
receptors BRK-1 and BRK-2 described below using previously described methods (T. Nohno, T. Ishikawa, T. Saito, K. Hosokawa, S. Noji, D.H. Wolsing, and J.
S.
Rosenbaum, J. Biological Chemistry 270:22522-22526 {1995); B.B. Koenig et al., Molecular and Cellular Biology, I4: 5961-5974 {1994)). If antibodies specific for a type I receptor precipitate not only the type I receptor crosslinked to [
125I~-BMP-4, but also mActRIIB2 crosslinked to (125I~_gMP-4, this indicates that the two receptors must be forming a complex, as expected for type I and type II
receptors having the same ligand-binding specificity.
The BRK-1 rabbit polyclonal antibody #1353 is raised against the E. coli produced extracellular domain and produced as described previously (B.B.
Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)). The BRK-2 rabbit polyclonal antibody JM#2 was raised against the intracellular juxtamembrane peptide A R P R Y S I G L E Q D E T Y I P P C (AA: 155-172) conjugated by standard methods to keyhole limpet, and used to immunize three New Zealand White rabbits (Berkeley Antibody Company, Richmond, CA 94806-1965). The resulting antisera are evaluated for their ability to recognize the original peptide coated on plastic, using an antibody capture ELISA via the COOH cysteine. For both of these antibodies, antisera was IgG purified over a Pierce Immunopure Plus Immobilized Protein A column (Pierce Chemical Company, Rockford, IL) using the Immunopure (A) IgG Purification Kit (product # 44667) as described by the manufacturer.
Complex formation between BRK-1 and ActRIIB, is demonstrated in two different experiments. The first experiment is performed in RIB/L-17 cells which do not express receptors to the levels observed in the COS-1 cells (D. Vivien, L.
Attisano, J.L. Wrana, and J. Massague, J. Biological Chemistry, 270:7134-7141(1995)). In the RIB/L-17 cells, complex formation is observed between either the BRK-1 or the BRK-2 type I receptor and the BRK-3 type II receptor, as previously described in COS-1 cells (T. Nohno, T. Ishikawa, T. Saito, K.
Hosokawa, S. Noji, D.H. Wolsing, and J. S. Rosenbaum, J. Biological Chemistry 270:22522-22526 (1995)). As is observed previously, in BRK-1 immunoprecipitates, the intensity of affinity labeling to the BRK-1 band is unchanged in the BRK-1 + BRK-3 co-expressing cells vs. that observed in the cells only expressing BRK-1; whereas it is considerably darker to the BRK-2 band in the BRK-2 immunoprecipitates from BRK-2 + BRK-3 co-expressing cells, vs. that observed in the cells only expressing BRK-2. This change in labeling intensity has been interpreted as being reflective of either an increased affinity or crosslinking efficiency when BRK-2, but not BRK-1 is present in a complex with the BRK-3 type II receptor (T. Nohno, T. Ishikawa, T. Saito, K. Hosokawa, S. Noji, D.H.
Wolsing, and J. S. Rosenbaum, J. Biological Chemistry 270:22522-22526 (1995)).
In RIBIL-17 cells, an increased intensity of labeling of the BRK-1, but not the BRK-2 type I receptor band is observed in type I receptor immunoprecipitates when these receptors are coexpressed with ActRIIB,. In addition, affinity labeling of a band corresponding to the molecular weight of ActRIIBz (L. Attisano, J.L. Wrana, S.
Cheifetz, and J. Massague, Cell 68: 97-108 (1992)) is observed in BRK-1 immunoprecipitates in cells co-expressing BRK-1 + ActRIIBz, but not in BRK-1 immunoprecipitates in cells expressing only the BRK-1 receptor. This indicates that BRK-1 forms a complex with ActRIIB2 in cells co-expressing both of these receptors. Complex formation is not observed between BRK-2 and ActRIIB2, as affinity labeling of the band corresponding to the molecular weight of ActRIIB2 is not observed in the BRK-2 immunoprecipitates in cells co-expressing BRK-2 and ActRIIB2. Furthermore, affinity labeling of a band corresponding to the molecular weight of ActRIIB4 (L. Attisano, J.L. Wrana, S. Cheifetz, and J. Massague, Cell 68:
97-108 (1992)) is not observed in type I receptor immunoprecipitates in cells co-expressing either type I receptor with ActRIIB4, indicating that ActRIIB4 does not form a complex with either BRK-1 or BRK-2 in the presence of [lzsIJBMP-4.
A similar experiment is performed in COS-1 cells, and complex formation between BRK-1 and ActRIIBz, but not BRK-1 + ActRIIB4 is demonstrated using the same criterion as that applied in the experiment in the RIB/L-17 cells. In addition, in the COS-1 cells, which express receptors to a higher level than that observed in the RIB/L-17 cells (D. Vivien, L. Attisano, J.L. Wrana, and J. Massague, J.
Biological Chemistry, 270:7134-7141(1995)), complex formation is also demonstrated with BRK-2 + ActRIIB2_ These data confirm that the eight amino acids present in the extracellular juxtamembrane region of ActRIIB, and ActRIIBz (L. Attisano, J.L. Wrana, S. Cheifetz, and J. Massague, Cell 68: 97-108 (1992)) are critical for binding of BMP-4 to the ActRIIB type II receptor. Furthermore, the data indicate that either BRK-1 or BRK-2 are competetent to form a complex with ActRIIB2 in the presence of ['zsI]BMP-4 ligand, but that complex formation between BRK-2 and ActRIIB2 requires higher levels of receptor expression than that nececessary for complex formation between BRK-l and ActRIIBz.
An additional experiment indicates that the BRK-2 + ActRIIBz complex is also capable of binding BMP-2. In this experiment COS-1 cells are co-transfected with BRK-2 and ActRIIBz and are affinity labeled with [lzsl]BMP-2 as previously described (B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)). As described above for ['zsI]BMP-4, affinity labeling of a band corresponding to the molecular weight of ActRIIBz is observed in BRK-2 immunoprecipitates in cells co-expressing BRK-2 + ActRIIB2, indicating that BMP-2 ligand is capable of binding to the BRK-2 + ActRIIBz complex when these receptors are overexpressed in COS-1 cells.
~~camnle TlcP of -ActRIIB -1 ActRIIB~ RRK 7 ;" a nsgandding ssaX
+ BRK or ~- bin a for m-the identificati on ~P eptorgonssrc and s of rec a antagonist Bh Identification of ligands that interact with mActRIIB2 complexed to a type I
BMP receptor can be achieved through the use of assays that are designed to measure the interaction of the ligands with this BMP receptor complex. A receptor binding assay that uses the m-ActRIIB2 + BRK-1 or mActRIIB2 + BRK-2 complex and is adapted to handle large numbers of samples is carried out as follows.
COS-1 cells are transfected with the cDNAs for m-ActRIIB2, using the construct pJT6-mActRIIB2, and either BRK-1, using the construct pJT4-J159F for BRK-1 expression, or BRK-2, using the construct pJT3-BRK-2 for BRK-2 expression, as described in Example 4 above, except that the cells are grown in a 12 well culture dish or a 96-well microtitre plate. The DNA mixture used to transfect the cells contains the receptors in the concentrations described above in Example 4. At 36-72 hours after transfection, the cells are washed once with binding buffer (50 mM HEPES, pH
7.4, 128 mM NaCI, 5 mM KCL, 5 mM MgS04, I.2 mM CaCl2, 2 mg/ml BSA), then equilibrated in the same buffer at 4°C for 60 min with gentle shaking.
After equilibration, the buffer is aspirated, and to each well is added 4°C
binding buffer containing [125I)BMP-4 tracer (100-400 pM) in the presence or absence of varying concentrations of test compounds (i.e., putative ligands), for a period of 4 hours at 4°C
with gentle shaking. For determination of nonspecific binding and complete displacement from the BMP receptor complex, BMP-2 is added at a final concentration of 10 nM. To prevent degradation of ligand, a protease inhibitor cocktail is also added, to give a final concentration of 10 ~.g/ml leupeptin, 10 ~,g/ml antipain, 50 ~,g/ml aprotinin, 100 ~.g/ml benzamidine, 100 ~,g/ml soybean trypsin inhibitor, 10 ~cglml bestatin, 10 ~cg/ml pepstatin, and 300 ~,M phenylmethyisulfonyl fluoride (PMSF). At the end of the incubation period, the buffer is aspirated, and the cells are rinsed 4 times with washing buffer (50 mM HEPES, pH 7.4, I28 mM NaCI, 5 mM KC1, 5 mM
MgS04, 1.2 mM CaCl2, 0.5 mg/ml BSA). After the final wash is aspirated, RIPS
buffer (20 mM Tris Base, 100mM NaCI, 1 mM EDTA, 0.5 % NP-40, 0.5 %
Deoxycholic Acid, 0.1 % SDS, 10 mM NaI, 1 % BSA, pH 8Ø) is added to each well WO 98/52038 PCT/US98/095i9 and incubated at room temperature for 15-30 min. The solubilized cells are then transferred to fresh tubes and counted in a Packard Model 5005 COBRA Gamma Counter (Packard Instruments, Meriden, CT).
Test compounds which interact with the mActRIIB2 + BRK-1 or mActRIIB2 +
BRK-2 receptor complex are observed to compete for binding to the receptor complex with the [125I)BMP-4 tracer, such that less (125I]BMP-4 tracer is bound in the presence of the test compound in comparison to the binding observed when the tracer is incubated in the absence of the novel compound. A decrease in binding of the [125I~BMP-4 tracer by > 30% at the highest concentration of the test compound that is studied demonstrates that the test compound binds to the mActRIIB2 + BRK-1 or mActRIIB2 + BRK-2 receptor complex.
in a Complex With T,Xne I BMP Receptors Since several laboratories have previously demonstrated use of the R1B/L-17 mink lung epithelial cells (L. Attisano, J. Carcamo, F. Ventura, F.M.B.
Weis, J. Massague, and J.L. Wrana, Cel175:67I-680 (1993); J. Carcamo, F.M.B. Weis, F. Ventura, R. Wieser, J.L. Wrana, L. Attisano, and J. Massague, Molecular and Cellular Biology 14:3810-3821 (1994)) in concert with the p3TP-Lux promoter construct (J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J.
Doody, M. Laiho, X.-F. Wang, and J. Massague, Cell 71:1003-1014 (1992)) to measure signaling of TGF-[i (L. Attisano, J. Carcamo, F. Ventura, F.M.B. Weis, J.
Massague, and J.L. Wrana, Cell 75:671-680 (1993); J.L. Wrana, L. Attisano, J.
Carcamo, A. Zentella, J. Doody, M. Laiho, X.-F. Wang, and J. Massague, Cell 71:1003-1014 (1992)), activin (L. Attisano, J.L. Wrana, E. Montalvo, and J.
Massague, Molecular and Cellular Biology 16:1066-1073 (1996); J. Carcamo, F.M.B. Weis, F. Ventura, R. Wieser, J.L. Wrana, L. Attisano, and J. Massague, Molecular and Cellular Biology 14:3810-3821 (1994); S.A. Willis, C.M.

Zimmerman, L. Li, and L.S. Mathews, Molecular Endocrinology 10:367-379 (1996)) and BMP (F. Liu, F. Ventura, J. Doody, and J. Massague, Molecular and Cellular Biology, 15: 3479-3486 (1995); B.L. Rosenzweig, T. Imamura, T.
Okadome, G.N. Cox, H. Yamashita, P. Ten Dijke, C.-H. Heldin, and K.
Miyazono, Proceedings of the National Academy of Sciences, U. S. A. , 92: 7632-7636 (1995); H. Yamashita, P. ten Dijke, D. Huylebroeck, T.K. Sampath, M.
Andries, J.C. Smith, C.-H. Heldin, and K. Miyazono, J. Cell Biology 130:217-226 (1995)) receptor complexes, we also used this reporter system to demonstrate the use of mActRIIB2 + BRK-1 in a signaling assay that can then be used for the identification of BMP receptor agonists and antagonists.
R-1B/L17 cells are transfected with various BMP receptor pairs, the 3TP-Lux reporter plasmid (J.L. Wrana, L. Attisano, J. Carcamo, A. Zentella, J.
Doody, M. Laiho, X.-F. Wang, and J. Massague, Cell 71:1003-1014 (1992)), and a ~3-galactosidase reporter construct driven by the CMV promoter (Clontech, Palo Alto, CA) as described in Example 4, and, after 24 hours, are plated at 2x105 cells per well in six well standard tissue culture plates (Corning, San Diego, CA). After allowing cells to recover for 3 to 4 hours in 10%FBS-MEM, the growth media is replaced with 0.1 %FBS-MEM for 2 to 4 hours. BMPs are then applied in increasing concentrations in 0.1 %FBS-MEM for 18 hours prior to cell harvesting. Cells are harvested and assayed for luciferase activity using the Dual Light System (Tropix, Bedford, MA) as described by the manufacturer. To eliminate variation from well to well and assay to assay, reported luciferase activity values are normalized to the ~-galactosidase values reported for the same aliquot (determined using the Dual Light System (Tropix, Bedford, MA) as described by the manufacturer), and all data are expressed as arbitrary units.
Demonstration that signaling is achieved with the BRK-1 + ActRIIB2, but not the BRK-1 + ActRIIB4 complex is demonstrated using the 3TP-Lux reporter system in two separate experiments. In the first experiment, the type I and type II

receptors are expressed either individually or in combination in the RIB/L-17 cells in the presence of the 3TP-Lux or (3-galactosidase reporter genes. This experiment demonstrates that neither the type I nor the type II receptors are capable of signaling on their own, as has previously been observed with BRK-1, BRK-2, and BRK-3 in this system (F. Liu, F. Ventura, J. Doody, and J.
Massague, Molecular and Cellular Biology, 15: 3479-3486 (1995); B.L.
Rosenzweig, T. Imamura, T. Okadome, G.N. Cox, H. Yamashita, P. Ten Dijke, C.-H. Heldin, and K. Miyazono, Proceedings of the National Academy of Sciences, U.S.A., 92: 7632-7636 (1995); H. Yamashita, P. ten Dijke, D.
Huylebroeck, T.K. Sampath, M. Andries, J.C. Smith, C.-H. Heldin, and K.
Miyazono, J. Cell Biology 130:217-226 (1995)). However, the previous studies did not address BMP-mediated signaling through these type I receptors in the presence of the ActRIIB type II receptor, and the data presented here demonstrate that the BRK-1 + ActRIIB2 complex, but not the BRK-1 + ActRIIB4 complex, produces a BMP-4-stimulated increase in 3TP-Lux reporter activity. These data indicate for the first time that BRK-I + ActRIiB2 represent a signaling receptor complex for BMP-4 in this assay, and that the eight amino acids in the extracellular -juxtamembrane region of ActRIIB~ or ActRIIB, that are absent in the ActRIIB3 and ActRIIB4 isoforms (L. Attisano, J.L. Wrana, S. Cheifetz, and J.
Massague, Cell 68: 97-108 (1992)) are critical for this activity. It is also interesting that BRK-2 + ActRIIB2 produce a much lower degree of stimulation in this assay than is observed with BRK-1 + ActRIIB2 at this single concentration of BMP-4. In the second experiment, complete dose-response curves are compared for BMP-4 in RIB cells co-expressing either BRK-1 + ActRIIB~ or BRK-1 +
ActRIIB4 in addition to the 3TP-Lux and ~i-galactosidase reporter constructs.
This experiment demonstrates an ECso for BMP-4 of 6.4 x 10-11 M at the BRK-1 +
ActRIIB2 complex but only a negligable response at the BRK-1 + ActRIIB4 complex.

Since BMP-2 and BMP-4 both hind to the BRK-1 receptor {B.B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)), it is necessary to demonstrate that BMP-2 is capable signaling through the BRK-1 + ActRIIB2 complex in the 3TP-Lux reporter assay. This is demonstrated in a third experiment, where it is demonstrated that BMP-4 exhibits an ECS° of 1.5 x 10-~°
M and BMP-2 exhibits an ECS° of 2.8 x 10-1° M. The experiment also demonstrates a similar maximal response (5-fold) for these two ligands. These data demonstrate that the BRK-1 + ActRIIB2 complex is a signaling receptor complex for either BMP-2 or BMP-4, and that these BMP ligands exhibit similar potency and efficacy at this receptor complex.
Test compounds which are agonists of the BRK-1 +ActRIIB2 receptor complex will cause an increase in reporter activity in RIB cells co-expressing the BRK-1 and ActRIIB2 receptors in combination with the 3TP-Lux and ~-galactosidase reporter genes, quantited via the arbitrary light units the level of luciferase activity produced by activation of the Iuciferase enzyme normalized to the arbitrary light units produced by activation of the ~i-galactosidase enzyme as described for BMP-2 or BMP-4 in Example 9 above. In this manner, RIB/L-17 cells expressing co-expressing the BRK-1 and ActRIIB2 receptors in combination with the 3TP-Lux and ~3-galactosidase reporter genes are produced as described in Examples 4 and 9 above are exposed to various concentrations of unknown agents, and the cells are evaluated for their response, quantited via the arbitrary light units the level of luciferase activity produced by activation of the luciferase enzyme normalized to the arbitrary light units produced by activation of the ~3-galactosidase enzyme as described for BMP-2 or BMP-4 in Example 9 above.
Those compounds which produce an activity in RIB/L-17 cells expressing BRK-1 + ActRIIB2 receptors in combination with the 3TP-Lux and ~i-galactosidase genes, but not in RIBIL-17 cells which express only the 3TP-Lux and ~3-galactosidase genes are said to act as agonists of the BRK-I + ActRIIB2 receptor complex.
In order to test for antagonist activity, test compounds are added to RIB/L-17 cells expressing BRK-1 + ActRIIB2 receptors in combination with the 3TP-Lux and ~3-galactosidase genes in the presence of a fixed concentration of BMP-or another BMP receptor agonist. Test compounds which are antagonists of BRK-1 +ActRIIB2 complex will cause a decrease in the reporter activity of the 3TP-Lux construct, quantited via the arbitrary light units the level of luciferase activity produced by activation of the luciferase enzyme normalized to the arbitrary light units produced by activation of the (3-galactosidase enzyme as described for BMP-2 or BMP-4 in Example 9 above, when activity is compared to that observed in cells exposed to BMP-4 or another BMP receptor agonist in the absence of the added test compound.
U. S. Patent application serial number 081462,467 by Rosenbaum, which is also incorporated herein by reference and is used to supplement any disclosure in U. S. Patent Application Serial number 08/334,179, filed November 4, 1994 by Rosenbaum and Nohno is hereby incorporated herein by reference. All other publications mentioned hereinabove are hereby incorporated in their entirety by reference.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to one skilled in the art and are to be included in the spirit and purview of this application and scope of the appended claims.

WO 98!52038 PCT/US98I09519 SEQUENCE LISTING
(1} GENERAL INFORMATION:
(i) APPLICANT: Rosenbaum, Jan S.
(ii) TITLE OF INVENTION: THE USE OF A BONE MORPHOGENETIC PROTEIN
(BMP) RECEPTOR COMPLEX FOR SCREENING CELLULAR
DIFFERENTIATION ACTIVES AND CELLS CO-TRANSFECTED WITH AN
ACTIVIN/BMP TYPE II RECEPTOR AND A BMP TYPE I RECEPTOR
(iii) NUMBER OF SEQUENCES: 22 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: The Procter & Gamble Company (B) STREET: 8700 Mason-Montgomery Road (C) CITY: Mason (D) STATE: OH
(E) COUNTRY: USA
(F) ZIP: 45040-9462 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hake, Richard A.
(B} REGISTRATION NUMBER: 37,343 (C) REFERENCE/DOCKET NUMBER: 10959 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 513/622-0087 (B} TELEFAX: 513/622-0270 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1722 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS

WO 98/52038 PCTlUS98/09519 (B) LOCATION: 44..1654 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:

Met Thr Ala Pro Trp AlaCTC Leu TGG TCG TGC AlaGly GGG
5 GCC 10 Leu Gly Leu Ser Ala Trp Ser Cys Gly Leu 15 20 Ala Arg GlyGlu AlaGluThr GAG ATC Tyr TyrAsnAla AsnTrp 25 Arg Cys 35 Glu Ile Glu LeuGlu ArgThrAsn GlnSerGly LeuGlu ArgCysGlu GlyGlu Gln AspLys ArgLeuHis CysTyrAla SerTrp ArgAsnSer SerGly Thr IleGlu LeuValLys LysGlyCys TrpLeu AspAspPhe AsnCys Tyr AspArg GlnGluCys ValAlaThr GluGlu AsnProGln ValTyr Phe CysCys CysGluGly AsnPheCys AsnGlu ArgPheThr HisLeu Pro GluPro GlyGlyPro GluValThr TyrGlu ProProPro ThrAla CCC ACCCTG CTCACGGTG CTGGCCTAC TCGCTG CTGCCCATT GGAGGC 48?
Pro ThrLeu LeuThrVal LeuAlaTyr SerLeu LeuProIle GlyGly Leu SerLeu IleValLeu LeuAlaPhe TrpMet TyrArgHis ArgLys CCC Tyr GlyGTG AspIleHis GluVal ArgGlnCys GlnArg Pro His Pro Val 175 180 GCA GCG TCC AAG TTG Pro GGG Cys Asp Phe Pro AGA Ala Ser Lys Leu GAC
GGC
Trp Ala Gly Arg Arg Asp Gly Phe Gln Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu Gln Leu Leu Glu Ile Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Met Asn Asp Phe Val Ala Val Lys Ile Phe Pro Leu Gln Asp Lys Gln Ser Trp Gln Ser Glu Arg Glu Ile Phe Ser Thr Pro Gly Met Lys His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu Lys Arg Gly Ser Asn Leu Glu Val Glu Leu Trp Leu Ile Thr Ala Phe His Asp Lys Gly Ser Leu Thr Asp Tyr Leu Lys Gly Asn Ile Ile Thr Trp Asn Glu Leu Cys His Val Ala Glu Thr Met Ser Arg Gly Leu Ser Tyr Leu His Glu Asp Val Pro Trp Cys Arg Gly Glu Gly His Lys Pro Ser Ile Ala His Arg Asp Phe Lys Ser Lys Asn Val Leu Leu Lys Ser Asp Leu Thr Ala Val Leu Ala Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Val SerArgCys LysAla AlaAspGly ProVal AspGluTyr MetLeu Pro PheGluGlu GluIle GlyGlnHis ProSer LeuGluGlu LeuGln Glu VaIValVal HisLys LysMetArg ProThr IleLysAsp HisTrp Leu LysHisPro GlyLeu AlaGlnLeu CysVal ThrIleGlu GluCys Trp AspHisAsp AlaGlu AlaArgLeu SerAla GlyCysVal GluGlu Arg ValSerLeu IleArg ArgSerVal AsnGly ThrThrSer AspCys Leu ValSerLeu ValThr SerValThr AsnVal AspLeuLeu ProLys TC TAAGCCCGGGACA CGTAGCGT CT TGGATCTG 169=
CTCCAGACTC
AG

GAG TCCAGCA

Glu SerSerIle AAGP.AAAAAA CAA 1722 AAAGTAAACG
TACTC

(2) INFORMATION FORSEQ ID N0:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 537 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Thr Ala Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn g5 90 95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Pro Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu Pro Ile Gly Gly Leu Ser Leu Ile Val Leu Leu Ala Phe Trp Met Tyr Arg His Arg Lys Pro Pro Tyr Gly His Val Asp Ile His Glu Val Arg Gln Cys Gln Arg Trp Ala Gly Arg Arg Asp Gly Cys Ala Asp Ser Phe Lys Pro Leu Pro Phe Gln Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu Gln Leu Leu Glu Ile Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Met Asn Asp Phe Val Ala Val Lys Ile Phe Pro Leu Gln Asp Lys Gln Ser Trp Gln Ser Glu Arg Glu Ile Phe Ser Thr Pro Gly Met Lys His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu Lys Arg Gly Ser Asn Leu Glu Val Glu Leu Trp Leu Ile Thr Ala Phe His Asp Lys Gly Ser Leu Thr Asp Tyr Leu Lys Gly Asn Ile Ile Thr Trp Asn Glu Leu Cys His Val Ala Glu Thr Met Ser Arg Gly Leu Ser Tyr Leu His Glu Asp Val Pro Trp Cys Arg Gly Glu Gly His Lys Pro Ser Ile Ala His Arg Asp Phe Lys Ser Lys Asn Val Leu WO 98!52038 PCT/US98/09519 Leu Lys Ser Asp Leu Thr Ala Val Leu Ala Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Val Ser Arg Cys Lys Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Glu Leu Gln Glu Val Val Val His Lys Lys Met Arg Pro Thr Ile Lys Asp His Trp Leu Lys His Pro Gly Leu Ala Gln Leu Cys Val Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Glu Glu Arg Val Ser Leu Ile Arg Arg Ser Val Asn Gly Thr Thr Ser Asp Cys Leu Val Ser Leu Val Thr Ser Val Thr Asn Val Asp Leu Leu Pro Lys Glu Ser Ser Ile (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1651 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 44..1582 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

Met Thr Ala Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Pro Gly G1y Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu Pro Ile Gly Gly TCT TTC ATG

Leu SerLeuIle ValLeu LeuAlaPhe Trp Tyr ArgHis ArgLys Met Pro ProTyrGly HisVal AspIleHis GluAspPro GlyPro ProPro Pro SerProLeu ValGly LeuLysPro LeuGlnLeu LeuGlu IleLys Ala ArgGlyArg PheGly CysValTrp LysAlaGln LeuMet AsnAsp Phe ValAlaVal LysIle PheProLeu GlnAspLys GlnSer TrpGln Ser GluArgGlu IlePhe SerThrPro GlyMetLys HisGlu AsnLeu Leu GlnPheIle AlaAla GluLysArg GlySerAsn LeuGlu ValGlu Leu TrpLeuIle ThrAla PheHisAsp LysGlySer LeuThr AspTyr Leu LysGlyAsn IleIle ThrTrpAsn GluLeuCys HisVal AlaGlu Thr MetSerArg GlyLeu SerTyrLeu HisGluAsp ValPro TrpCys Arg GlyGluGly HisLys ProSerIle AlaHisArg AspPhe LysSer CTG

Lys AsnValLeu LeuLys SerAspLeu ThrAlaVal LeuAla AspPhe GTT TTT

Gly LeuAla Arg GluProGly LysProPro GlyAsp ThrHis Val Phe CAG GAG GGA
GTT
GGC
ACC
AGA
CGG
TAC

Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Val Ser Arg Cys Lys Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Glu Leu Gln Glu Val Val Val His Lys Lys Met Arg Pro Thr Ile Lys Asp His Trp Leu Lys His Pro Gly Leu Ala Gln Leu Cys Val Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Glu Glu Arg Val Ser Leu Ile Arg Arg Ser Val Asn Gly Thr Thr Ser Asp Cys Leu Val Ser Leu Val Thr Ser Val Thr Asn Val Asp Leu Leu Pro Lys Glu Ser Ser Ile 505 51.0 (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 513 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Thr Ala Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr WO 98152038 PCT/US98l09519 Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Pro Gly Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro Thr Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu Pro Ile Gly Gly Leu Ser Leu Ile Val Leu Leu A1a Phe Trp Met Tyr Arg His Arg Lys Pro Pro Tyr Gly His Val Asp Ile His Glu Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu Gln Leu Leu Glu Ile Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Met Asn Asp Phe Val Ala Val Lys Ile Phe Pro Leu Gln Asp Lys Gln Ser Trp Gln Ser Glu Arg Glu Zle Phe Ser Thr Pro Gly Met Lys His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu Lys Arg Gly Ser Asn Leu Glu Val Glu Leu Trp Leu Ile Thr Ala Phe His Asp Lys Gly Ser Leu Thr Asp Tyr Leu Lys Gly Asn Ile Ile Thr Trp Asn Glu Leu Cys His Val Ala Glu Thr Met Ser Arg Gly Leu Ser Tyr Leu His Glu Asp Val Pro Trp Cys Arg Gly Glu Gly His Lys Pro Ser Ile Ala His Arg Asp Phe Lys Ser Lys Asn Val Leu Leu Lys Ser Asp Leu Thr Ala Val Leu Ala Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Val Ser Arg Cys Lys Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Glu Leu Gln Glu Val Val Val His Lys Lys Met Arg Pro Thr Ile Lys Asp His Trp Leu Lys His Pro Gly Leu Ala Gln Leu Cys Val Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Glu Glu Arg Val Ser Leu Ile Arg Arg Ser Val Asn Gly Thr Thr Ser Asp Cys Leu Val Ser Leu Val Thr Ser Val Thr Asn Val Asp Leu Leu Pro Lys Glu Ser Ser Ile (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1699 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 44..1630 (xi) SEQUENCE DESCRIPTION: 5EQ ID N0:5:

Met Thr Ala Pro Trp AlaAlaLeu AlaLeuLeu TrpGly SerLeuCys AlaGly SerGly Arg GlyGluAla GluThrArg GluCys IleTyrTyr AsnAla AsnTrp Glu LeuGluArg ThrAsnGln SerGly LeuGluArg CysGlu GlyGlu Gln AspLysArg LeuHisCys TyrAla SerTrpArg AsnSer SerGly Thr IleGluLeu ValLysLys GlyCys TrpLeuAsp AspPhe AsnCys ~5 80 Tyr AspArgGlriGluCysVal AlaThr GluGluAsn ProGln ValTyr Phe CysCysCys GluGlyAsn PheCys AsnGluArg PheThr HisLeu Pro GluProGly GIyProGlu AlaPro ThrLeuLeu ThrVal LeuAla Tyr SerLeuLeu ProIleGly GlyLeu SerLeuIle ValLeu LeuAla Phe Trp Met Tyr Arg His Arg Lys Pro Pro Tyr Gly His Val Asp Ile His Glu Val Arg Gln Cys Gln Arg Trp Ala Gly Arg Arg Asp Gly Cys Ala Asp Ser Phe Lys Pro Leu Pro Phe Gln Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu Gln Leu Leu Glu Ile Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Met Asn Asp Phe Val Ala Val Lys Ile Phe Pro Leu Gln Asp Lys Gln Ser Trp Gln Ser Glu Arg Glu Ile Phe Ser Thr Pro Gly Met Lys His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu Lys Arg Gly Ser Asn Leu Glu Val Glu Leu Trp Leu Ile Thr Ala Phe His Asp Lys Gly Ser Leu Thr Asp Tyr Leu Lys Gly Asn Ile Ile Thr Trp Asn Glu Leu Cys His Val Ala Glu Thr Met Ser Arg Gly Leu Ser Tyr Leu His Glu Asp Val Pro Trp Cys Arg Gly Glu Gly His Lys Pro Ser Ile Ala His Arg Asp Phe Lys Ser Lys Asn Val Leu Leu Lys Ser Asp Leu Thr Ala Val Leu Ala Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro Gly Asp Thr His GlyGln ValGly ThrArgArg TyrMetAla ProGlu ValLeuGlu Gly AlaIle AsnPhe GlnArgAsp AlaPheLeu ArgIle AspMetTyr Ala MetGly LeuVal LeuTrpGlu LeuValSer ArgCys LysAlaAla Asp GlyPro ValAsp GluTyrMet LeuProPhe GluGlu GluIleGly Gln HisPro SerLeu GluGluLeu GlnGluVal ValVal HisLysLys Met ArgPro ThrIle LysAspHis TrpLeuLys HisPro GlyLeuAla Gln LeuCys ValThr IleGluGlu CysTrpAsp HisAsp AlaGluAla Arg LeuSer AlaGly CysValGlu GluArgVal SerLeu IleArgArg Ser ValAsn GlyThr ThrSerAsp CysLeuVal SerLeu ValThrSer Val ThrAsn ValAsp LeuLeuPro LysGluSer 5erIle CGTAGCGTCT CTCCAGAC TC AAAGTAAACC GTACTCCAA

AGTGGATCTG
AAGAAAAAAA

(2)INFORMATION FORSEQID N0:6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 529 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:

Met Thr Ala Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Giu Arg Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Pro Gly Gly Pro Glu Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu Pro Ile Gly Gly Leu Ser Leu Ile Val Leu Leu Ala Phe Trp Met Tyr Arg His Arg Lys Pro Pro Tyr Gly His Val Asp Ile His Glu Val Arg Gln Cys Gln Arg Trp Ala Gly Arg Arg Asp Gly Cys Ala Asp Ser Phe Lys Pro Leu Pro Phe Gln Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu Gln Leu Leu Glu Ile Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Met Asn Asp Phe Val Ala Val Lys Ile Phe Pro Leu Gln Asp Lys Gln Ser Trp Gln Ser Glu Arg Glu Ile Phe Ser Thr Pro Gly Met Lys His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu Lys Arg Gly Ser Asn Leu Glu Val Glu Leu Trp Leu Ile Thr Ala Phe His Asp Lys Gly Ser Leu Thr Asp Tyr Leu Lys Gly Asn Ile Ile Thr Trp Asn Glu Leu Cys His Val Ala Glu Thr Met Ser Arg Gly Leu Ser Tyr Leu His Glu Asp Val Pro Trp Cys Arg Gly Glu Gly His Lys Pro Ser Ile Ala His Arg Asp Phe Lys Ser Lys Asn Val Leu Leu Lys Ser Asp Leu Thr Ala Val Leu Ala Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Val Ser Arg Cys Lys Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Glu Leu Gln Glu Val Val Val His Lys Lys Met Arg Pro Thr Ile Lys Asp His Trp Leu Lys His Pro Gly Leu Ala Gln Leu Cys Val Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Glu Glu Arg Val Ser Leu Ile Arg Arg Ser Val Asn Gly Thr Thr Ser Asp Cys Leu Val Ser Leu Val Thr Ser Val Thr Asn Val Asp Leu Leu Pro Lys Glu Ser Ser Ile (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1628 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii} MOLECULE TYPE: DNA (genomic) ( ix ) FEATUR.E
(A) NAME/KEY: CDS
(B) LOCATION: 44..1558 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

Met Thr Ala Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Pro Gly Gly Pro Glu Ala Pro Thr Leu Leu Thr VaI Leu Ala Tyr Ser Leu Leu Pro Ile Gly Gly Leu Ser Leu Ile Val Leu Leu Ala Phe Trp Met Tyr Arg His Arg Lys Pro Pro Tyr Gly His Val Asp Ile His Glu Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Val Gly Leu Lys Asp Met Tyr Ala Met Gly Leu Val Leu Tr Pro LeuGln LeuLeuGlu IleLys AlaArgGly ArgPheGly CysVal Trp LysAla GlnLeuMet AsnAsp PheValAla ValLysIle PhePro Leu GlnAsp LysGlnSer TrpGln SerGluArg GluIlePhe SerThr Pro GlyMet LysHisGlu AsnLeu LeuGlnPhe IleAlaAla GluLys Arg GlySer AsnLeuGlu ValGlu LeuTrpLeu IleThrAla PheHis Rsp LysGly SerLeuThr AspTyr LeuLysGly AsnIleIle ThrTrp Asn GluLeu CysHisVal AlaGlu ThrMetSer ArgGlyLeu SerTyr Leu HisGlu AspValPro TrpCys ArgGlyGlu GlyHisLys ProSer Ile AlaHis ArgAspPhe LysSer LysAsnVal LeuLeuLys SerAsp Leu ThrAla ValLeuAla AspPhe GlyLeuAla ValArgPhe GluPro Gly LysPro ProGlyAsp ThrHis GlyGlnVal GlyThrArg ArgTyr Met AlaPro GluValLeu GluGly AlaIleAsn PheGlnArg AspAla Phe LeuArg IleAspMet TyrAla MetGlyLeu ValLeuTrp GluLeu TCT GCT CCT

Val SerArg CysLysAla AlaAsp Gly Val AspGluTyr MetLeu Pro TTC GAG ATT CCT GAG CAG
TCG

Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Glu Leu Gln AAG

GluValVal ValHis Lys MetArg ProThr IleLysAsp HisTrp Lys GCC

LeuLysHis ProGly Leu GlnLeu CysVal ThrIleGlu GluCys Ala GCT

TrpAspHis AspAla Glu ArgLeu SerAla GlyCysVal GluGlu Ala AGG

ArgValSer LeuIle Arg SerVal AsnGly ThrThrSer AspCys Arg TCC

LeuVal5er LeuVal Thr ValThr AsnVal AspLeuLeu ProLys Ser CTCCAGACTC
AGTGGATCTG

GluSerSer Ile AAGAP.AAAAA AAAGTAAACA CGTACTCCAA 1628 (2) INFORMATION FOR SEQ ID NO: B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 505 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii} MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION. SEQ ID N0:8:
Met Thr Ala Pro Trp Ala Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys Ala Gly Ser Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro Glu Pro Gly Gly Pro Glu Ala Pro Thr Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu Pro Ile Gly Gly Leu Ser Leu Ile Val Leu Leu Ala Phe Trp Met Tyr Arg His Arg Lys Pro Pro Tyr Gly His Val Asp Ile His Glu Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu Gln Leu Leu Glu Ile Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Met Asn Asp Phe Val Ala Val Lys Ile Phe Pro Leu Gln Asp Lys Gln Ser Trp Gln Ser Glu Arg Glu Ile Phe Ser Thr Pro Gly Met Lys His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu Lys Arg Gly Ser Asn Leu Glu Val Glu Leu Trp Leu Ile Thr Ala Phe His Asp Lys Gly Ser Leu Thr Asp Tyr Leu Lys Gly Asn Ile Ile Thr Trp Asn Glu Leu Cys His Val Ala Glu Thr Met Ser Arg Gly Leu Ser Tyr Leu His Glu Asp Val Pro Trp Cys Arg Gly Glu Gly His Lys Pro Ser Ile Ala His Arg Asp Phe Lys Ser Lys Asn Val Leu Leu Lys Ser Asp Leu Thr Ala Val Leu Ala Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Val Ser Arg Cys Lys Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Glu Leu Gln Glu Val Val Val His Lys Lys Met Arg Pro Thr Ile Lys Asp His Trp Leu Lys His Pro Gly Leu Ala Gln Leu Cys Val Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Glu Glu Arg Val Ser Leu Ile Arg Arg Ser Val Asn Gly Thr Thr Ser Asp Cys Leu Val Ser Leu Val Thr Ser Val Thr Asn Val Asp Leu Leu Pro Lys Glu Ser Ser Ile (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3601 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(409..3522) (xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:9:

ATG ACT
TCC

Met Thr Ser SerLeuGln ArgPro TrpArgVal ProTrpLeu ProTrpThr IleLeu LeuValSer ThrAla AlaAlaSer GlnAsnGln GluArgLeu CysAla PheLysAsp ProTyr GlnGlnAsp LeuGlyIle GlyGluSer ArgIle SerHisGlu AsnGly ThrIleLeu CysSerLys GlySerThr CysTyr GlyLeuTrp GluLys SerLysGly AspIleAsn LeuValLys GlnGly i Il Gl AspPro GlnGluCys HisTyrGlu GluCys CysTrpSer s e y H

Val Val Thr Thr Thr Pro Pro Ser Ile Gln Asn Gly Thr Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys Asn Val Asn Phe Thr Glu Asn Phe Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser Phe Asn Arg Asp Glu Thr Ile Ile Ile Ala Leu Ala Ser Val Ser Val Leu Ala Val Leu Ile Val Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr Gly Asp Arg Lys Gln Gly Leu His Ser Met Asn Met Met Glu Ala Ala Ala Ser Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu Glu Leu Ile Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys Gly Ser Leu Asp Glu Arg Pro Val Ala Val Lys Val Phe Ser Phe Ala Asn Arg Gln Asn Phe Ile Asn Glu Lys Asn Ile Tyr Arg Val Pro Leu Met Glu His Asp Asn Ile Ala Arg Phe Ile Val Gly Asp Glu Arg Val Thr Ala Asp Gly Arg Met Glu Tyr 260 265 2?0 275 Leu Leu Val Met Glu Tyr Tyr Pro Asn Gly Ser Leu Cys Lys Tyr Leu Ser Leu His Thr Ser Asp Trp Val Ser Ser Cys Arg Leu Ala His Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro Arg Gly Asp TAT ATT AAC AAT
AAA

His LysPro Ala SerHisArg AspLeu SerArg Val Tyr Ile Asn Asn GTG GGA
AAA

Leu LysAsn Asp ThrCysVal IleSer AspPheGly LeuSer Val Gly AGG AAT

Met LeuThr Gly ArgLeuVal ArgPro GlyGluGlu AspAsn Arg Asn GCC GTT

Ala IleSer Glu GlyThrIle ArgTyr MetAlaPro GluVal Ala Val GAA AAC

Leu GlyAla Val LeuArgAsp CysGlu SerAlaLeu LysGln Glu Asn GAC CTT

Val MetTyr Ala GlyLeuIle TyrTrp GluIlePhe MetArg Asp Leu ACA CCA

Cys AspLeu Phe GlyGluSer ValPro GluTyrGln MetAla Thr Pro TTT ACAGAG GTT AACCATCCC ACTTTT GAGGATATG CAGGTT 1?61 CAG GGA

Phe ThrGlu Val AsnHisPro ThrPhe GluAspMet GlnVal Gln Gly GTG AAA

Leu SerArg Glu GlnArgPro LysPhe ProGluAla TrpLys Val Lys AAT GTG

Glu SerLeu Ala ArgSerLeu LysGlu ThrIleGlu AspCys Asn Val GAC GAG

Trp GlnAsp Ala AlaArgLeu ThrAla GlnCysAla GluGlu Asp Glu ATG ATG ATT

Arg AlaGlu Leu MetIleTrp GluArg AsnLysSer ValSer Met Met ACA AAT ATG ACT ATG GAA

Pro ValAsn Pro Ser Ala Gln Asn Arg AsnLeu Thr Met Thr Met Glu CAT CCA
AAT GAT
AGG
CGT
GTG
CCA
AAA
ATT
GGT
CCT

Ser Asn Tyr TyrSer His Arg Pro Arg Asp Val Pro Lys Ile Gly Pro Ser Ser Ser Tyr Ile Glu Asp Ser Ile His His Thr Asp Ser Ile Val Lys Asn Ile Ser Ser Glu His Ser Met Ser Ser Thr Pro Leu Thr Ile Gly Glu Lys Asn Arg Asn Ser Ile Asn Tyr Glu Arg Gln Gln Ala Gln Ala Arg Ile Pro Ser Pro Glu Thr Ser Val Thr Ser Leu Ser Thr Asn Thr Thr Thr Thr Asn Thr Thr Giy Leu Thr Pro Ser Thr Gly Met Thr TCT ATG GAT ACC

ThrIleSer GluMetPro TyrPro GluThr AsnLeuHis ThrThr Asp ACC

AsnValAla GlnSerIle GlyPro ProVal CysLeuGln LeuThr Thr CTA

GluGluAsp LeuGluThr AsnLys AspPro LysGluVal AspLys Leu AAT

AsnLeuLys GluSerSer AspGlu LeuMet GluHisSer LeuLys Asn Gln Phe Ser Gly Pro Asp Pro Leu Ser Ser Thr Ser Ser Ser Leu Leu Tyr Pro Leu Ile Lys Leu Ala Val Glu Ala Thr Gly Gln Gln Asp Phe Thr Gln Thr Ala Asn Gly Gln Ala Cys Leu Ile Pro Asp Val Leu Pro Thr Gln Ile Tyr Pro Leu Pro Lys Gln Gln Asn Leu Pro Lys Arg Pro Thr Ser Leu Pro Leu Asn Thr Lys Asn Ser Thr Lys Glu Pro Arg Leu LysPhe GlySerLys HisLys SerAsnLeu LysGln ValGluThr Gly ValAla LysMetAsn ThrIle AsnAlaAla GluPro HisValVal Thr ValThr MetAsnGly ValAla GlyArgAsn HisSer ValAsnSer His AlaAla ThrThrGln TyrAla AsnGlyThr ValLeu SerGIyGln Thr ThrAsn IleValThr HisArg AlaGlnGlu MetLeu GlnAsnGln Phe IleGly GluAspThr ArgLeu AsnIleAsn SerSer ProAspGlu His g55 860 865 GluPro LeuLeuArg ArgGlu GlnGlnAla GlyHis AspGluGly Val LeuAsp ArgLeuVal AspArg ArgGluArg ProLeu GluGlyGly Arg ThrAsn SerAsnAsn AsnAsn SerAsnPro CysSer GluGlnAsp Val LeuAla GlnGlyVal ProSer ThrAlaAla AspPro GlyProSer Lys ProArg ArgAlaGln ArgPro AsnSerLeu AspLeu SerAlaThr Asn ValLeu AspGlySer SerIle GlnIleGly GluSer ThrGlnAsp Gly LysSer GlySerGly GluLys IleLysLys ArgVaI LysThrPro Tyr SerLeu LysArgTrp ArgPro SerThrTrp ValIle SerThrGlu Ser AGT

Leu Asp CysGlu Val Asn Asn Asn Gly AsnArg AlaVal His Ser Ser _ AAA TCC AGCACT GCT GTT TAC CTT GCA GGAGGC ACTGCT ACA ACC 3489 GAA

Lys Ser SerThr Ala Val Tyr Leu Ala GlyGly ThrAla Thr Thr Glu ATG GTG TCTAAA GAT ATA GGA ATG AAC CTGTGAAATGTTT

TGT TCAAGCCTAT

Met Val SerLys Asp Ile Gly Met Asn Leu Cys GGAGTGAAAT TATTTTTTGC ATCATTTAAA ATGTTTAAAA

CATGCAGAAG AAAAAAAAA

(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1038 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Thr Ser Ser Leu Gln Arg Pro Trp Arg Val Pro Trp Leu Pro Trp Thr Ile Leu Leu Val Ser Thr Ala Ala Ala Ser Gln Asn Gln Glu Arg Leu Cys Ala Phe Lys Asp Pro Tyr Gln G1n Asp Leu Gly Ile Gly Glu Ser Arg Ile Ser His Glu Asn Gly Thr Ile Leu Cys Ser Lys Gly Ser Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp Ile Asn Leu Val Lys Gln Gly Cys Trp Ser His Ile Gly Asp Pro Gln Glu Cys His Tyr g5 90 95 Glu Glu Cys Val Val Thr Thr Thr Pro Pro Ser Ile Gln Asn Gly Thr Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys Asn VaI Asn Phe Thr Glu Asn Phe Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser Phe Asn Arg Asp Glu Thr Ile Ile Ile Ala Leu Ala Ser Val Ser Val Leu Ala Val Leu Ile Val Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr Gly Asp Arg Lys Gln Gly Leu His Ser Met Asn Met Met Glu Ala Ala Ala Ser Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu Glu Leu Ile Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys Gly Ser Leu Asp Glu Arg Pro Val Ala Val Lys Val Phe Ser Phe Ala Asn Arg Gln Asn Phe Ile Asn Glu Lys Asn IIe Tyr Arg Val Pro Leu Met Glu His Asp Asn Ile Ala Arg Phe Ile Val Gly Asp Glu Arg Val Thr Ala Asp Gly Arg Met Glu Tyr Leu Leu Val Met Glu Tyr Tyr Pro Asn Gly Ser Leu Cys Lys Tyr Leu Ser Leu His Thr Ser Asp Trp Val Ser Ser Cys Arg Leu Ala His Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro Arg Gly Asp His Tyr Lys Pro Ala Ile Ser His Arg Asp Leu Asn Ser Arg Asn Val Leu Val Lys Asn Asp Gly Thr Cys Val Ile Ser Asp Phe Gly Leu Ser Met Arg Leu Thr Gly Asn Arg Leu Val Arg Pro Gly Glu Glu Asp Asn Ala Ala Ile Ser Glu Val Gly Thr Ile Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Val Asn Leu Arg Asp Cys Glu Ser Ala Leu Lys Gln Val Asp Met Tyr Ala Leu Gly Leu Ile Tyr Trp Glu Ile Phe Met Arg Cys Thr Asp Leu Phe Pro Gly Glu Ser Val Pro Glu Tyr Gln Met Ala Phe Gln Thr Glu Val Gly Asn His Pro Thr Phe Glu Asp Met Gln Val Leu Val Ser Arg Glu Lys Gln Arg Pro Lys Phe Pro Glu Ala Trp Lys Glu Asn Ser Leu Ala Val Arg Ser Leu Lys Glu Thr Ile Glu Asp Cys Trp Asp Gln Asp Ala Glu Ala Arg Leu Thr Ala Gln Cys Ala Glu Glu Arg Met Ala Glu Leu Met Met Ile Trp Glu Arg Asn Lys Ser Val Ser Pro Thr Val Asn Pro Met Ser Thr Ala Met Gln Asn Glu Arg Asn Leu Ser His Asn Arg Arg Val Pro Lys Ile Gly Pro Tyr Pro Asp Tyr Ser Ser Ser Ser Tyr Ile Glu Asp Ser Ile His His Thr Asp Ser Ile Val Lys Asn Ile Ser Ser Glu His Ser Met Ser Ser Thr Pro Leu Thr Ile Gly Glu Lys Asn Arg Asn Ser Ile Asn Tyr Glu Arg Gln Gln Ala Gln Ala Arg Ile Pro Ser Pro Glu Thr Ser Val Thr Ser Leu Ser Thr Asn Thr Thr Thr Thr Asn Thr Thr Gly Leu Thr Pro Ser Thr Gly Met Thr Thr Ile Ser Glu Met Pro Tyr Pro Asp Glu Thr Asn Leu His Thr Thr Asn Val Ala Gln Ser Ile Gly Pro Thr Pro Val Cys Leu Gln Leu Thr Glu Glu Asp Leu Glu Thr Asn Lys Leu Asp Pro Lys Glu VaI Asp Lys Asn Leu Lys Glu Ser Ser Asp Glu Asn Leu Met Glu His Ser Leu Lys Gln Phe Ser Gly Pro Asp Pro Leu Ser Ser Thr Ser Ser Ser Leu Leu Tyr Pro Leu Ile Lys Leu Ala Val Glu Ala Thr Gly Gln Gln Asp Phe Thr Gln Thr Ala Asn Gly Gln Ala Cys Leu Ile Pro Asp Val Leu Pro Thr Gln Ile Tyr Pro Leu Pro Lys Gln Gln Asn Leu Pro Lys Arg Pro Thr Ser Leu Pro Leu Asn Thr Lys Asn Ser Thr Lys Glu Pro Arg Leu Lys Phe Gly Ser Lys His Lys Ser Asn Leu Lys Gln Val Glu Thr Gly Val Ala Lys Met Asn Thr Ile Asn Ala Ala Glu Pro His Val Val Thr Val Thr Met Asn Gly Val Ala Gly Arg Asn His Ser Val Asn Ser His Ala Ala Thr Thr Gln Tyr Ala Asn Gly Thr Val Leu Ser Gly Gln Thr Thr Asn Ile Val Thr His Arg Ala Gln Glu Met Leu Gln Asn Gln Phe Ile Gly Glu Asp Thr Arg Leu Asn Ile Asn Ser Ser Pro Asp Glu His Glu Pro Leu Leu Arg Arg Glu Gln Gln Ala Gly His Asp Glu Gly Val Leu Asp Arg Leu Val Asp Arg Arg Glu Arg Pro Leu Glu Gly Gly Arg Thr Asn Ser Asn Asn Asn Asn Ser Asn Pro Cys Ser Glu Gln Asp Val Leu Ala Gln Gly Val Pro Ser Thr Ala Ala Asp Pro Gly Pro Ser Lys Pro Arg Arg Ala Gln Arg Pro Asn Ser Leu Asp Leu Ser Ala Thr Asn Val Leu Asp Gly 5er Ser Ile Gln Ile Giy Glu Ser Thr Gln Asp Gly Lys Ser Gly Ser Gly Glu Lys Ile Lys Lys Arg Val Lys Thr Pro Tyr Ser Leu Lys Arg Trp Arg Pro Ser Thr Trp Val Ile Ser Thr Glu Ser Leu Asp Cys Glu Val Asn Asn Asn Gly Ser Asn Arg Ala Val His Ser Lys Ser Ser Thr Ala Val Tyr Leu Ala Glu Gly Gly Thr Ala Thr Thr Met Val Ser Lys Asp Ile Gly Met Asn Cys Leu (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3508 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(17..3130) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Met Thr Ser Ser Leu His Arg Pro Phe Arg Val Pro Trp Leu Leu Trp Ala Val Leu Leu Val Ser Thr Thr Ala Ala Ser Gln Asn Gln Glu Arg Leu Cys Ala Phe Lys Asp Pro Tyr Gln Gln Asp Leu Gly Ile Gly Glu Ser Arg Ile Ser His Glu Asn Gly Thr Ile Leu Cys Ser Lys Gly Ser Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp Ile Asn Leu Val Lys Gln Gly Cys Trp Ser His Ile Gly Asp Pro Gln Glu Cys His Tyr Glu Glu Cys Val Val Thr Thr Thr Pro Pro Ser Ile Gln Asn Gly Thr Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys Asn Val Asn Phe Thr Glu Asn Phe Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser Phe Asn Arg Asp Glu Thr Ile Ile Ile Ala Leu GTT GTG GTT ATA

Ala Ser Ser Leu Ala Leu IleVal AlaLeu CysPheGly Val Val Val TAC AGA TTG GGA GACCGGAAA CAGGGT CTTCAC AGCATGAAC 57?
ATG ACA

Tyr Arg LeuThrGly AspArgLys GlnGly LeuHis SerMetAsn Met Met MetGlu AlaAlaAla AlaGluPro SerLeu AspLeu AspAsnLeu Lys LeuLeu GluLeuIle GlyArgGly ArgTyr GlyAla ValTyrLys Gly SerLeu AspGluArg ProValAla ValLys ValPhe SerPheAla Asn ArgGln AsnPheIle AsnGluLys AsnIle TyrArg ValProLeu Met GluHis AspAsnIle AlaArgPhe IleVal GlyAsp GluArgLeu Thr AlaAsp GlyArgMet GluTyrLeu LeuVal MetGlu TyrTyrPro Asn GlySer LeuCysLys TyrLeuSer LeuHis ThrSer AspTrpVal Ser SerCys ArgLeuAla HisSerVal ThrArg GlyLeu AlaTyrLeu His ThrGlu LeuProArg GlyAspHis TyrLys ProAla IleSerHis AAT AAG

Arg AspLeu AsnSerArg ValLeu ValLys AsnAsp GlyAlaCys Asn ATC TTA ATG CTA
ACT

Val IleSer AspPheGly Ser Arg Gly AsnArg Leu Met Leu Leu Thr CGC GGG GAA ACA
GAT
AAT
GCG
GCT
ATA
AGT

Val Pro Glu Asn Glu ValGly Arg Gly Glu Ala Thr Asp Ala Ile Ser Ile Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Val Asn Leu Arg Asp Cys Glu Ser Ala Leu Lys Gln Val Asp Met Tyr Ala Leu Gly Leu Ile Tyr Trp Glu Val Phe Met Arg Cys Thr Asp Leu Phe Pro Gly Glu Ser Val Pro Asp Tyr Gln Met Ala Phe Gln Thr Glu Val Gly Asn His Pro Thr Phe Glu Asp Met Gln Val Leu Val Ser Arg Glu Lys Gln Arg Pro Lys Phe Pro Glu Ala Trp Lys Glu Asn Ser Leu Ala Val Arg Ser Leu Lys Glu Thr Ile Glu Asp Cys Trp Asp Gln Asp Ala Glu Ala Arg Leu Thr Ala Gln Cys Ala Glu Glu Arg Met Ala Glu Leu Met Met Ile Trp Glu Arg Asn Lys Ser Val Ser Pro Thr Val Asn Pro Met Ser Thr Ala Met Gln Asn Glu Arg Asn Leu Ser His Asn Arg Arg Val Pro Lys Ile Gly Pro Tyr Pro Asp Tyr Ser Ser Ser 5er Tyr Ile Glu Asp Ser Ile His His Thr Asp Ser Ile Val Lys Asn Ile Ser Ser Glu His Ser Met Ser Ser Thr Pro Leu Thr Ile Gly Glu Lys Asn Arg Asn Ser Ile Asn Tyr Glu Arg Gln Gln Ala Gln Ala Arg Ile Pro Ser Pro Glu Thr 59p 595 600 Ser ValThrSer LeuSer ThrAsnThr ThrThr ThrAsnThr ThrGly Leu ThrProSer ThrGly MetThrThr IleSer GluMetPro TyrPro Asp GluThrHis LeuHis AlaThrAsn ValAla GlnSerIle GlyPro Thr ProValCys LeuGln LeuThrGlu GluAsp LeuGluThr AsnLys Leu AspProLys GluVal AspLysAsn LeuLys GluSerSer AspGlu Asn LeuMetGlu HisSer LeuLysGln PheSer GlyProAsp ProLeu Ser SerThrSer SerSer LeuLeuTyr ProLeu IleLysLeu AlaVal Glu ValThrGly GlnGln AspPheThr GlnAla AlaAsnGly GlnAla Cys LeuIlePro AspVal ProProAla GlnIle TyrProLeu ProLys Gln GlnAsnLeu ProLys ArgProThr SerLeu ProLeuAsn ThrLys Asn SerThrLys GluPro ArgLeuLys PheGly AsnLysHis LysSer Asn LeuLysGln ValGlu ThrGlyVal AlaLys MetAsnThr IleAsn Ala AlaGluPro HisVal ValThrVal ThrMet AsnGlyVal AlaGly AGC CAT

Arg HisAsn ValAsn Ser Ala AlaThr ThrGlnTyr AlaAsn Ser His Gly Ala Val Pro Ala Gly Gln Ala Ala Asn Ile Val Ala His Arg Ser Gln Glu Met Leu Gln Asn Gln Phe Ile Gly Glu Asp Thr Arg Leu Asn Ile Asn Ser Ser Pro Asp Glu His Glu Pro Leu Leu Arg Arg Glu Gln Gln Ala Gly His Asp Glu Gly VaI Leu Asp Arg Leu Val Asp Arg Arg Glu Arg Pro Leu Glu Giy Gly Arg Thr Asn Ser Asn Asn Asn Asn Ser g95 900 905 Asn Pro Cys Ser Glu Gln Asp Ile Leu Thr Gln Gly Val Thr Ser Thr Ala Ala Asp Pro Gly Pro Ser Lys Pro Arg Arg Ala Gln Arg Pro Asn Ser Leu Asp Leu Ser Ala Thr Asn Ile Leu Asp Gly Ser Ser Ile Gln Ile Gly Glu Ser Thr Gln Asp Gly Lys Ser Gly Ser Gly Glu Lys Ile Lys Arg Arg Val Lys Thr Pro Tyr Ser Leu Lys Arg Trp Arg Pro Ser Thr Trp Val Ile Ser Thr Glu Pro Leu Asp Cys Glu VaI Asn Asn Asn ggp 995 1000 Gly Ser Asp Arg Ala Val His Ser Lys Ser Ser Thr Ala Val Tyr Leu Ala Glu Gly Gly Thr Ala Thr Thr Thr Val Ser Lys Asp Ile Gly Met Asn Cys Leu CATCATTTAAACATGCAGAAGACATTTAAA AP.AAAAACTGCTTTAACCTC CTGTCAGCAC3230 (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1038 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Thr Ser Ser Leu His Arg Pro Phe Arg Val Pro Trp Leu Leu Trp Ala Val Leu Leu Val Ser Thr Thr Ala Ala Ser Gln Asn Gln Glu Arg Leu Cys Ala Phe Lys Asp Pro Tyr Gln Gln Asp Leu Gly Ile Gly Glu Ser Arg Ile Ser His Glu Asn Gly Thr Ile Leu Cys Ser Lys Gly Ser Thr Cys Tyr Gly Leu Trp Glu Lys Ser Lys Gly Asp Ile Asn Leu Val Lys Gln Gly Cys Trp Ser His Ile Gly Asp Pro Gln Glu Cys His Tyr Glu Glu Cys Val Val Thr Thr Thr Pro Pro Ser Ile Gln Asn Gly Thr Tyr Arg Phe Cys Cys Cys Ser Thr Asp Leu Cys Asn Val Asn Phe Thr Glu Asn Phe Pro Pro Pro Asp Thr Thr Pro Leu Ser Pro Pro His Ser Phe Asn Arg Asp Glu Thr Ile Ile Ile A1a Leu Ala Ser Val Ser Val Leu Ala Val Leu Ile Val Ala Leu Cys Phe Gly Tyr Arg Met Leu Thr Gly Asp Arg Lys Gln Gly Leu His Ser Met Asn Met Met Glu Ala Ala Ala Ala Glu Pro Ser Leu Asp Leu Asp Asn Leu Lys Leu Leu Glu Leu Ile Gly Arg Gly Arg Tyr Gly Ala Val Tyr Lys Gly Ser Leu Asp Glu Arg Pro Val Ala VaI Lys VaI Phe Ser Phe Ala Asn Arg Gln Asn Phe Ile Asn Glu Lys Asn Ile Tyr Arg Val Pro Leu Met Glu His Asp Asn Ile Ala Arg Phe Ile Val Gly Asp Glu Arg Leu Thr Ala Asp Gly Arg 260 265 2?0 Met Glu Tyr Leu Leu Val Met Glu Tyr Tyr Pro Asn Gly Ser Leu Cys Lys Tyr Leu Ser Leu His Thr Ser Asp Trp Val Ser Ser Cys Arg Leu Ala His Ser Val Thr Arg Gly Leu Ala Tyr Leu His Thr Glu Leu Pro Arg Gly Asp His Tyr Lys Pro Ala Ile Ser His Arg Asp Leu Asn Ser Arg Asn Val Leu Val Lys Asn Asp Gly Ala Cys Val Ile Ser Asp Phe Gly Leu Ser Met Arg Leu Thr Gly Asn Arg Leu Val Arg Pro Gly Glu Glu Asp Asn Ala Ala Ile Ser Glu VaI Gly Thr Ile Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Val Asn Leu Arg Asp Cys Glu Ser Ala Leu Lys Gln Val Asp Met Tyr Ala Leu Gly Leu Ile Tyr Trp Glu Val Phe Met Arg Cys Thr Asp Leu Phe Pro Gly Glu Ser Val Pro Asp Tyr Gln Met Ala Phe Gln Thr Glu Val Gly Asn His Pro Thr Phe Glu Asp Met Gln Val Leu Val Ser Arg Glu Lys Gln Arg Pro Lys Phe Pro Glu Ala Trp Lys Glu Asn Ser Leu Ala Val Arg Ser Leu Lys Glu Thr Ile Glu Asp Cys Trp Asp Gln Asp Ala Glu Ala Arg Leu Thr Ala Gln Cys Ala Glu Glu Arg Met Ala Glu Leu Met Met Ile Trp Glu Arg Asn Lys Ser Val Ser Pro Thr Val Asn Pro Met Ser Thr Ala Met Gln Asn Glu Arg Asn Leu Ser His Asn Arg Arg Val Pro Lys Ile Gly Pro Tyr Pro Asp Tyr Ser Ser Ser Ser Tyr Ile Glu Asp Ser Ile His His Thr Asp Ser Ile Val Lys Asn Ile Ser Ser Glu His Ser Met Ser Ser Thr Pro Leu Thr Ile Gly Glu Lys Asn Arg Asn Ser Ile Asn Tyr Glu Arg Gln Gln Aia Gln Ala Arg Ile Pro Ser Pro Glu Thr Ser Val Thr Ser Leu Ser Thr Asn Thr Thr Thr Thr Asn Thr Thr Gly Leu Thr Pro Ser Thr Gly Met Thr Thr Ile Ser Glu Met Pro Tyr Pro Asp Glu Thr His Leu His Ala Thr Asn Val Aia Gln Ser I1e Gly Pro Thr Pro Val Cys Leu Gln Leu Thr Glu Glu Asp Leu Glu Thr Asn Lys Leu Asp Pro Lys Glu Val Asp Lys Asn Leu Lys Glu Ser Ser Asp Glu Asn Leu Met Glu His Ser Leu Lys Gln Phe Ser Gly Pro Asp Pro Leu Ser Ser Thr Ser Ser Ser Leu Leu Tyr Pro Leu Ile Lys Leu Ala Val Glu Val Thr Gly Gln Gln Asp Phe Thr Gln Ala Ala Asn Gly Gln Ala Cys Leu Ile Pro Asp Val Pro Pro Ala Gln Ile Tyr Pro Leu Pro Lys Gln Gln Asn Leu Pro Lys Arg Pro Thr Ser Leu Pro Leu Asn Thr Lys Asn Ser Thr Lys Glu Pro Arg Leu Lys Phe Gly Asn Lys His Lys Ser Asn Leu Lys Gln Val Glu Thr Gly Val Ala Lys Met Asn Thr Ile Asn Ala Ala Glu Pro His Val Val Thr Val Thr Met Asn Gly Val Ala Gly Arg Ser His Asn Val Asn Ser His Ala Ala Thr Thr Gln Tyr Ala Asn Gly Ala Val Pro Ala Gly Gln Ala Ala Asn Ile Val Ala His Arg Ser Gln Glu Met Leu Gln Asn Gln Phe Ile Gly Glu Asp Thr Arg Leu Asn Ile Asn Ser Ser Pro Asp Glu His Glu Pro Leu Leu Arg Arg Glu Gln Gln Ala Gly His Asp Glu Gly Val Leu Asp Arg Leu Val Asp Arg Arg Glu Arg Pro Leu Glu G1y Gly Arg Thr Asn Ser Asn Asn Asn Asn Ser Asn Pro Cys Ser Glu Gln Asp Ile Leu Thr Gln Gly Val Thr Ser Thr Ala Ala Asp Pro Gly Pro Ser Lys Pro Arg Arg Aia Gln Arg Pro Asn Ser Leu Asp Leu Ser Ala Thr Asn Ile Leu Asp Gly Ser Ser Ile Gln Ile Gly Glu Ser Thr Gln Asp Gly Lys Ser Gly Ser Gly Glu Lys Ile Lys Arg Arg Val Lys Thr Pro Tyr Ser Leu Lys Arg Trp Arg Pro Ser Thr Trp Val Ile Ser Thr Glu Pro Leu Asp Cys Glu Val Asn Asn Asn Gly Ser Asp Arg Ala Val His Ser Lys Ser Ser Thr Ala Val Tyr Leu Ala Glu Gly Gly Thr Ala Thr Thr Thr Val Ser Lys Asp Ile Gly Met Asn Cys Leu (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2402 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii} MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(11..1606) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:

Met Thr Gln Leu Tyr Thr Tyr Ile Arg Leu Leu Gly Ala Cys Leu Phe Ile Ile Ser His Val Gln Gly Gln Asn Leu Asp Ser Met Leu His Gly Thr Gly Met Lys Ser Asp Leu Asp Gln Lys Lys Pro Glu Asn Gly Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys TyrCys SerGly HisCysPro AspAspAla IleAsn AsnThrCys Ile ThrAsn GlyHis CysPheAla IleIleGlu GluAsp AspGlnGly Glu ThrThr LeuThr SerGlyCys MetLysTyr GluGly SerAspPhe Gln g5 100 105 CysLys AspSer ProLysAla GlnLeuArg ArgThr IleGluCys Cys ArgThr AsnLeu CysAsnGln TyrLeuGln ProThr LeuProPro Val ValIle GlyPro PhePheAsp GlySerIle ArgTrp LeuValVal Leu Ile Ser Met Ala Val Cys Ile Val Ala Met Ile Ile Phe Ser Ser Cys Phe Cys Tyr Lys His Tyr Cys Lys Ser Ile Ser Ser Arg Gly Arg Tyr Asn Arg Asp Leu Glu Gln Asp Glu Ala Phe Ile Pro Val Gly Glu Ser Leu Lys Asp Leu Ile Asp Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly Leu Pro Leu Leu Val Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Arg Gln Val Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg Gly Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu A1a Ser Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp Phe Leu Lys Cys Ala Thr Leu Asp Thr Arg Ala Leu Leu Lys Leu Ala Tyr Ser Ala Ala Cys Gly Leu Cys His Leu His Thr Glu Ile Tyr Gly Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys Asn Ile Leu Ile Lys Lys Asn Gly Ser Cys Cys Ile Ala Asp Leu Gly LeuAlaVal TTC AsnSerAsp AAT GTT Asp IlePro 385 Lys 390 Thr Glu 395 Phe Asn Val AAT ThrArg ValGly ThrLysArg TyrMet AlaProGlu ValLeu Leu 400 405 410 Asn Asp GluSerLeu AsnLys AsnHisPhe GlnPro TyrIleMet AlaAsp Ile TyrSerPhe GlyLeu IleIleTrp GluMet AlaArgArg CysIle Thr GlyGlyIle VaIGlu GluTyrGln LeuPro TyrTyrAsn MetVal Pro SerAspPro SerTyr GluAspMet ArgGlu ValValCys ValLys Arg LeuArgPro IleVal SerAsnArg TrpAsn SerAspGlu CysLeu Arg AlaValLeu LysLeu MetSerGlu CysTrp AlaHisAsn ProAla Ser ArgACA AlaLeu ArgIleLys LysThr LeuAlaATG Val 510 Leu Lys Thr 515 520 Met 525 CAG AAG ATT
TGACAATTAA
ACAATTTTGA
GGGAGAATTT

Glu Ser Asp Val Gln Lys Ile (2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 532 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Met Thr Gln Leu Tyr Thr Tyr Ile Arg Leu Leu Gly Ala Cys Leu Phe Ile Ile Ser His Val Gln Gly Gln Asn Leu Asp Ser Met Leu His Gly Thr Gly Met Lys Ser Asp Leu Asp Gln Lys Lys Pro Glu Asn Gly Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys Tyr Cys Ser Gly His Cys Pro Asp Asp Ala Ile Asn Asn Thr Cys Ile Thr Asn Gly His Cys Phe Ala Ile Ile Glu Glu Asp Asp Gln Gly Glu Thr Thr Leu Thr Ser Gly Cys Met Lys Tyr Glu Gly Ser Asp Phe Gln Cys Lys Asp Ser Pro Lys Ala Gln Leu Arg Arg Thr Ile Glu Cys Cys Arg Thr Asn Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro Val Val Ile Gly Pro Phe Phe Asp Gly Ser Ile Arg Trp Leu Val Val Leu Ile Ser Met Ala Val Cys Ile Val Ala Met Ile Ile Phe Ser Ser Cys Phe Cys Tyr Lys His Tyr Cys Lys Ser Ile Ser Ser Arg Gly Arg Tyr Asn Arg Asp Leu Glu Gln Asp Glu Ala Phe Ile Pro Val Gly Glu Ser Leu Lys Asp Leu Ile Asp Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly Leu Pro Leu Leu Val Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Arg Gln Val Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg Gly Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp Phe Leu Lys Cys Ala Thr Leu Asp Thr Arg Ala Leu Leu Lys Leu Ala Tyr Ser Ala Ala Cys Gly Leu Cys His Leu His Thr Glu Ile Tyr Gly Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys Asn Ile Leu Ile Lys Lys Asn Gly Ser Cys Cys Ile Ala Asp Leu Gly Leu Ala Val Lys Phe Asn Ser Asp Thr Asn Glu Val Asp Ile Pro Leu Asn Thr Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu Asp Glu Ser Leu Asn Lys Asn His Phe Gln Pro Tyr Ile Met Ala Asp Ile Tyr Ser Phe Gly Leu Ile Ile Trp Glu Met Ala Arg Arg Cys Ile Thr Gly Gly Ile Val Glu Glu Tyr Gln Leu Pro Tyr Tyr Asn Met Val Pro Ser Asp Pro Ser Tyr Glu Asp Met Arg Glu Val Val Cys Val Lys Arg Leu Arg Pro Ile Val Ser Asn Arg Trp Asn Ser Asp Glu Cys Leu Arg Ala Val Leu Lys Leu Met Ser Glu Cys Trp Ala His Asn Pro Ala Ser Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu Ala Lys Met Val Glu Ser Gln Asp Val Lys Ile (2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2252 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(355..1860) (xi) SEQUENCE DESCRIPTION:
SEQ ID N0:15:

Met TTG GAG

Pro Leu Leu Ser Ser Ser Lys Ser Met Ser Arg Lys Glu Asp Leu Glu CCT AAG

Ser Glu Gly Thr Ala Pro Ala Pro Gln Lys Leu Ser Cys Gln Pro Lys GAC AAC

Cys His His His Cys Pro Glu Ser Val Ser Thr Cys Ser Thr Asp Asn Asp Gly Tyr Cys Phe Thr Ile Ile Glu Glu Asp Asp Ser Gly Gly His Leu Val Thr Lys Gly Cys Leu Gly Leu Glu Gly Ser Asp Phe Gln Cys ACT ATT CAC AGA ATT ACA

Arg AspThrPro IleProHis GlnArgArg SerIle GluCysCys Thr Gly GlnAspTyr CysAsnLys HisLeuHis ProThr LeuProPro Leu Lys AsnArgAsp PheAlaGlu GlyAsnIle HisHis LysAlaLeu Leu Ile SerValThr ValCysSer IleLeuLeu ValLeu IleIleIle Phe Cys TyrPheArg TyrLysArg GlnGluAla ArgPro ArgTyrSer Ile Gly LeuGluGln AspGluThr TyrIlePro ProGly GluSerLeu Lys Asp LeuIleGlu GlnSerGln SerSerGly SerGly SerGlyLeu Pro Leu LeuValGln ArgThrIle AlaLysGln IleGln MetValLys Gln Ile GlyLysGly ArgTyrGly GluValTrp MetGly LysTrpArg Gly Glu LysValAla ValLysVal PhePheThr ThrGlu GluAlaSer Trp Phe ArgGluThr GluIleTyr GlnThrVal LeuMet ArgHisGlu Asn Ile LeuGlyPhe IleAlaAla AspIleLys GlyThr GlySerTrp Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp Tyr Leu Lys Ser Thr Thr Leu Asp Thr Lys Gly Met Leu Lys Leu Ala Tyr Ser Ser Val Ser Gly Leu Cys His Leu His Thr Gly Ile Phe Ser AAA AAA
AGT

ThrGlnGly LysProAla IleAla HisArgAsp Leu Ser LysAsn Lys GAT

IleLeuVal LysLysAsn GlyThr CysCysIle Ala Leu GlyLeu Asp ATC

AlaValLys PheIleSer AspThr AsnGluVal Asp Pro ProAsn Ile GTG

ThrArgVal GlyThrLys ArgTyr MetProPro Glu Leu AspGlu Val GCT

SerLeuAsn ArgAsnHis PheGln SerTyrIle Met Asp MetTyr Ala Ser Phe Gly Leu Ile Leu Trp Glu Ile Ala Arg Arg Cys Val Ser Gly Gly Ile Val Glu Glu Tyr Gln Leu Pro Tyr His Asp Leu Val Pro Ser Asp Pro Ser Tyr Glu Asp Met Arg Glu Ile Val Cys Ile Lys Arg Leu Arg Pro Ser Phe Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg Gln Met Gly Lys Leu Met Met Glu Cys Trp Ala His Asn Pro Ala Ser Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met Ser Glu Ser ATT TGATGGAGCA
AAAACAGCTC
CTTCTCGTGA
AGACCCATGG

Gln Asp Lys Leu Ile TTATGAAAATAAAACCCTTTGGTTAGAAGAAAAA.AAGATGTATATTGTTACA 2252 (2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 502 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Pro Leu Leu Ser Ser Ser Lys Leu Ser Met Glu Ser Arg Lys Giu Asp Ser Glu Gly Thr Ala Pro Ala Pro Pro Gln Lys Lys Leu Ser Cys Gln Cys His His His Cys Pro Glu Asp Ser Val Asn Ser Thr Cys Ser Thr Asp Gly Tyr Cys Phe Thr Ile Ile Glu Glu Asp Asp Ser Gly Gly His Leu Val Thr Lys Gly Cys Leu Gly Leu Glu Gly Ser Asp Phe Gln Cys Arg Asp Thr Pro Ile Pro His Gln Arg Arg Ser Ile Glu Cys Cys Thr Gly Gln Asp Tyr Cys Asn Lys His Leu His Pro Thr Leu Pro Pro Leu Lys Asn Arg Asp Phe Ala Glu Gly Asn Ile His His Lys Ala Leu Leu Ile Ser Val Thr Val Cys Ser Ile Leu Leu Val Leu Ile Ile Ile Phe Cys Tyr Phe Arg Tyr Lys Arg Gln Glu Ala Arg Pro Arg Tyr Ser Ile Gly Leu Glu Gln Asp Glu Thr Tyr Ile Pro Pro Gly Glu Ser Leu Lys Asp Leu Ile Glu Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly Leu Pro Leu Leu Val Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Lys Gln Ile Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg Gly Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp Tyr Leu Lys Ser Thr Thr Leu Asp Thr Lys Gly Met Leu Lys Leu Ala Tyr Ser Ser Val Ser Gly Leu Cys His Leu His Thr Gly Ile Phe Ser Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu Gly Leu Ala Val Lys Phe Ile Ser Asp Thr Asn Glu Val Asp Ile Pro Pro Asn Thr Arg Val Gly Thr Lys Arg Tyr Met Pro Pro Glu Val Leu Asp Glu Ser Leu Asn Arg Asn His Phe Gln Ser Tyr Ile Met Ala Asp Met Tyr Ser Phe Gly Leu Ile Leu Trp Glu Ile Ala Arg Arg Cys Val Ser Gly Gly Ile Val Glu Glu Tyr Gln Leu Pro Tyr His Asp Leu Val Pro Ser Asp Pro Ser Tyr Glu Asp Met Arg Glu Ile Val Cys Ile Lys Arg Leu Arg Pro Ser Phe Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg Gln Met Gly Lys Leu Met Met Glu Cys Trp Ala His Asn Pro Ala Ser Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met Ser Glu Ser Gln Asp Ile Lys Leu (2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C1 STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:

(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:

(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B} TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:

(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:

(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:

(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1B base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:

Claims (11)

What is claimed:

1. A method for determining whether a compound is capable of binding to a BMP receptor kinase protein complex, wherein the complex is comprised of a) a BMP;
b) a BMP receptor kinase protein selected from BRK-2 (SEQ ID NO:16) or BRK-1 (SEQ ID NO:14), a soluble fragment of BRK-2, a soluble fragment of BRK-1, an incomplete receptor kinase fragment of BRK-1, an incomplete receptor kinase fragment of of BRK-2;
c) an ActRIIB receptor comprising amino acids 124 to 131 of SEQ ID NO:
2, or SEQ ID NO: 4, or a soluble fragment thereof, or an incomplete receptor kinase fragment thereof;
the method comprising introducing a sample comprising the compound to the complex and allowing the compound to bind to the complex.

2. The method of Claim 1 wherein the BMP binding is determined using a cell co-transfected with an expression vector comprising a DNA sequence that codes for BMP receptor kinase protein BRK-1, SEQ ID NO:13, or a DNA
sequence that codes for a soluble fragment thereof, or codes for an incomplete receptor kinase fragment thereof, and an expression vector comprising a DNA sequence that codes for the ActRIIB protein, selected from SEQ ID NO:1 or SEQ ID NO:3 or a DNA sequence that codes for a soluble fragment thereof, or codes for an incomplete receptor kinase fragment thereof.

3. The method of any preceding claim, wherein the BMP receptor kinase protein is selected from BRK-1 which has the amino acid sequence SEQ ID
NO:14 or a soluble fragment of BRK-1, or an incomplete receptor kinase fragment of BRK-1; and BRK-2 which has the amino acid sequence SEQ ID
NO:16 or a soluble fragment of BRK-2, or an incomplete receptor kinase fragment of BRK-2.

4. The method of any preceding claim, wherein the ActRIIB protein has the amino acid sequence is ActRIII1 and has the amino acid sequence SEQ ID
NO:2 or is ActRIIB2 and has the amino acid sequence SEQ ID NO:4, or a soluble fragment thereof, or an incomplete receptor kinase fragment thereof.

5. The method of Claim 1, wherein the BMP is BMP-2 or BMP-4.

6. A host cell co-transfected with an expression vector comprising a DNA
sequence that codes for BMP receptor kinase protein BRK-1, SEQ ID
NO:13, or a DNA sequence that codes for a soluble fragment thereof, or codes for an incomplete receptor kinase fragment thereof, and an expression vector comprising a DNA sequence that codes for the ActRIIB protein, selected from SEQ ID NO:1 or SEQ ID NO:3 or a DNA sequence that codes for a soluble fragment thereof, or codes for an incomplete receptor kinase fragment thereof; such proteins or fragment useful in the method of any preceding claims.

7. A method for determining the concentration of a BMP receptor ligand in a clinical sample, the method comprising:
a. combining the clinical sample comprising the ligand with a BMP
receptor kinase protein complex and a labeled BMP;
b. allowing the labeled BMP to bind to the complex in the presence of the sample; and c. comparing with a standard curve prepared with known concentration of a BMP ligand;
wherein the BMP receptor kinase protein complex is comprised of a BMP
receptor kinase protein selected from BRK-2 (SEQ ID NO:16) or BRK-1 (SEQ ID NO:14), a soluble fragment of BRK-2, a soluble fragment of BRK-1, an incomplete receptor kinase fragment of BRK-1, or an incomplete receptor kinase fragment of BRK-2; and an ActRIIB receptor comprising amino acids 124 to 131 of SEQ ID NO: 2, or SEQ ID NO: 4, a soluble fragment thereof, or an incomplete receptor kinase fragment thereof.

8. The method of any preceding claim wherein the BMP binding is determined using a cell co-transfected with an expression vector comprising a DNA
sequence that codes for BMP receptor kinase protein BRK-1, SEQ ID
NO:13, or a DNA sequence that codes for a soluble fragment thereof, or codes for an incomplete receptor kinase fragment thereof, and an expression vector comprising a DNA sequence that codes for the ActRIIB protein, selected from SEQ ID NO:1 or SEQ ID NO:3 or a DNA sequence that codes for a soluble fragment thereof, or codes for an incomplete receptor kinase fragment thereof.

9. The method of any preceding claim, wherein the BMP receptor kinase protein is the soluble fragment of BRK-2 or BRK-1 (SEQ ID NO:16) or the soluble fragment of BRK-1 or BRK-2 (SEQ ID NO:14).

10. A method for determining whether a test compound produces a signal upon binding to a BMP receptor protein complex, the method comprising:
(a) labeling BMP receptor protein complex expressing cells with 32p, wherein the cells have been transfected with a DNA sequence coding for a BMP, a BMP receptor kinase protein BRK-2 (SEQ ID NO:15) or BRK-1 (SEQ ID NO:13), an ActRIIB receptor comprising DNA
encoding amino acids 124 to 131 of SEQ ID NO: 2, or SEQ ID NO: 4, (b) culturing:
(i} a first set of the cells in the presence of the test compound, and (ii) a second set of the cells in the absence of the test compound;
(c) quantitating via autoradiography any phosphorylated proteins produced from step (b); and (d) comparing the amount of phosphorylated proteins quantitated in step (c) from the first set of cells to the amount of phosphorylated proteins quantitated in step (c) for the second set of cells.

11. A method for determining whether a test compound produces a signal upon binding to a BMP receptor protein complex, the method comprising:
(a) transfecting BMP receptor protein complex expressing cells with a luciferase reporter gene in conjunction with a beta-galactosidase gene, wherein the cells have been transfected with a DNA sequence coding for a BMP, a BMP receptor kinase protein BRK-2 (SEQ ID NO:15) or BRK-1 (SEQ ID NO:13), an ActRIIB receptor comprising DNA
encoding amino acids I24 to 131 of SEQ ID NO: 2, or SEQ ID NO: 4;
(b) culturing (i) a first set of the cells in the presence of the test compound, and (ii) a second set of the cells in the absence of the test compound;

(c) quantitating via the arbitrary light units the level of luciferase activity produced by activation of the luciferase enzyme that results from stimulation of the reporter construct produced from step (b); and (d} comparing the amount of arbitrary light units quantitated in step (c) from the first set of cells to the amount of arbitrary light units quantitated in step (c) for the second set of cells.