US20030017528A1 - Human orphan G protein-coupled receptors - Google Patents

Human orphan G protein-coupled receptors Download PDF

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US20030017528A1
US20030017528A1 US09/875,076 US87507601A US2003017528A1 US 20030017528 A1 US20030017528 A1 US 20030017528A1 US 87507601 A US87507601 A US 87507601A US 2003017528 A1 US2003017528 A1 US 2003017528A1
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Ruoping Chen
Huong Dang
Chen Liaw
I-Lin Lin
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Arena Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Definitions

  • the invention disclosed in this patent document relates to transmembrane receptors, and more particularly to endogenous, orphan, human G protein-coupled receptors (“GPCRs”).
  • GPCRs G protein-coupled receptors
  • GPCR G protein-coupled receptor
  • Receptors including GPCRs, for which the endogenous ligand has been identified are referred to as “known” receptors, while receptors for which the endogenous ligand has not been identified are referred to as “orphan” receptors.
  • GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, 60% of all prescription pharmaceuticals have been developed. This distinction is not merely semantic, particularly in the case of GPCRs.
  • the orphan GPCRs are to the pharmaceutical industry what gold was to California in the late 19 th century—an opportunity to drive growth, expansion, enhancement and development.
  • GPCRs share a common structural motif. All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane-1 (TM-1), transmebrane-2 (TM-2), etc.).
  • the transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or “extracellular” side, of the cell membrane (these are referred to as “extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively).
  • transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or “intracellular” side, of the cell membrane (these are referred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively).
  • the “carboxy” (“C”) terminus of the receptor lies in the intracellular space within the cell, and the “amino” (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
  • GPCRs are “promiscuous” with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, T., 43 Life Sciences 1095 (1988). Although other G proteins exist, currently, Gq, Gs, Gi, and Go are G proteins that have been identified. Endogenous ligand-activated GPCR coupling with the G-protein begins a signaling cascade process (referred to as “signal transduction”). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. It is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.
  • GPCRs exist in the cell membrane in equilibrium between two different conformations: an “inactive” state and an “active” state.
  • a receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to produce a biological response.
  • Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
  • a receptor may be stabilized in an active state by an endogenous ligand or a compound such as a drug.
  • FIGS. 1A and 1B provide reference “grids” for certain dot-blots provided herein (see also, FIG. 2A and 2B, respectively).
  • FIGS. 2A and 2B provide reproductions of the results of certain dot-blot analyses resulting from hCHN3 and hCHN8, respectively (see also, FIGS. 1A and 1B, respectively).
  • FIG. 3 provides a reproduction of the results of RT-PCR analysis of hRUP3.
  • FIG. 4 provides a reproduction of the results of RT-PCR analysis of hRUP4.
  • FIG. 5 provides a reproduction of the results of RT-PCR analysis of hRUP6.
  • AMINO ACID ABBREVIATIONS used herein are set out in Table 1: TABLE 1 ALANINE ALA A ARGININE ARG R ASPARAGINE ASN N ASPARTIC ACID ASP D CYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE GLN Q GLYCINE GLY G HISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L LYSINE LYS K METHIONINE MET M PHENYLALANINE PHE F PROLINE PRO P SERINE SER S THREONINE THR T TRYPTOPHAN TRP W TYROSINE TYR Y VALINE VAL V
  • COMPOSITION means a material comprising at least one component.
  • ENDOGENOUS shall mean a material that a mammal naturally produces.
  • ENDOGENOUS in reference to, for example and not limitation, the term “receptor,” shall mean that which is naturally produced by a mammal (for example, and not limitation, a human) or a virus.
  • the term NON-ENDOGENOUS in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human) or a virus.
  • HOST CELL shall mean a cell capable of having a Plasmid and/or Vector incorporated therein.
  • a Plasmid is typically replicated as a autonomous molecule as the Host Cell replicates (generally, the Plasmid is thereafter isolated for introduction into a eukaryotic Host Cell); in the case of a eukaryotic Host Cell, a Plasmid is integrated into the cellular DNA of the Host Cell such that when the eukaryotic Host Cell replicates, the Plasmid replicates.
  • the Host Cell is eukaryotic, more preferably, mammalian, and most preferably selected from the group consisting of 293, 293T and COS-7 cells.
  • LIGAND shall mean an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor.
  • NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurring molecule specific for an endogenous naturally occurring ligand wherein the binding of a ligand to a receptor activates an intracellular signaling pathway.
  • ORPHAN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has not been identified or is not known.
  • PLASMID shall mean the combination of a Vector and cDNA.
  • a Plasmid is introduced into a Host Cell for the purposes of replication and/or expression of the cDNA as a protein.
  • VECTOR sin reference to cDNA shall mean a circular DNA capable of incorporating at least one cDNA and capable of incorporation into a Host Cell.
  • Receptor homology is useful in terms of gaining an appreciation of a role of the disclosed receptors within the human body. Additionally, such homology can provide insight as to possible endogenous ligand(s) that may be natural activators for the disclosed orphan GPCRs.
  • any search for therapeutic compounds should start by screening compounds against the ligand-independent active state.
  • GPCRs can be “active” in their endogenous state even without the binding of the receptor's endogenous ligand thereto.
  • Such naturally-active receptors can be screened for the direct identification (i.e., without the need for the receptor's endogenous ligand) of, in particular, inverse agonists.
  • the receptor can be “activated” via, e.g., mutation of the receptor to establish a non-endogenous version of the receptor that is active in the absence of the receptor's endogenous ligand.
  • Screening candidate compounds against an endogenous or non-endogenous, constitutively activated version of the human orphan GPCRs disclosed herein can provide for the direct identification of candidate compounds which act at this cell surface receptor, without requiring use of the receptor's endogenous ligand.
  • By determining areas within the body where the endogenous version of human GPCRs disclosed herein is expressed and/or over-expressed it is possible to determine related disease/disorder states which are associated with the expression and/or over-expression of the receptor; such an approach is disclosed in this patent document.
  • the DNA sequence of the human orphan GPCR can be used to make a probe for (a) dot-blot analysis against tissue-mRNA, and/or (b) RT-PCR identification of the expression of the receptor in tissue samples.
  • the presence of a receptor in a tissue source, or a diseased tissue, or the presence of the receptor at elevated concentrations in diseased tissue compared to a normal tissue can be preferably utilized to identify a correlation with a treatment regimen, including but not limited to, a disease associated with that disease.
  • Receptors can equally well be localized to regions of organs by this technique. Based on the known functions of the specific tissues to which the receptor is localized, the putative functional role of the receptor can be deduced.
  • G protein receptor When a G protein receptor becomes constitutively active (i.e., active in the absence of endogenous ligand binding thereto), it binds to a G protein (e.g., Gq, Gs, Gi, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, constitutively activated receptors continue to exchange GDP to GTP.
  • a non-hydrolyzable analog of GTP [ 35 S]GTP ⁇ S, can be used to monitor enhanced binding to membranes which express constitutively activated receptors.
  • candidate compounds are identified using the “generic” G protein-coupled receptor assay (i.e., an assay to select compounds that are agonists, partial agonists, or inverse agonists), further screening to confirm that the compounds have interacted at the receptor site is preferred.
  • a compound identified by the “generic” assay may not bind to the receptor, but may instead merely “uncouple” the G protein from the intracellular domain.
  • Gs stimulates the enzyme adenylyl cyclase. Gi (and Go), on the other hand, inhibit this enzyme.
  • Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, S constitutively activated GPCRs that couple the Gs protein are associated with increased cellular levels of cAMP.
  • constitutively activated GPCRs that couple the Gi (or Go) protein are associated with decreased cellular levels of cAMP. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron To Brain (3 rd Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992).
  • assays that detect cAMP can be utilized to determine if a candidate compound is, e.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP).
  • a candidate compound e.g., an inverse agonist to the receptor
  • a variety of approaches known in the art for measuring cAMP can be utilized; a most preferred approach relies upon the use of anti-cAMP antibodies in an ELISA-based format.
  • Another type of assay that can be utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes.
  • Cyclic AMP drives gene expression by promoting the binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) which then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene.
  • Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter gene, e.g., ⁇ -galactosidase or luciferase.
  • a constitutively activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and expression of the reporter protein.
  • the reporter protein such as ⁇ -galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).
  • Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP 2 , releasing two intracellular messengers: diacycloglycerol (DAG) and inistol 1,4,5-triphoisphate (IP 3 ). Increased accumulation of IP 3 is associated with activation of Gq- and Go-associated receptors.
  • DAG diacycloglycerol
  • IP 3 inistol 1,4,5-triphoisphate
  • Assays that detect IP 3 accumulation can be utilized to determine if a candidate compound is, e.g., an inverse agonist to a Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of IP 3 ).
  • Gq-associated receptors can also been examined using an AP1 reporter assay in that Gq-dependent phospholipase C causes activation of genes containing AP1 elements; thus, activated Gq-associated receptors will evidence an increase in the expression of such genes, whereby inverse agonists thereto will evidence a decrease in such expression, and agonists will evidence an increase in such expression.
  • Commercially available assays for such detection are available.
  • an endogenous, constitutively activated orphan GPCR, or a non-endogenous, constitutively activated orphan GPCR, for screening of candidate compounds for the direct identification of inverse agonists, agonists and partial agonists provides a unique challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto. Thus, it is often useful that an approach be utilized that can enhance the signal obtained by the activated receptor.
  • a preferred approach is the use of a GPCR Fusion Protein.
  • a GPCR is or has been constitutively activated, using the assay techniques set forth above (as well as others), it is possible to determine the predominant G protein that couples with the endogenous GPCR. Coupling of the G protein to the GPCR provides a signaling pathway that can be assessed. Because it is most preferred that screening take place by use of a mammalian expression system, such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the constitutively activated orphan GPCR will continuously signal.
  • this signal be enhanced such that in the presence of, e.g., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist.
  • the GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the GPCR.
  • the GPCR Fusion Protein is preferred for screening with a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is most preferably utilized in such screening techniques, although the GPCR Fusion Protein can also be (and preferably is) used with an endogenous, constitutively activated GPCR. This is important in facilitating a significant “signal to noise” ratio; such a significant ratio is import preferred for the screening of candidate compounds as disclosed herein.
  • GPCR Fusion Protein The construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator.
  • the criteria of importance for such a GPCR Fusion Protein construct is that the GPCR sequence and the G protein sequence both be in-frame (preferably, the sequence for the GPCR is upstream of the G protein sequence) and that the “stop” codon of the GPCR must be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed.
  • the GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art).
  • G protein that couples to the GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct. Because there are only a few G proteins that have been identified, it is preferred that a construct comprising the sequence of the G protein (i.e., a universal G protein construct) be available for insertion of an endogenous GPCR sequence therein; this provides for efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences.
  • human orphan GPCRs disclosed herein may be for the direct identification of candidate compounds as inverse agonists, agonists or partial agonists (preferably for use as pharmaceutical agents), these versions of human GPCRs can also be utilized in research settings.
  • in vitro and in vivo systems incorporating GPCRs can be utilized to further elucidate and understand the roles these receptors play in the human condition, both normal and diseased, as well as understanding the role of constitutive activation as it applies to understanding the signaling cascade.
  • the value in human orphan GPCRs is that its utility as a research tool is enhanced in that by determining the location(s) of such receptors within the body, the GPCRs can be used to understand the role of these receptors in the human body before the endogenous ligand therefor is identified.
  • Other uses of the disclosed receptors will become apparent to those in the art based upon, inter alia, a review of this patent document.
  • hGPCRZ7 Mouse AA775870 1,125 bp 15 16 GPCR27 hARE-1 TDAG 1689643 999 bp 17 18 AI090920 hARE-2 GPCR27 68530 1,122 bp 19 20 AA359504 hPPR1 Bovine 238667 1,053 bp 21 22 PPR1 H67224 hG2A Mouse See Example 2(a), 1,113 bp 23 24 1179426 below hCHN3 N.A. EST 36581 1,113 bp 25 26 (full length) hCHN4 TDAG 1184934 1,077 bp 27 28 AA804531 hCHN6 N.A.
  • Mouse EST clone 1179426 was used to obtain a human genomic clone containing all but three amino acid hG2A coding sequences.
  • the 5′end of this coding sequence was obtained by using 5′RACETM, and the template for PCR was Clontech's Human Spleen Marathon-readyTM cDNA.
  • the disclosed human G2A was amplified by PCR using the G2A cDNA specific primers for the first and second round PCR as shown in SEQ.ID.NO.: 39 and SEQ.ID.NO.:40 as follows: 5′-CTGTGTACAGCAGTTCGCAGAGTG-3′ (SEQ.ID.NO.:39; 1 st round PCR) 5′-GAGTGCCAGGCAGAGCAGGTAGAC-3′ (SEQ.ID.NO.:40; second round PCR).
  • PCR was performed using AdvantageTM GC Polymerase Kit (Clontech; manufacturing instructions will be followed), at 94° C. for 30 sec followed by 5 cycles of 94° C. for 5 sec and 72° C. for 4 min; and 30 cycles of 94° for 5 sec and 70° for 4 min.
  • An approximate 1.3 Kb PCR fragment was purified from agarose gel, digested with Hind III and Xba I and cloned into the expression vector pRC/CMV2 (Invitrogen). The cloned-insert was sequenced using the T7 SequenaseTM kit (USB Amersham; manufacturer instructions will be followed) and the sequence was compared with the presented sequence. Expression of the human G2A will be detected by probing an RNA dot blot (Clontech; manufacturer instructions will be followed) with the P 32 -labeled fragment.
  • hCHN9 is a partial cDNA clone having only an initiation codon; i.e., the termination codon was missing.
  • hCHN9 was used to “blast” against the data base (nr)
  • the 3′ sequence of hCHN9 was 100% homologous to the 5′ untranslated region of the leukotriene B4 receptor cDNA, which contained a termination codon in the frame with hCHN9 coding sequence.
  • PCR was performed using primers based upon the 5′ sequence flanking the initiation codon found in hCHN9 and the 3′ sequence around the termination codon found in the LTB4R 5′ untranslated region.
  • the 5′ primer sequence utilized was as follows: 5′-CCCGAATTCCTGCTTGCTCCCAGCTTGGCCC-3′ (SEQ.ID.NO.:41; sense) and 5′-TGTGGATCCTGCTGTCAAAGGTCCCATTCCGG-3′ (SEQ.ID.NO.:42; antisense).
  • PCR was performed using thymus cDNA as a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides.
  • the cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 10 sec.
  • a 1.1 kb fragment consistent with the predicted size was obtained from PCR. This PCR fragment was subcloned into pCMV (see below) and sequenced (see, SEQ.ID.NO.: 33).
  • hRUP4 was cloned by RT-PCR with human brain cDNA (Clontech) as templates: 5′-TCACAATGCTAGGTGTGGTC-3′ (SEQ.ID.NO.:43; sense) and 5′-TGCATAGACAATGGGATTACAG-3′ (SEQ.ID.NO.:44; antisense).
  • PCR was performed using TaqPlusTM PrecisionTM polymerase (Stratagene; manufacturing instructions will be followed) by the following cycles: 94° C. for 2 min; 94° C. 30 sec; 55° C. for 30 sec, 72° C. for 45 sec, and 72° C. for 10 min. Cycles 2 through 4 were repeated 30 times.
  • PCR products were separated on a 1% agarose gel and a 500 bp PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the T7 DNA SequenaseTM kit (Amsham) and the SP6/T7 primers (Stratagene). Sequence analysis revealed that the PCR fragment was indeed an alternatively spliced form of AI307658 having a continuous open reading frame with similarity to other GPCRs.
  • oligonucleotide primer sets 5′-CAAGGATGAAGGTGGTGTAGA-3′ (SEQ.ID.NO.:48; oligo 3) 5′-GTGTAGATCTTCTGGTGCACAGG-3′ (SEQ.ID.NO.:49; oligo 4)
  • [0067] were used for 3′- and 5′-race PCR with a human brain Marathon-ReadyTM cDNA (Clontech, Cat# 7400-1) as template, according to manufacture's instructions.
  • DNA fragments generated by the RACE PCR were cloned into the pCRII-TOPOTM vector (Invitrogen) and sequenced using the SP6/T7 primers (Stratagene) and some internal primers.
  • the 3′ RACE product contained a poly(A) tail and a completed open reading frame ending at a TAA stop codon.
  • the 5′ RACE product contained an incomplete 5′ end; i.e., the ATG initiation codon was not present.
  • oligo 3 Based on the new 5′ sequence, oligo 3 and the following primer: 5′-GCAATGCAGGTCATAGTGAGC-3′ (SEQ.ID.NO.:50; oligo 5)
  • [0069] were used for the second round of 5′ RACE PCR and the PCR products were analyzed as above.
  • a third round of 5′ RACE PCR was carried out utilizing antisense primers: 5′-TGGAGCATGGTGACGGGAATGCAGAAG-3′ (SEQ.ID.NO.:51; oligo 6) 5′-GTGATGAGCAGGTCACTGAGCGCCAAG-3′ (SEQ.ID.NO.:52; oligo 7).
  • oligo 4 as primers and sequence analysis of the 650 bp PCR product generated from human brain and heart cDNA templates (Clontech, Cat# 7404-1). The completed 3′ sequence was confirmed by RT-PCR using oligo 2 and the following antisense primer: 5′-TTGGGTTACAATCTGAAGGGCA-3′ (SEQ.ID.NO.:54; oligo 9)
  • hRUP5 The full length hRUP5 was cloned by RT-PCR using a sense primer upstream from ATG, the initiation codon (SEQ.ID.NO.: 55), and an antisense primer containing TCA as the stop codon (SEQ.ID.NO.: 56), which had the following sequences: 5′-ACTCCGTGTCCAGCAGGACTCTG-3′ (SEQ.ID.NO.:55) 5′-TGCGTGTTCCTGGACCCTCACGTG3′ (SEQ.ID.NO.:56)
  • Advantage cDNA polymerase (Clontech) was used for the amplification in a 50 ul reaction by the following cycle with step 2 through step 4 repeated 30 times: 94° C. for 30 sec; 94° for 15 sec; 69° for 40 sec; 72° C. for 3 min; and 72° C. fro 6 min.
  • a 1.4 kb PCR fragment was isolated and cloned with the pCRII-TOPOTM vector (Invitrogen) and completely sequenced using the T7 DNA SequenaseTM kit (Amsham). See, SEQ.ID.NO.: 9.
  • hRUP6 The full length hRUP6 was cloned by RT-PCR using primers: 5′-CAGGCCTTGGATTTTAATGTCAGGGATGG-3′ (SEQ.ID.NO.:57) and 5′-GGAGAGTCAGCTCTGAAAGAATTCAGG-3′ (SEQ.ID.NO.:58);
  • RUP7 was cloned by RT-PCR using primers: 5′-TGATGTGATGCCAGATACTAATAGCAC-3′ (SEQ.ID.NO.:59; sense) and 5′-CCTGATTCATTTAGGTGAGATTGAGAC-3′ (SEQ.ID.NO.:60; antisense)
  • AdvantageTM cDNA polymerase (Clontech) was used for the amplification in a 50 ul reaction by the following cycle with step 2 to step 4 repeated 30 times: 94° C. for 2 minutes; 94° C. for 15 seconds; 60° C. for 20 seconds; 72° C. for 2 minutes; 72° C. for 10 minutes.
  • a 1.25 Kb PCR fragment was isolated and cloned into the pCRII-TOPOTM vector (Invitrogen) and completely sequenced using the ABI Big Dye TerminatorTM kit (P.E. Biosystem). See, SEQ.ID.NO.: 13.
  • hARE-5 was cloned by PCR using the hARE5 specific primers 5′-CAGCGCAGGGTGAAGCCTGAGAGC-3′ SEQ.ID.NO.:69 (sense, 5′ of initiation codon ATG) and 5′-GGCACCTGCTGTGACCTGTGCAGG-3′ SEQ.ID.NO.:70 (antisense, 3′ of stop codon TGA)
  • a 1.1 Kb PCR fragment of predicated size was isolated and cloned into the pCRII-TOPOTM vector (Invitrogen) and completely sequenced (SEQ.ID.NO.:5) using the T7 DNA SequenaseTM kit (Amsham).
  • hARE4 The full length hARE4 was cloned by PCR using the hARE4 specific primers 5′-CTGGTGTGCTCCATGGCATCCC-3′SEQ.ID.NO.:67 (sense, 5′ of initiation codon ATG) and 5′GTAAGCCTCCCAGAACGAGAGG-3′ SEQ.ID.NO.:68 (antisense, 3′ of stop codon TGA) and
  • a 1.12 Kb PCR fragment of predicated size was isolated and cloned into the pCRII-TOPOTM vector (Invitrogen) and completely sequenced (SEQ.ID.NO.:3) using the T7 DNA SequenaseTM kit (Amsham).
  • hARE-3 The full length hARE-3 was cloned by PCR using the hARE-3 specific primers 5′-gatcaagcttCCATCCTACTGAAACCATGGTC-3′ SEQ.ID.NO.:65 (sense, lower case nucleotides represent Hind III overhang, ATG as initiation codon) and 5′-gatcagatctCAGTrCCAATATTCACACCACCGTC-3′ SEQ.ID.NO.:66 (antisense, lower case nucleotides represent Xba I overhang, TCA as stop codon) and human genomic DNA as template.
  • TaqPlus PrecisionTM DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4 repeated 35 times: 94° C., 3 minutes; 94° C., 1 minute; 55° C., 1 minute; 72° C., 2 minutes; 72° C., 10 minutes.
  • a 1.3 Kb PCR fragment of predicated size was isolated and digested with Hind III and Xba I, cloned into the pRC/CMV2 vector (Invitrogen) at the Hind III and Xba I sites and completely sequenced (SEQ.ID.NO.: 1) using the T7 DNA SequenaseTM kit (Amsham).
  • hRUP3 The full length hRUP3 was cloned by PCR using the hRUP3 specific primers 5′GTCCTGCCACTTCGAGACATGG-3′ SEQ.ID.NO.:71 (sense, ATG as initiation condon) and 5′-GAAACTTCTCTGCCTTACCGTC-3′ SEQ.ID.NO.:72 (antisense, 3′ of stop condon TAA) and human genomic DNA as template.
  • TaqPlus PrecisionTM DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4 repeated 35 times: 94° C., 3 minutes; 94° C., 1 minute; 58° C., 1 minute; 72° C., 2 minutes; 72° C., 10 minutes
  • a 1.0 Kb PCR fragment of predicated size was isolated and cloned into the pCRII-TOPOTM vector (Invitrogen) and completely sequenced (SEQ.ID.NO.: 7)using the T7 DNA sequenase kit (Amsham).
  • mammalian cells Although a variety of cells are available to the art for the expression of proteins, it is most preferred that mammalian cells be utilized. The primary reason for this is predicated upon practicalities, i.e., utilization of, e.g., yeast cells for the expression of a GPCR, while possible, introduces into the protocol a non-mammalian cell which may not (indeed, in the case of yeast, does not) include the receptor-coupling, genetic-mechanism and secretary pathways that have evolved for mammalian systems—thus, results obtained in non-mammalian cells, while of potential use, are not as preferred as that obtained from mammalian cells. Of the mammalian cells, COS-7, 293 and 293T cells are particularly preferred, although the specific mammalian cell utilized can be predicated upon the particular needs of the artisan.
  • the general procedure for expression of the disclosed GPCRs is as follows.
  • tube A will be prepared by mixing 20 kg DNA (e.g., pCMV vector; pCMV vector with receptor cDNA, etc.) in 1.2 ml serum free DMEM (Irvine Scientific, Irvine, Calif.); tube B will be prepared by mixing 120 ⁇ l lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B are admixed by inversions (several times), followed by incubation at room temperature for 30-45 min.
  • DNA e.g., pCMV vector; pCMV vector with receptor cDNA, etc.
  • tube B will be prepared by mixing 120 ⁇ l lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM.
  • Tubes A and B are admixed by inversions (several times), followed by incubation at room temperature for 30-45 min.
  • the admixture can be referred to as the “transfection mixture”.
  • Plated 293T cells are washed with 1XPBS, followed by addition of 10ml serum free DMEM.
  • 2.4 ml of the transfection mixture will then be added to the cells, followed by incubation for 4hrs at 37° C./5% CO 2 .
  • the transfection mixture was then be removed by aspiration, followed by the addition of 25 ml of DMEM/10% Fetal Bovine Serum. Cells will then be incubated at 37° C./5% CO 2 . After 72 hr incubation, cells can then be harvested and utilized for analysis.
  • RNA Master BlotTM (Clontech, #7770-1) was hybridized with the endogenous human GPCR radiolabeled probe and washed under stringent conditions according manufacturer's instructions.
  • RT-PCR was performed using hRUP3-specific primers and human multiple tissue cDNA panels (MTC, Clontech) as templates.
  • Taq DNA polymerase (Stratagene) was utilized for the PCR reaction, using the following reaction cycles in a 40ul reaction: 94° C. for 2 min; 94° C. for 15 sec; 55° C. for 30 sec; 72° C. for 1 min; 72° C., for 10 min.
  • Primers were as follows: 5′-GACAGGTACCTTGCCATCAAG-3′ (SEQ.ID.NO.:61; sense) 5′-CTGCACAATGCCAGTGATAAGG-3′ (SEQ.ID.NQ.:62; antisense).
  • hRUP3 As is supported by the data of FIG. 3, of the 16 human tissues in the cDNA panel utilized (brain, colon, heart, kidney, lung, ovary, pancreas, placenta, prostate, skeleton, small intestine, spleen, testis, thymus leukocyte, and liver) a single hRUP3 band is evident only from the pancreas. Additional comparative analysis of the protein sequence of hRUP3 with other GPCRs suggest that hRUP3 is related to GPCRs having small molecule endogenous ligand such that it is predicted that the endogenous ligand for hRUP3 is a small molecule.
  • RT-PCR was performed using hRUP4 oligo's 8 and 4 as primers and the human multiple tissue cDNA panels (MTC, Clontech) as templates.
  • Taq DNA polymerase 10 (Stratagene) was used for the amplification in a 40 ul reaction by the following cycles: 94° C. for 30 seconds, 94° C. for 10 seconds, 55° C. for 30 seconds, 72° C. for 2 minutes, and 72° C. for 5 minutes with cycles 2 through 4 repeated 30 times.
  • hRUP4 mRNA was found expressed in many human tissues, with the strongest expression in heart and kidney. (see, FIG. 4).
  • a 300 bp fragment derived from the 5′ end of hRUP4 was used as a probe for the Southern Blot analysis.
  • the probe was labeled with 32 P-dCTP using the Prime-It IITM Random Primer Labeling Kit (Stratagene) and purified using the ProbeQuantTM G-50 micro columns (Amersham). Hybridization was done overnight at 42° C. following a 12 hr pre-hybridization. The blot was finally washed at 65° C. with 0.1 ⁇ SSC. The Southern blot did confirm the PCR fragments as hRUP4.
  • RT-PCR was performed using the following hRUP5 specific primers: 5′-CTGACTTCTTGTTCCTGGCAGCAGCGG-3′ (SEQ.ID.NO.:63; sense) 5′-AGACCAGCCAGGGCACGCTGAAGAGTG-3′ (SEQ.ID.NO.:64; antisense)
  • RT-PCR was applied to confirm the expression and to determine the tissue distribution of hRUP6.
  • Oligonucleotides used based on an alignment of AC005871 and GPR66 segments, had the following sequences: 5′-CCAACACCAGCATCCATGGCATCAAG-3′ (SEQ.ID.NO.:73; sense), 5′-GGAGAGTCAGCTCTGAAAGAATTCAGG-3′ (SEQ.ID.NO.:74; antisense)
  • PCR was performed using TaqPlus PrecisionTM polymerase (Stratagene; manufacturing instructions will be followed) in a 40 ul reaction by the following cycles: 94° C. for 30 sec; 94° C. 5 sec; 66° C. for 40 sec, 72° C. for 2.5 min, and 72° C. for 7 min. Cycles 2 through 4 were repeated 30 times.
  • the Vector utilized be pCMV.
  • This vector was deposited with the American Type Culture Collection (ATCC) on Oct. 13, 1998 (10801 University Boulevard., Manassas, Va. 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be. The ATCC has assigned the following deposit number to pCMV: ATCC #203351.

Abstract

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to endogenous, human orphan G protein-coupled receptors.

Description

  • This patent document claims priority benefit of each of the following applications, all filed with the United States Patent and Trademark Office via U.S. Express Mail on the indicated filing dates: U.S. Provisional No. 60/121,852, filed; Feb. 26, 1999 claiming the benefit of U.S. Provisional No. 60/109,213, filed Nov. 20, 1998; U.S. Provisional No. 60/120,416, filed Feb. 16, 1999; U.S. [0001] Provisional Number 60/123,946, filed Mar. 12, 1999; U.S. Provisional No. 60/123,949, filed Mar. 12, 1999; U.S. Provisional No. 60/136,436, filed May 28, 1999; U.S. Provisional No. 60/136,439, filed May 28, 1999; U.S. Provisional No. 60/136,567, filed May 28, 1999; U.S. Provisional No. 60/137,127, filed May 28, 1999; U.S. Provisional Number 60/137,131, filed May 28, 1999; U.S. Provisional No. 141,448, filed Jun. 29, 1999 claiming priority from U.S. Provisional No. 60/136,437, filed May 28, 1999, U.S. Provisional Number ______ (Arena Pharmaceuticals, Inc. docket number CHN10-1), filed Sep. 29, 1999; U.S. Provisional No. 60/156,333, filed Sep. 29, 1999; U.S. Provisional No. 60/156,555, filed Sep. 29, 1999; U.S. Provisional No. 60/156,634, filed Sep. 29, 1999; U.S. Provisional Number ______ (Arena Pharmaceuticals, Inc. docket number RUP6-1), filed Oct. 1, 1999; U.S. Provisional Number ______ (Arena Pharmaceuticals, Inc. docket number RUP7-1), filed Oct. 1, 1999; U.S. Provisional Number ______ (Arena Pharmaceuticals, Inc. docket number CHN6-1), filed Oct. 1, 1999; U.S. Provisional Number ______ (Arena Pharmaceuticals, Inc. docket number RUP5-1), filed Oct. 1, 1999; U.S. Provisional Number ______ (Arena Pharmaceuticals, Inc. docket number CHN9-1), filed Oct. 1, 1999. This patent document is related to U.S. Ser. No. 09/170,496 filed Oct. 13, 1999, and U.S. Serial Number unknown (Woodcock Washburn Kurtz Mackiewicz & Norris, LLP docket number AREN-0054 ) filed on Oct. 12, 1999 (via U.S. Express Mail) both being incorporated herein by reference. This patent document also is related to U.S. Ser. No. 09/364,425; filed Jul. 30, 1999, which is incorporated by reference in its entirety. Each of the foregoing applications are incorporated herein by reference in their entirety.
    • FIELD OF THE INVENTION
  • The invention disclosed in this patent document relates to transmembrane receptors, and more particularly to endogenous, orphan, human G protein-coupled receptors (“GPCRs”). [0002]
    • BACKGROUND OF THE INVENTION
  • Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR or GPCRs) class. It is estimated that there are some 100,000 genes within the human genome, and of these, approximately 2% or 2,000 genes, are estimated to code for GPCRs. Receptors, including GPCRs, for which the endogenous ligand has been identified are referred to as “known” receptors, while receptors for which the endogenous ligand has not been identified are referred to as “orphan” receptors. GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, 60% of all prescription pharmaceuticals have been developed. This distinction is not merely semantic, particularly in the case of GPCRs. Thus, the orphan GPCRs are to the pharmaceutical industry what gold was to California in the late 19[0003] th century—an opportunity to drive growth, expansion, enhancement and development.
  • GPCRs share a common structural motif. All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane-1 (TM-1), transmebrane-2 (TM-2), etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or “extracellular” side, of the cell membrane (these are referred to as “extracellular” [0004] regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or “intracellular” side, of the cell membrane (these are referred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively). The “carboxy” (“C”) terminus of the receptor lies in the intracellular space within the cell, and the “amino” (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
  • Generally, when an endogenous ligand binds with the receptor (often referred to as “activation” of the receptor), there is a change in the conformation of the intracellular region that allows for coupling between the intracellular region and an intracellular “G-protein.” It has been reported that GPCRs are “promiscuous” with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, T., 43 [0005] Life Sciences 1095 (1988). Although other G proteins exist, currently, Gq, Gs, Gi, and Go are G proteins that have been identified. Endogenous ligand-activated GPCR coupling with the G-protein begins a signaling cascade process (referred to as “signal transduction”). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. It is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.
  • Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an “inactive” state and an “active” state. A receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to produce a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response. A receptor may be stabilized in an active state by an endogenous ligand or a compound such as a drug. [0006]
    • SUMMARY OF THE INVENTION
  • Disclosed herein are human endogenous orphan G protein-coupled receptors.[0007]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGS. 1A and 1B provide reference “grids” for certain dot-blots provided herein (see also, FIG. 2A and 2B, respectively). [0008]
  • FIGS. 2A and 2B provide reproductions of the results of certain dot-blot analyses resulting from hCHN3 and hCHN8, respectively (see also, FIGS. 1A and 1B, respectively). [0009]
  • FIG. 3 provides a reproduction of the results of RT-PCR analysis of hRUP3. [0010]
  • FIG. 4 provides a reproduction of the results of RT-PCR analysis of hRUP4. [0011]
  • FIG. 5 provides a reproduction of the results of RT-PCR analysis of hRUP6.[0012]
    • DETAILED DESCRIPTION
  • The scientific literature that has evolved around receptors has adopted a number of terms to refer to ligands having various effects on receptors. For clarity and consistency, the following definitions will be used throughout this patent document. To the extent that these definitions conflict with other definitions for these terms, the following definitions shall control: [0013]
  • AMINO ACID ABBREVIATIONS used herein are set out in Table 1: [0014]
    TABLE 1
    ALANINE ALA A
    ARGININE ARG R
    ASPARAGINE ASN N
    ASPARTIC ACID ASP D
    CYSTEINE CYS C
    GLUTAMIC ACID GLU E
    GLUTAMINE GLN Q
    GLYCINE GLY G
    HISTIDINE HIS H
    ISOLEUCINE ILE I
    LEUCINE LEU L
    LYSINE LYS K
    METHIONINE MET M
    PHENYLALANINE PHE F
    PROLINE PRO P
    SERINE SER S
    THREONINE THR T
    TRYPTOPHAN TRP W
    TYROSINE TYR Y
    VALINE VAL V
  • COMPOSITION means a material comprising at least one component. [0015]
  • ENDOGENOUS shall mean a material that a mammal naturally produces. ENDOGENOUS in reference to, for example and not limitation, the term “receptor,” shall mean that which is naturally produced by a mammal (for example, and not limitation, a human) or a virus. By contrast, the term NON-ENDOGENOUS in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human) or a virus. [0016]
  • HOST CELL shall mean a cell capable of having a Plasmid and/or Vector incorporated therein. In the case of a prokaryotic Host Cell, a Plasmid is typically replicated as a autonomous molecule as the Host Cell replicates (generally, the Plasmid is thereafter isolated for introduction into a eukaryotic Host Cell); in the case of a eukaryotic Host Cell, a Plasmid is integrated into the cellular DNA of the Host Cell such that when the eukaryotic Host Cell replicates, the Plasmid replicates. Preferably, for the purposes of the invention disclosed herein the Host Cell is eukaryotic, more preferably, mammalian, and most preferably selected from the group consisting of 293, 293T and COS-7 cells. [0017]
  • LIGAND shall mean an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor. [0018]
  • NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurring molecule specific for an endogenous naturally occurring ligand wherein the binding of a ligand to a receptor activates an intracellular signaling pathway. [0019]
  • ORPHAN RECEPTOR shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has not been identified or is not known. [0020]
  • PLASMID shall mean the combination of a Vector and cDNA. Generally, a Plasmid is introduced into a Host Cell for the purposes of replication and/or expression of the cDNA as a protein. [0021]
  • VECTOR sin reference to cDNA shall mean a circular DNA capable of incorporating at least one cDNA and capable of incorporation into a Host Cell. [0022]
  • The order of the following sections is set forth for presentational efficiency and is not intended, nor should be construed, as a limitation on the disclosure or the claims to follow. [0023]
  • Identification of Human GPCRs [0024]
  • The efforts of the Human Genome project have led to the identification of a plethora of information regarding nucleic acid sequences located within the human genome; it has been the case in this endeavor that genetic sequence information has been made available without an understanding or recognition as to whether or not any particular genomic sequence does or may contain open-reading frame information that translate human proteins. Several methods of identifying nucleic acid sequences within the human genome are within the purview of those having ordinary skill in the art. For example, and not limitation, a variety of GPCRs, disclosed herein, were discovered by reviewing the GenBank™ database, while other GPCRs were discovered by utilizing a nucleic acid sequence of a GPCR, previously sequenced, to conduct a BLAST™ search of the EST database. Table A, below, lists the disclosed endogenous orphan GPCRs along with a GPCR's respective homologous GPCR: [0025]
    TABLE A
    Reference
    Open To
    Disclosed Reading Per Cent Homologous
    Human Accession Frame Homology To GPCR
    Orphan Number (Base Designated (Accession
    GPCRS Identified Pairs) GPCR No.)
    hARE-3 AL033379 1,260 bp 52.3% LPA-R U92642
    hARE-4 AC006087 1,119 bp 36% P2Y5 AF000546
    hARE-5 AC006255 1,104 bp 32% Oryzias D43633
    latipes
    hGPR27 AA775870 1,128 bp
    hARE-1 AI090920   999 bp 43% KIAA0001 D13626
    hARE-2 AA359504 1,122 bp 53% GPR27
    hPPR1 H67224 1,053 bp 39% EBI1 L31581
    hG2A AA754702 1,113 bp 31% GPR4 L36148
    hRUP3 AL035423 1,005 bp 30% Drosophila 2133653
    melanogaster
    hRUP4 AI307658 1,296 bp 32% pNPGPR NP_004876
    28% and 29% AAC41276
    Zebra fish Ya and
    and Yb, AAB94616
    respectively
    hRUP5 AC005849 1,413 bp 25% DEZ Q99788
    23% FMLPR P21462
    hRUP6 AC005871 1,245 bp 48% GPR66 NP_006047
    hRUP7 AC007922 1,173 bp 43% H3R AF140538
    hCHN3 EST 36581 1,113 bp 53% GPR27
    hCHN4 AA804531 1,077 bp 32% thrombin 4503637
    hCHN6 EST 2134670 1,503 bp 36% edg-1 NP_001391
    hCHN8 EST 764455 1,029 bp 47% KIAA0001 D13626
    hCHN9 EST 1541536 1,077 bp 41% LTB4R NM_000752
    hCHN10 EST 1365839 1,055 bp 35% P2Y NM_002563
  • Receptor homology is useful in terms of gaining an appreciation of a role of the disclosed receptors within the human body. Additionally, such homology can provide insight as to possible endogenous ligand(s) that may be natural activators for the disclosed orphan GPCRs. [0026]
  • B. Receptor Screening [0027]
  • Techniques have become more readily available over the past few years for endogenous-ligand identification (this, primarily, for the purpose of providing a means of conducting receptor-binding assays that require a receptor's endogenous ligand) because the traditional study of receptors has always proceeded from the a priori assumption (historically based) that the endogenous ligand must first be identified before discovery could proceed to find antagonists and other molecules that could affect the receptor. Even in cases where an antagonist might have been known first, the search immediately extended to looking for the endogenous ligand. This mode of thinking has persisted in receptor research even after the discovery of constitutively activated receptors. What has not been heretofore recognized is that it is the active state of the receptor that is most useful for discovering agonists, partial agonists, and inverse agonists of the receptor. For those diseases which result from an overly active receptor or an under-active receptor, what is desired in a therapeutic drug is a compound which acts to diminish the active state of a receptor or enhance the activity of the receptor, respectively, not necessarily a drug which is an antagonist to the endogenous ligand. This is because a compound that reduces or enhances the activity of the active receptor state need not bind at the same site as the endogenous ligand. Thus, as taught by a method of this invention, any search for therapeutic compounds should start by screening compounds against the ligand-independent active state. [0028]
  • As is known in the art, GPCRs can be “active” in their endogenous state even without the binding of the receptor's endogenous ligand thereto. Such naturally-active receptors can be screened for the direct identification (i.e., without the need for the receptor's endogenous ligand) of, in particular, inverse agonists. Alternatively, the receptor can be “activated” via, e.g., mutation of the receptor to establish a non-endogenous version of the receptor that is active in the absence of the receptor's endogenous ligand. [0029]
  • Screening candidate compounds against an endogenous or non-endogenous, constitutively activated version of the human orphan GPCRs disclosed herein can provide for the direct identification of candidate compounds which act at this cell surface receptor, without requiring use of the receptor's endogenous ligand. By determining areas within the body where the endogenous version of human GPCRs disclosed herein is expressed and/or over-expressed, it is possible to determine related disease/disorder states which are associated with the expression and/or over-expression of the receptor; such an approach is disclosed in this patent document. [0030]
  • With respect to creation of a mutation that may evidence constitutive activation of human orphan GPCRs disclosed herein is based upon the distance from the proline residue at which is presumed to be located within TM6 of the GPCR typically nears the TM6/IC3 interface (such proline residue appears to be quite conserved). By mutating the amino acid residue located 16 amino acid residues from this residue (presumably located in the IC3 region of the receptor) to, most preferably, a lysine residue, such activation may be obtained. Other amino acid residues may be useful in the mutation at this position to achieve this objective. [0031]
  • C. Disease/Disorder Identification and/or Selection [0032]
  • Preferably, the DNA sequence of the human orphan GPCR can be used to make a probe for (a) dot-blot analysis against tissue-mRNA, and/or (b) RT-PCR identification of the expression of the receptor in tissue samples. The presence of a receptor in a tissue source, or a diseased tissue, or the presence of the receptor at elevated concentrations in diseased tissue compared to a normal tissue, can be preferably utilized to identify a correlation with a treatment regimen, including but not limited to, a disease associated with that disease. Receptors can equally well be localized to regions of organs by this technique. Based on the known functions of the specific tissues to which the receptor is localized, the putative functional role of the receptor can be deduced. [0033]
  • D. Screening of Candidate Compounds [0034]
  • 1. Generic GPCR screening assay techniques [0035]
  • When a G protein receptor becomes constitutively active (i.e., active in the absence of endogenous ligand binding thereto), it binds to a G protein (e.g., Gq, Gs, Gi, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, constitutively activated receptors continue to exchange GDP to GTP. A non-hydrolyzable analog of GTP, [[0036] 35S]GTPγS, can be used to monitor enhanced binding to membranes which express constitutively activated receptors. It is reported that [35S]GTPγS can be used to monitor G protein coupling to membranes in the absence and presence of ligand. An example of this monitoring, among other examples well-known and available to those in the art, was reported by Traynor and Nahorski in 1995. The preferred use of this assay system is for initial screening of candidate compounds because the system is generically applicable to all G protein-coupled receptors regardless of the particular G protein- that interacts with the intracellular domain of the receptor.
  • 2. Specific GPCR screening assay techniques [0037]
  • Once candidate compounds are identified using the “generic” G protein-coupled receptor assay (i.e., an assay to select compounds that are agonists, partial agonists, or inverse agonists), further screening to confirm that the compounds have interacted at the receptor site is preferred. For example, a compound identified by the “generic” assay may not bind to the receptor, but may instead merely “uncouple” the G protein from the intracellular domain. [0038]
  • a. Gs and Gi. [0039]
  • Gs stimulates the enzyme adenylyl cyclase. Gi (and Go), on the other hand, inhibit this enzyme. Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, S constitutively activated GPCRs that couple the Gs protein are associated with increased cellular levels of cAMP. On the other hand, constitutively activated GPCRs that couple the Gi (or Go) protein are associated with decreased cellular levels of cAMP. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, [0040] From Neuron To Brain (3rd Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP can be utilized to determine if a candidate compound is, e.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP). A variety of approaches known in the art for measuring cAMP can be utilized; a most preferred approach relies upon the use of anti-cAMP antibodies in an ELISA-based format. Another type of assay that can be utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes. Cyclic AMP drives gene expression by promoting the binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) which then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene. Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter gene, e.g., β-galactosidase or luciferase. Thus, a constitutively activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and expression of the reporter protein. The reporter protein such as β-galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).
  • Go and Gq. [0041]
  • Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP[0042] 2, releasing two intracellular messengers: diacycloglycerol (DAG) and inistol 1,4,5-triphoisphate (IP3). Increased accumulation of IP3 is associated with activation of Gq- and Go-associated receptors. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron To Brain (3rd Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992). Assays that detect IP3 accumulation can be utilized to determine if a candidate compound is, e.g., an inverse agonist to a Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of IP3). Gq-associated receptors can also been examined using an AP1 reporter assay in that Gq-dependent phospholipase C causes activation of genes containing AP1 elements; thus, activated Gq-associated receptors will evidence an increase in the expression of such genes, whereby inverse agonists thereto will evidence a decrease in such expression, and agonists will evidence an increase in such expression. Commercially available assays for such detection are available.
  • 3. GPCR Fusion Protein [0043]
  • The use of an endogenous, constitutively activated orphan GPCR, or a non-endogenous, constitutively activated orphan GPCR, for screening of candidate compounds for the direct identification of inverse agonists, agonists and partial agonists provides a unique challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto. Thus, it is often useful that an approach be utilized that can enhance the signal obtained by the activated receptor. A preferred approach is the use of a GPCR Fusion Protein. [0044]
  • Generally, once it is determined that a GPCR is or has been constitutively activated, using the assay techniques set forth above (as well as others), it is possible to determine the predominant G protein that couples with the endogenous GPCR. Coupling of the G protein to the GPCR provides a signaling pathway that can be assessed. Because it is most preferred that screening take place by use of a mammalian expression system, such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the constitutively activated orphan GPCR will continuously signal. In this regard, it is preferred that this signal be enhanced such that in the presence of, e.g., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist. [0045]
  • The GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the GPCR. The GPCR Fusion Protein is preferred for screening with a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is most preferably utilized in such screening techniques, although the GPCR Fusion Protein can also be (and preferably is) used with an endogenous, constitutively activated GPCR. This is important in facilitating a significant “signal to noise” ratio; such a significant ratio is import preferred for the screening of candidate compounds as disclosed herein. [0046]
  • The construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator. The criteria of importance for such a GPCR Fusion Protein construct is that the GPCR sequence and the G protein sequence both be in-frame (preferably, the sequence for the GPCR is upstream of the G protein sequence) and that the “stop” codon of the GPCR must be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed. The GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). We have a preference (based upon convenience) of use of a spacer in that some restriction sites that are not used will, effectively, upon expression, become a spacer. Most preferably, the G protein that couples to the GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct. Because there are only a few G proteins that have been identified, it is preferred that a construct comprising the sequence of the G protein (i.e., a universal G protein construct) be available for insertion of an endogenous GPCR sequence therein; this provides for efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences. [0047]
  • E. Other Utility [0048]
  • Although a preferred use of the human orphan GPCRs disclosed herein may be for the direct identification of candidate compounds as inverse agonists, agonists or partial agonists (preferably for use as pharmaceutical agents), these versions of human GPCRs can also be utilized in research settings. For example, in vitro and in vivo systems incorporating GPCRs can be utilized to further elucidate and understand the roles these receptors play in the human condition, both normal and diseased, as well as understanding the role of constitutive activation as it applies to understanding the signaling cascade. The value in human orphan GPCRs is that its utility as a research tool is enhanced in that by determining the location(s) of such receptors within the body, the GPCRs can be used to understand the role of these receptors in the human body before the endogenous ligand therefor is identified. Other uses of the disclosed receptors will become apparent to those in the art based upon, inter alia, a review of this patent document. [0049]
    • EXAMPLES
  • The following examples are presented for purposes of elucidation, and not limitation, of the present invention. While specific nucleic acid and amino acid sequences are disclosed herein, those of ordinary skill in the art are credited with the ability to make minor modifications to these sequences while achieving the same or substantially similar results reported below. Unless otherwise indicated below, all nucleic acid sequences for the disclosed endogenous orphan human GPCRs have been sequenced and verified. For purposes of equivalent receptors, those of ordinary skill in the art will readily appreciate that conservative substitutions can be made to the disclosed sequences to obtain a functionally equivalent receptor. [0050]
    • Example 1 ENDOGENOUS HUMAN GPCRS
  • 1. Identification of Human GPCRs [0051]
  • Several of the disclosed endogenous human GPCRs were identified based upon a review of the GenBank database information. While searching the database, the following cDNA clones were identified as evidenced below. [0052]
    Disclosed Nucleic Amino
    Human Complete DNA Open Reading Acid Acid
    Orphan Accession Sequence Frame SEQ. ID. SEQ. ID.
    GPCRs Number (Base Pairs) (Base Pairs) NO. NO.
    hARE-3 AL033379 111,389 bp 1,260 bp 1 2
    hARE-4 AC006087 226,925 bp 1,119 bp 3 4
    hARE-5 AC006255 127,605 bp 1,104 bp 5 6
    hRUP3 AL035423 140,094 bp 1,005 bp 7 8
    hRUP5 AC005849 169,144 bp 1,413 bp 9 10
    hRUP6 AC005871 218,807 bp 1,245 bp 11 12
    hRUP7 AC007922 158,858 bp 1,173 bp 13 14
  • Other disclosed endogenous human GPCRs were identified by conducting a BLAST search of EST database (dbest) using the following EST clones as query sequences. The following EST clones identified were then used as a probe to screen a human genomic library. [0053]
    Disclosed Open Nucleic Amino
    Human EST Clone/ Reading Acid Acid
    Orphan Query Accession No. Frame SEQ. ID. SEQ. ID.
    GPCRs (Sequence) Identified (Base Pairs) NO. NO.
    hGPCRZ7 Mouse AA775870 1,125 bp 15 16
    GPCR27
    hARE-1 TDAG 1689643   999 bp 17 18
    AI090920
    hARE-2 GPCR27  68530 1,122 bp 19 20
    AA359504
    hPPR1 Bovine  238667 1,053 bp 21 22
    PPR1 H67224
    hG2A Mouse See Example 2(a), 1,113 bp 23 24
    1179426 below
    hCHN3 N.A. EST 36581 1,113 bp 25 26
    (full length)
    hCHN4 TDAG 1184934 1,077 bp 27 28
    AA804531
    hCHN6 N.A. EST 2134670 1,503 bp 29 30
    (full length)
    hCHN8 KIAA0001 EST 764455 1,029 bp 31 32
    hCHN9 1365839 EST 1541536 1,077 bp 33 34
    hCHN10 Mouse EST Human 1365839 1,005 bp 35 36
    1365839
    hRUF4 NA. AI307658 1,296 bp 37 38
  • 2. Full Length Cloning [0054]
  • a. hG2A (Seq. Id. Nos. 23 & 24) [0055]
  • Mouse EST clone 1179426 was used to obtain a human genomic clone containing all but three amino acid hG2A coding sequences. The 5′end of this coding sequence was obtained by using 5′RACE™, and the template for PCR was Clontech's Human Spleen Marathon-ready™ cDNA. The disclosed human G2A was amplified by PCR using the G2A cDNA specific primers for the first and second round PCR as shown in SEQ.ID.NO.: 39 and SEQ.ID.NO.:40 as follows: [0056]
    5′-CTGTGTACAGCAGTTCGCAGAGTG-3′ (SEQ.ID.NO.:39; 1st round PCR)
    5′-GAGTGCCAGGCAGAGCAGGTAGAC-3′ (SEQ.ID.NO.:40; second round PCR).
  • PCR was performed using Advantage™ GC Polymerase Kit (Clontech; manufacturing instructions will be followed), at 94° C. for 30 sec followed by 5 cycles of 94° C. for 5 sec and 72° C. for 4 min; and 30 cycles of 94° for 5 sec and 70° for 4 min. An approximate 1.3 Kb PCR fragment was purified from agarose gel, digested with Hind III and Xba I and cloned into the expression vector pRC/CMV2 (Invitrogen). The cloned-insert was sequenced using the T7 Sequenase™ kit (USB Amersham; manufacturer instructions will be followed) and the sequence was compared with the presented sequence. Expression of the human G2A will be detected by probing an RNA dot blot (Clontech; manufacturer instructions will be followed) with the P[0057] 32-labeled fragment.
  • b. hCHN9 (Seq. Id. Nos. 33 & 34) [0058]
  • Sequencing of the EST clone 1541536 indicated that hCHN9 is a partial cDNA clone having only an initiation codon; i.e., the termination codon was missing. When hCHN9 was used to “blast” against the data base (nr), the 3′ sequence of hCHN9 was 100% homologous to the 5′ untranslated region of the leukotriene B4 receptor cDNA, which contained a termination codon in the frame with hCHN9 coding sequence. To determine whether the 5′ untranslated region of LTB4R cDNA was the 3′ sequence of hCHN9, PCR was performed using primers based upon the 5′ sequence flanking the initiation codon found in hCHN9 and the 3′ sequence around the termination codon found in the [0059] LTB4R 5′ untranslated region. The 5′ primer sequence utilized was as follows:
    5′-CCCGAATTCCTGCTTGCTCCCAGCTTGGCCC-3′ (SEQ.ID.NO.:41; sense) and
    5′-TGTGGATCCTGCTGTCAAAGGTCCCATTCCGG-3′ (SEQ.ID.NO.:42; antisense).
  • PCR was performed using thymus cDNA as a template and rTth polymerase (Perkin Elmer) with the buffer system provided by the manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4 nucleotides. The cycle condition was 30 cycles of 94° C. for 1 min, 65° C. for 1 min and 72° C. for 1 min and 10 sec. A 1.1 kb fragment consistent with the predicted size was obtained from PCR. This PCR fragment was subcloned into pCMV (see below) and sequenced (see, SEQ.ID.NO.: 33). [0060]
  • c. hRUP 4 (Seq. Id. Nos. 37 & 38) [0061]
  • The full length hRUP4 was cloned by RT-PCR with human brain cDNA (Clontech) as templates: [0062]
    5′-TCACAATGCTAGGTGTGGTC-3′ (SEQ.ID.NO.:43; sense) and
    5′-TGCATAGACAATGGGATTACAG-3′ (SEQ.ID.NO.:44; antisense).
  • PCR was performed using TaqPlus™ Precision™ polymerase (Stratagene; manufacturing instructions will be followed) by the following cycles: 94° C. for 2 min; 94° C. 30 sec; 55° C. for 30 sec, 72° C. for 45 sec, and 72° C. for 10 min. [0063] Cycles 2 through 4 were repeated 30 times.
  • The PCR products were separated on a 1% agarose gel and a 500 bp PCR fragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen) and sequenced using the T7 DNA Sequenase™ kit (Amsham) and the SP6/T7 primers (Stratagene). Sequence analysis revealed that the PCR fragment was indeed an alternatively spliced form of AI307658 having a continuous open reading frame with similarity to other GPCRs. The completed sequence of this PCR fragment was as follows: [0064]
    5′-TCACAATGCTAGGTGTGGTCTGGCTGGTGGCAGTCATCGTAGGATCACCCATGTGGCAC (SEQ.ID.NO.:45)
    GTGCAACAACTTGAGATCAAATATGACTTCCTATATGAAAAGGAACACATCTGCTGCTTAGAA
    GAGTGGACCAGCCCTGTGCACCAGAAGATCTACACCACCTTCATCCTTGTCATCCTCTTCCTCC
    TGCCTCTTATGGTGATGCTTATTCTGTACGTAAAATTGGTTATGAACTTTGGATAAAGAAAAGA
    GTTGGGGATGGTTCAGTGCTTCGAACTATTCATGGAAAAGAAATGTCCAAAATAGCCAGGAAG
    AAGAAACGAGCTGTCATTATGATGGTGACAGTGGTGGCTCTCTTTGCTGTGTGCTGGGCACCA
    TTCCATGTTGTCCATATGATGATTGAATACAGTAATTTTGAAAAGGAATATGATGATGTCACA
    ATCAAGATGATTTTTGCTATCGTGCAAATTATTGGATTTTCCAACTCCATCTGTAATCCCATTG
    TCTATGCA-3′
  • Based on the above sequence, two sense oligonucleotide primer sets: [0065]
    5′-CTGCTTAGAAGAGTGGACCAG-3′ (SEQ.ID.NO.:46;
    oligo 1),
    5′-CTGTGCACCAGAAGATCTACAC-3′ (SEQ.ID.NO.:47;
    oligo 2)
  • and two antisense oligonucleotide primer sets: [0066]
    5′-CAAGGATGAAGGTGGTGTAGA-3′ (SEQ.ID.NO.:48;
    oligo 3)
    5′-GTGTAGATCTTCTGGTGCACAGG-3′ (SEQ.ID.NO.:49;
    oligo 4)
  • were used for 3′- and 5′-race PCR with a human brain Marathon-Ready™ cDNA (Clontech, Cat# 7400-1) as template, according to manufacture's instructions. DNA fragments generated by the RACE PCR were cloned into the pCRII-TOPO™ vector (Invitrogen) and sequenced using the SP6/T7 primers (Stratagene) and some internal primers. The 3′ RACE product contained a poly(A) tail and a completed open reading frame ending at a TAA stop codon. The 5′ RACE product contained an incomplete 5′ end; i.e., the ATG initiation codon was not present. [0067]
  • Based on the new 5′ sequence, [0068] oligo 3 and the following primer:
    5′-GCAATGCAGGTCATAGTGAGC-3′ (SEQ.ID.NO.:50;
    oligo 5)
  • were used for the second round of 5′ RACE PCR and the PCR products were analyzed as above. A third round of 5′ RACE PCR was carried out utilizing antisense primers: [0069]
    5′-TGGAGCATGGTGACGGGAATGCAGAAG-3′ (SEQ.ID.NO.:51; oligo 6)
    5′-GTGATGAGCAGGTCACTGAGCGCCAAG-3′ (SEQ.ID.NO.:52; oligo 7).
  • The sequence of the 5′ RACE PCR products revealed the presence of the initiation codon ATG, and further round of 5′ RACE PCR did not generate any more 5′ sequence. The completed 5′ sequence was confirmed by RT-PCR using [0070] sense primer
    5′-GCAATGCAGGCGCTTAACATTAC-3′ (SEQ.ID.NO.:53;
    oligo 8)
  • and [0071] oligo 4 as primers and sequence analysis of the 650 bp PCR product generated from human brain and heart cDNA templates (Clontech, Cat# 7404-1). The completed 3′ sequence was confirmed by RT-PCR using oligo 2 and the following antisense primer:
    5′-TTGGGTTACAATCTGAAGGGCA-3′ (SEQ.ID.NO.:54;
    oligo 9)
  • and sequence analysis of the 670 bp PCR product generated from human brain and heart cDNA templates. (Clontech, Cat# 7404-1). [0072]
  • d. hRUP5 (Seq. Id. Nos. 9 & 10) [0073]
  • The full length hRUP5 was cloned by RT-PCR using a sense primer upstream from ATG, the initiation codon (SEQ.ID.NO.: 55), and an antisense primer containing TCA as the stop codon (SEQ.ID.NO.: 56), which had the following sequences: [0074]
    5′-ACTCCGTGTCCAGCAGGACTCTG-3′ (SEQ.ID.NO.:55)
    5′-TGCGTGTTCCTGGACCCTCACGTG3′ (SEQ.ID.NO.:56)
  • and human peripheral leukocyte cDNA (Clontech) as a template. Advantage cDNA polymerase (Clontech) was used for the amplification in a 50 ul reaction by the following cycle with [0075] step 2 through step 4 repeated 30 times: 94° C. for 30 sec; 94° for 15 sec; 69° for 40 sec; 72° C. for 3 min; and 72° C. fro 6 min. A 1.4 kb PCR fragment was isolated and cloned with the pCRII-TOPO™ vector (Invitrogen) and completely sequenced using the T7 DNA Sequenase™ kit (Amsham). See, SEQ.ID.NO.: 9.
  • e. hRUP6 (Seq. Id. Nos. 11 & 12) [0076]
  • The full length hRUP6 was cloned by RT-PCR using primers: [0077]
    5′-CAGGCCTTGGATTTTAATGTCAGGGATGG-3′ (SEQ.ID.NO.:57) and
    5′-GGAGAGTCAGCTCTGAAAGAATTCAGG-3′ (SEQ.ID.NO.:58);
  • and human thymus Marathon-Ready™ cDNA (Clontech) as a template. Advantage cDNA polymerase (Clontech, according to manufacturer's instructions) was used for the amplification in a 50 ul reaction by the following cycle: 94° C. for 30 sec; 94° C. for 5 sec; 66° C. for 40 sec; 72° C. for 2.5 sec and 72° C. for 7 min. [0078] Cycles 2 through 4 were repeated 30 times. A 1.3 Kb PCR fragment was isolated and cloned into the pCRII-TOPO™ vector (Invitrogen) and completely sequenced (see, SEQ.ID.NO.: 11) using the ABI Big Dye Terminator™ kit (P.E. Biosystem).
  • hRUP7 (Seq. Id. Nos. 13 & 14) [0079]
  • The full length RUP7 was cloned by RT-PCR using primers: [0080]
    5′-TGATGTGATGCCAGATACTAATAGCAC-3′ (SEQ.ID.NO.:59; sense) and
    5′-CCTGATTCATTTAGGTGAGATTGAGAC-3′ (SEQ.ID.NO.:60; antisense)
  • and human peripheral leukocyte cDNA (Clontech) as a template. Advantage™ cDNA polymerase (Clontech) was used for the amplification in a 50 ul reaction by the following cycle with [0081] step 2 to step 4 repeated 30 times: 94° C. for 2 minutes; 94° C. for 15 seconds; 60° C. for 20 seconds; 72° C. for 2 minutes; 72° C. for 10 minutes. A 1.25 Kb PCR fragment was isolated and cloned into the pCRII-TOPO™ vector (Invitrogen) and completely sequenced using the ABI Big Dye Terminator™ kit (P.E. Biosystem). See, SEQ.ID.NO.: 13.
  • g. hARE-S (Seq. Id. Nos. 5 & 6) [0082]
  • The full length hARE-5 was cloned by PCR using the hARE5 [0083] specific primers
    5′-CAGCGCAGGGTGAAGCCTGAGAGC-3′ SEQ.ID.NO.:69 (sense, 5′ of initiation codon ATG)
    and
    5′-GGCACCTGCTGTGACCTGTGCAGG-3′ SEQ.ID.NO.:70 (antisense, 3′ of stop codon TGA)
  • and human genomic DNA as template. TaqPlus Precision™ DNA polymerase (Stratagene) was used for the amplification by the following cycle with [0084] step 2 to step 4 repeated 35 times: 96° C., 2 minutes; 96° C., 20 seconds; 58° C., 30 seconds; 72° C., 2 minutes; and 72° C., 10 minutes
  • A 1.1 Kb PCR fragment of predicated size was isolated and cloned into the pCRII-TOPO™ vector (Invitrogen) and completely sequenced (SEQ.ID.NO.:5) using the T7 DNA Sequenase™ kit (Amsham). [0085]
  • h. hARE4 (Seq. Id. Nos.: 3 & 4) [0086]
  • The full length hARE4 was cloned by PCR using the hARE4 [0087] specific primers 5′-CTGGTGTGCTCCATGGCATCCC-3′SEQ.ID.NO.:67 (sense, 5′ of initiation codon ATG) and 5′GTAAGCCTCCCAGAACGAGAGG-3′ SEQ.ID.NO.:68 (antisense, 3′ of stop codon TGA) and
  • human genomic DNA as template. Taq DNA polymerase (Stratagene) and 5% DMSO was used for the amplification by the following cycle with [0088] step 2 to step 3 repeated 35 times: 94° C., 3 minutes; 94° C., 30 seconds; 59° C., 2 minutes; 72° C., 10 minutes
  • A 1.12 Kb PCR fragment of predicated size was isolated and cloned into the pCRII-TOPO™ vector (Invitrogen) and completely sequenced (SEQ.ID.NO.:3) using the T7 DNA Sequenase™ kit (Amsham). [0089]
  • i. hARE-3 (Seq.Id.Nos.: 1 & 2) [0090]
  • The full length hARE-3 was cloned by PCR using the hARE-3 [0091] specific primers 5′-gatcaagcttCCATCCTACTGAAACCATGGTC-3′ SEQ.ID.NO.:65 (sense, lower case nucleotides represent Hind III overhang, ATG as initiation codon) and 5′-gatcagatctCAGTrCCAATATTCACACCACCGTC-3′ SEQ.ID.NO.:66 (antisense, lower case nucleotides represent Xba I overhang, TCA as stop codon) and human genomic DNA as template. TaqPlus Precision™ DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4 repeated 35 times: 94° C., 3 minutes; 94° C., 1 minute; 55° C., 1 minute; 72° C., 2 minutes; 72° C., 10 minutes.
  • A 1.3 Kb PCR fragment of predicated size was isolated and digested with Hind III and Xba I, cloned into the pRC/CMV2 vector (Invitrogen) at the Hind III and Xba I sites and completely sequenced (SEQ.ID.NO.: 1) using the T7 DNA Sequenase™ kit (Amsham). [0092]
  • j. hRUP3 (Seq. Id. Nos.:7 & 8) [0093]
  • The full length hRUP3 was cloned by PCR using the hRUP3 [0094] specific primers 5′GTCCTGCCACTTCGAGACATGG-3′ SEQ.ID.NO.:71 (sense, ATG as initiation condon) and 5′-GAAACTTCTCTGCCTTACCGTC-3′ SEQ.ID.NO.:72 (antisense, 3′ of stop condon TAA) and human genomic DNA as template. TaqPlus Precision™ DNA polymerase (Stratagene) was used for the amplification by the following cycle with step 2 to step 4 repeated 35 times: 94° C., 3 minutes; 94° C., 1 minute; 58° C., 1 minute; 72° C., 2 minutes; 72° C., 10 minutes
  • A 1.0 Kb PCR fragment of predicated size was isolated and cloned into the pCRII-TOPO™ vector (Invitrogen) and completely sequenced (SEQ.ID.NO.: 7)using the T7 DNA sequenase kit (Amsham). [0095]
    • Example 2 RECEPTOR EXPRESSION
  • Although a variety of cells are available to the art for the expression of proteins, it is most preferred that mammalian cells be utilized. The primary reason for this is predicated upon practicalities, i.e., utilization of, e.g., yeast cells for the expression of a GPCR, while possible, introduces into the protocol a non-mammalian cell which may not (indeed, in the case of yeast, does not) include the receptor-coupling, genetic-mechanism and secretary pathways that have evolved for mammalian systems—thus, results obtained in non-mammalian cells, while of potential use, are not as preferred as that obtained from mammalian cells. Of the mammalian cells, COS-7, 293 and 293T cells are particularly preferred, although the specific mammalian cell utilized can be predicated upon the particular needs of the artisan. The general procedure for expression of the disclosed GPCRs is as follows. [0096]
  • On day one, 1×10[0097] 7 293T cells per 150 mm plate were plated out. On day two, two reaction tubes will be prepared (the proportions to follow for each tube are per plate): tube A will be prepared by mixing 20 kg DNA (e.g., pCMV vector; pCMV vector with receptor cDNA, etc.) in 1.2 ml serum free DMEM (Irvine Scientific, Irvine, Calif.); tube B will be prepared by mixing 120 μl lipofectamine (Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B are admixed by inversions (several times), followed by incubation at room temperature for 30-45 min. The admixture can be referred to as the “transfection mixture”. Plated 293T cells are washed with 1XPBS, followed by addition of 10ml serum free DMEM. 2.4 ml of the transfection mixture will then be added to the cells, followed by incubation for 4hrs at 37° C./5% CO2. The transfection mixture was then be removed by aspiration, followed by the addition of 25 ml of DMEM/10% Fetal Bovine Serum. Cells will then be incubated at 37° C./5% CO2. After 72 hr incubation, cells can then be harvested and utilized for analysis.
    • Example 3 TISSUE DISTRIBUTION OF THE DISCLOSED HUMAN GPCRS
  • Several approaches can be used for determination of the tissue distribution of the GPCRs disclosed herein. [0098]
  • 1. Dot-Blot Analysis [0099]
  • Using a commercially available human-tissue dot-blot format, endogenous orphan GPCRs were probed for a determination of the areas where such receptors are localized. cDNA fragments from the GPCRs of Example 1 (radiolabelled) were (or can be) used as the probe: radiolabeled probe was (or can be) generated using the complete receptor cDNA (excised from the vector) using a Prime-It II™ Random Primer Labeling Kit (Stratagene, #300385), according to manufacturer's instructions. A human RNA Master Blot™ (Clontech, #7770-1) was hybridized with the endogenous human GPCR radiolabeled probe and washed under stringent conditions according manufacturer's instructions. The blot was exposed to Kodak BioMax™ Autoradiography film overnight at −80° C. Results are summarized for several receptors in Table B and C (see FIGS. 1A and 1B for a grid identifying the various tissues and their locations, respectively). Exemplary dot-blots are provided in FIG. 2A and 2B for results derived using hCHN3 and hCHN8, respectively. [0100]
    TABLE B
    Tissue Distribution
    ORPHAN GPCR (highest levels, relative to other tissues in the dot-blot)
    hGPCR27 Fetal brain, Putamen, Pituitary gland, Caudate nucleus
    hARE-1 Spleen, Peripheral leukocytes, Fetal spleen
    hPPR1 Pituitary gland, Heart, salivary gland, Small intestine,
    Testis
    hRUP3 Pancreas
    hCHN3 Fetal brain, Putamen, Occipital cortex
    hCHN9 Pancreas, Small intestine, Liver
    hCHN10 Kidney, Thryoid
  • [0101]
    TABLE C
    Tissue Distribution
    ORPHAN GPCR (highest levels, relative to other tissues in the dot-blot)
    hARE-3 Cerebellum left, Cerebellum right, Testis, Accumbens
    hGPCR3 Corpus collusum, Caudate nucleus, Liver, Heart, Inter-
    Ventricular Septum
    bARE-2 Cerebellum left, Cerebellum right, Substantia
    hCHN8 Cerebellum left, Cerebellum right, Kidney, Lung
  • 2. RT-PCR [0102]
  • a. hRUP3 [0103]
  • To ascertain the tissue distribution of hRUP3 mRNA, RT-PCR was performed using hRUP3-specific primers and human multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNA polymerase (Stratagene) was utilized for the PCR reaction, using the following reaction cycles in a 40ul reaction: 94° C. for 2 min; 94° C. for 15 sec; 55° C. for 30 sec; 72° C. for 1 min; 72° C., for 10 min. Primers were as follows: [0104]
    5′-GACAGGTACCTTGCCATCAAG-3′ (SEQ.ID.NO.:61; sense)
    5′-CTGCACAATGCCAGTGATAAGG-3′ (SEQ.ID.NQ.:62; antisense).
  • 20 ul of the reaction was loaded onto a 1% agarose gel; results are set forth in FIG. 3. [0105]
  • As is supported by the data of FIG. 3, of the 16 human tissues in the cDNA panel utilized (brain, colon, heart, kidney, lung, ovary, pancreas, placenta, prostate, skeleton, small intestine, spleen, testis, thymus leukocyte, and liver) a single hRUP3 band is evident only from the pancreas. Additional comparative analysis of the protein sequence of hRUP3 with other GPCRs suggest that hRUP3 is related to GPCRs having small molecule endogenous ligand such that it is predicted that the endogenous ligand for hRUP3 is a small molecule. [0106]
  • b. hRUP4 [0107]
  • RT-PCR was performed using hRUP4 oligo's 8 and 4 as primers and the human multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNA polymerase 10 (Stratagene) was used for the amplification in a 40 ul reaction by the following cycles: 94° C. for 30 seconds, 94° C. for 10 seconds, 55° C. for 30 seconds, 72° C. for 2 minutes, and 72° C. for 5 minutes with [0108] cycles 2 through 4 repeated 30 times.
  • 20 μl of the reaction were loaded on a 1% agarose gel to analyze the RT-PCR products, and hRUP4 mRNA was found expressed in many human tissues, with the strongest expression in heart and kidney. (see, FIG. 4). To confirm the authenticity of the PCR fragments, a 300 bp fragment derived from the 5′ end of hRUP4 was used as a probe for the Southern Blot analysis. The probe was labeled with [0109] 32P-dCTP using the Prime-It II™ Random Primer Labeling Kit (Stratagene) and purified using the ProbeQuant™ G-50 micro columns (Amersham). Hybridization was done overnight at 42° C. following a 12 hr pre-hybridization. The blot was finally washed at 65° C. with 0.1×SSC. The Southern blot did confirm the PCR fragments as hRUP4.
  • c. hRUP5 [0110]
  • RT-PCR was performed using the following hRUP5 specific primers: [0111]
    5′-CTGACTTCTTGTTCCTGGCAGCAGCGG-3′ (SEQ.ID.NO.:63; sense)
    5′-AGACCAGCCAGGGCACGCTGAAGAGTG-3′ (SEQ.ID.NO.:64; antisense)
  • and the human multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNA polymerase (Stratagene) was used for the amplification in a 40ul reaction by the following cycles: 94° C. for 30 sec, 94° C. for 10 sec, 62° C. for 1.5 min, 72° C. for 5 min, and with [0112] cycles 2 through 3 repeated 30 times. 20 μl of the reaction were loaded on a 1.5% agarose gel to analyze the RT-PCR products, and hRUP5 mRNA was found expressed only in the peripheral blood leukocytes (data not shown).
  • d. hRUP6 [0113]
  • RT-PCR was applied to confirm the expression and to determine the tissue distribution of hRUP6. Oligonucleotides used, based on an alignment of AC005871 and GPR66 segments, had the following sequences: [0114]
    5′-CCAACACCAGCATCCATGGCATCAAG-3′ (SEQ.ID.NO.:73; sense),
    5′-GGAGAGTCAGCTCTGAAAGAATTCAGG-3′ (SEQ.ID.NO.:74; antisense)
  • and the human multiple tissue cDNA panels (MTC, Clontech) were used as templates. PCR was performed using TaqPlus Precision™ polymerase (Stratagene; manufacturing instructions will be followed) in a 40 ul reaction by the following cycles: 94° C. for 30 sec; 94° C. 5 sec; 66° C. for 40 sec, 72° C. for 2.5 min, and 72° C. for 7 min. [0115] Cycles 2 through 4 were repeated 30 times.
  • ul of the reaction were loaded on a 1.2% agarose gel to analyze the RT-PCR products, and a specific 760 bp DNA fragment representing hRUP6 was expressed predominantly in the thymus and with less expression in the heart, kidney, lung, prostate small intestine and testis. (see, FIG. 5). [0116]
  • It is intended that each of the patents, applications, and printed publications mentioned in this patent document be hereby incorporated by reference in their entirety. [0117]
  • As those skilled in the art will appreciate, numerous changes and modifications may be made to the preferred embodiments of the invention without departing from the spirit of the invention. It is intended that all such variations fall within the scope of the invention and the claims that follow. [0118]
  • Although a variety of Vectors are available to those in the art, for purposes of utilization for both endogenous and non-endogenous human GPCRs, it is most preferred that the Vector utilized be pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on Oct. 13, 1998 (10801 University Blvd., Manassas, Va. 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be. The ATCC has assigned the following deposit number to pCMV: ATCC #203351. [0119]
  • 1 74 1 1260 DNA Homo sapiens 1 atggtcttct cggcagtgtt gactgcgttc cataccggga catccaacac aacatttgtc 60 gtgtatgaaa acacctacat gaatattaca ctccctccac cattccagca tcctgacctc 120 agtccattgc ttagatatag ttttgaaacc atggctccca ctggtttgag ttccttgacc 180 gtgaatagta cagctgtgcc cacaacacca gcagcattta agagcctaaa cttgcctctt 240 cagatcaccc tttctgctat aatgatattc attctgtttg tgtcttttct tgggaacttg 300 gttgtttgcc tcatggttta ccaaaaagct gccatgaggt ctgcaattaa catcctcctt 360 gccagcctag cttttgcaga catgttgctt gcagtgctga acatgccctt tgccctggta 420 actattctta ctacccgatg gatttttggg aaattcttct gtagggtatc tgctatgttt 480 ttctggttat ttgtgataga aggagtagcc atcctgctca tcattagcat agataggttc 540 cttattatag tccagaggca ggataagcta aacccatata gagctaaggt tctgattgca 600 gtttcttggg caacttcctt ttgtgtagct tttcctttag ccgtaggaaa ccccgacctg 660 cagatacctt cccgagctcc ccagtgtgtg tttgggtaca caaccaatcc aggctaccag 720 gcttatgtga ttttgatttc tctcatttct ttcttcatac ccttcctggt aatactgtac 780 tcatttatgg gcatactcaa cacccttcgg cacaatgcct tgaggatcca tagctaccct 840 gaaggtatat gcctcagcca ggccagcaaa ctgggtctca tgagtctgca gagacctttc 900 cagatgagca ttgacatggg ctttaaaaca cgtgccttca ccactatttt gattctcttt 960 gctgtcttca ttgtctgctg ggccccattc accacttaca gccttgtggc aacattcagt 1020 aagcactttt actatcagca caactttttt gagattagca cctggctact gtggctctgc 1080 tacctcaagt ctgcattgaa tccgctgatc tactactgga ggattaagaa attccatgat 1140 gcttgcctgg acatgatgcc taagtccttc aagtttttgc cgcagctccc tggtcacaca 1200 aagcgacgga tacgtcctag tgctgtctat gtgtgtgggg aacatcggac ggtggtgtga 1260 2 419 PRT Homo sapiens 2 Met Val Phe Ser Ala Val Leu Thr Ala Phe His Thr Gly Thr Ser Asn 1 5 10 15 Thr Thr Phe Val Val Tyr Glu Asn Thr Tyr Met Asn Ile Thr Leu Pro 20 25 30 Pro Pro Phe Gln His Pro Asp Leu Ser Pro Leu Leu Arg Tyr Ser Phe 35 40 45 Glu Thr Met Ala Pro Thr Gly Leu Ser Ser Leu Thr Val Asn Ser Thr 50 55 60 Ala Val Pro Thr Thr Pro Ala Ala Phe Lys Ser Leu Asn Leu Pro Leu 65 70 75 80 Gln Ile Thr Leu Ser Ala Ile Met Ile Phe Ile Leu Phe Val Ser Phe 85 90 95 Leu Gly Asn Leu Val Val Cys Leu Met Val Tyr Gln Lys Ala Ala Met 100 105 110 Arg Ser Ala Ile Asn Ile Leu Leu Ala Ser Leu Ala Phe Ala Asp Met 115 120 125 Leu Leu Ala Val Leu Asn Met Pro Phe Ala Leu Val Thr Ile Leu Thr 130 135 140 Thr Arg Trp Ile Phe Gly Lys Phe Phe Cys Arg Val Ser Ala Met Phe 145 150 155 160 Phe Trp Leu Phe Val Ile Glu Gly Val Ala Ile Leu Leu Ile Ile Ser 165 170 175 Ile Asp Arg Phe Leu Ile Ile Val Gln Arg Gln Asp Lys Leu Asn Pro 180 185 190 Tyr Arg Ala Lys Val Leu Ile Ala Val Ser Trp Ala Thr Ser Phe Cys 195 200 205 Val Ala Phe Pro Leu Ala Val Gly Asn Pro Asp Leu Gln Ile Pro Ser 210 215 220 Arg Ala Pro Gln Cys Val Phe Gly Tyr Thr Thr Asn Pro Gly Tyr Gln 225 230 235 240 Ala Tyr Val Ile Leu Ile Ser Leu Ile Ser Phe Phe Ile Pro Phe Leu 245 250 255 Val Ile Leu Tyr Ser Phe Met Gly Ile Leu Asn Thr Leu Arg His Asn 260 265 270 Ala Leu Arg Ile His Ser Tyr Pro Glu Gly Ile Cys Leu Ser Gln Ala 275 280 285 Ser Lys Leu Gly Leu Met Ser Leu Gln Arg Pro Phe Gln Met Ser Ile 290 295 300 Asp Met Gly Phe Lys Thr Arg Ala Phe Thr Thr Ile Leu Ile Leu Phe 305 310 315 320 Ala Val Phe Ile Val Cys Trp Ala Pro Phe Thr Thr Tyr Ser Leu Val 325 330 335 Ala Thr Phe Ser Lys His Phe Tyr Tyr Gln His Asn Phe Phe Glu Ile 340 345 350 Ser Thr Trp Leu Leu Trp Leu Cys Tyr Leu Lys Ser Ala Leu Asn Pro 355 360 365 Leu Ile Tyr Tyr Trp Arg Ile Lys Lys Phe His Asp Ala Cys Leu Asp 370 375 380 Met Met Pro Lys Ser Phe Lys Phe Leu Pro Gln Leu Pro Gly His Thr 385 390 395 400 Lys Arg Arg Ile Arg Pro Ser Ala Val Tyr Val Cys Gly Glu His Arg 405 410 415 Thr Val Val 3 1119 DNA Homo sapiens 3 atgttagcca acagctcctc aaccaacagt tctgttctcc cgtgtcctga ctaccgacct 60 acccaccgcc tgcacttggt ggtctacagc ttggtgctgg ctgccgggct ccccctcaac 120 gcgctagccc tctgggtctt cctgcgcgcg ctgcgcgtgc actcggtggt gagcgtgtac 180 atgtgtaacc tggcggccag cgacctgctc ttcaccctct cgctgcccgt tcgtctctcc 240 tactacgcac tgcaccactg gcccttcccc gacctcctgt gccagacgac gggcgccatc 300 ttccagatga acatgtacgg cagctgcatc ttcctgatgc tcatcaacgt ggaccgctac 360 gccgccatcg tgcacccgct gcgactgcgc cacctgcggc ggccccgcgt ggcgcggctg 420 ctctgcctgg gcgtgtgggc gctcatcctg gtgtttgccg tgcccgccgc ccgcgtgcac 480 aggccctcgc gttgccgcta ccgggacctc gaggtgcgcc tatgcttcga gagcttcagc 540 gacgagctgt ggaaaggcag gctgctgccc ctcgtgctgc tggccgaggc gctgggcttc 600 ctgctgcccc tggcggcggt ggtctactcg tcgggccgag tcttctggac gctggcgcgc 660 cccgacgcca cgcagagcca gcggcggcgg aagaccgtgc gcctcctgct ggctaacctc 720 gtcatcttcc tgctgtgctt cgtgccctac aacagcacgc tggcggtcta cgggctgctg 780 cggagcaagc tggtggcggc cagcgtgcct gcccgcgatc gcgtgcgcgg ggtgctgatg 840 gtgatggtgc tgctggccgg cgccaactgc gtgctggacc cgctggtgta ctactttagc 900 gccgagggct tccgcaacac cctgcgcggc ctgggcactc cgcaccgggc caggacctcg 960 gccaccaacg ggacgcgggc ggcgctcgcg caatccgaaa ggtccgccgt caccaccgac 1020 gccaccaggc cggatgccgc cagtcagggg ctgctccgac cctccgactc ccactctctg 1080 tcttccttca cacagtgtcc ccaggattcc gccctctga 1119 4 372 PRT Homo sapiens 4 Met Leu Ala Asn Ser Ser Ser Thr Asn Ser Ser Val Leu Pro Cys Pro 1 5 10 15 Asp Tyr Arg Pro Thr His Arg Leu His Leu Val Val Tyr Ser Leu Val 20 25 30 Leu Ala Ala Gly Leu Pro Leu Asn Ala Leu Ala Leu Trp Val Phe Leu 35 40 45 Arg Ala Leu Arg Val His Ser Val Val Ser Val Tyr Met Cys Asn Leu 50 55 60 Ala Ala Ser Asp Leu Leu Phe Thr Leu Ser Leu Pro Val Arg Leu Ser 65 70 75 80 Tyr Tyr Ala Leu His His Trp Pro Phe Pro Asp Leu Leu Cys Gln Thr 85 90 95 Thr Gly Ala Ile Phe Gln Met Asn Met Tyr Gly Ser Cys Ile Phe Leu 100 105 110 Met Leu Ile Asn Val Asp Arg Tyr Ala Ala Ile Val His Pro Leu Arg 115 120 125 Leu Arg His Leu Arg Arg Pro Arg Val Ala Arg Leu Leu Cys Leu Gly 130 135 140 Val Trp Ala Leu Ile Leu Val Phe Ala Val Pro Ala Ala Arg Val His 145 150 155 160 Arg Pro Ser Arg Cys Arg Tyr Arg Asp Leu Glu Val Arg Leu Cys Phe 165 170 175 Glu Ser Phe Ser Asp Glu Leu Trp Lys Gly Arg Leu Leu Pro Leu Val 180 185 190 Leu Leu Ala Glu Ala Leu Gly Phe Leu Leu Pro Leu Ala Ala Val Val 195 200 205 Tyr Ser Ser Gly Arg Val Phe Trp Thr Leu Ala Arg Pro Asp Ala Thr 210 215 220 Gln Ser Gln Arg Arg Arg Lys Thr Val Arg Leu Leu Leu Ala Asn Leu 225 230 235 240 Val Ile Phe Leu Leu Cys Phe Val Pro Tyr Asn Ser Thr Leu Ala Val 245 250 255 Tyr Gly Leu Leu Arg Ser Lys Leu Val Ala Ala Ser Val Pro Ala Arg 260 265 270 Asp Arg Val Arg Gly Val Leu Met Val Met Val Leu Leu Ala Gly Ala 275 280 285 Asn Cys Val Leu Asp Pro Leu Val Tyr Tyr Phe Ser Ala Glu Gly Phe 290 295 300 Arg Asn Thr Leu Arg Gly Leu Gly Thr Pro His Arg Ala Arg Thr Ser 305 310 315 320 Ala Thr Asn Gly Thr Arg Ala Ala Leu Ala Gln Ser Glu Arg Ser Ala 325 330 335 Val Thr Thr Asp Ala Thr Arg Pro Asp Ala Ala Ser Gln Gly Leu Leu 340 345 350 Arg Pro Ser Asp Ser His Ser Leu Ser Ser Phe Thr Gln Cys Pro Gln 355 360 365 Asp Ser Ala Leu 370 5 1107 DNA Homo sapiens 5 atggccaact ccacagggct gaacgcctca gaagtcgcag gctcgttggg gttgatcctg 60 gcagctgtcg tggaggtggg ggcactgctg ggcaacggcg cgctgctggt cgtggtgctg 120 cgcacgccgg gactgcgcga cgcgctctac ctggcgcacc tgtgcgtcgt ggacctgctg 180 gcggccgcct ccatcatgcc gctgggcctg ctggccgcac cgccgcccgg gctgggccgc 240 gtgcgcctgg gccccgcgcc atgccgcgcc gctcgcttcc tctccgccgc tctgctgccg 300 gcctgcacgc tcggggtggc cgcacttggc ctggcacgct accgcctcat cgtgcacccg 360 ctgcggccag gctcgcggcc gccgcctgtg ctcgtgctca ccgccgtgtg ggccgcggcg 420 ggactgctgg gcgcgctctc cctgctcggc ccgccgcccg caccgccccc tgctcctgct 480 cgctgctcgg tcctggctgg gggcctcggg cccttccggc cgctctgggc cctgctggcc 540 ttcgcgctgc ccgccctcct gctgctcggc gcctacggcg gcatcttcgt ggtggcgcgt 600 cgcgctgccc tgaggccccc acggccggcg cgcgggtccc gactccgctc ggactctctg 660 gatagccgcc tttccatctt gccgccgctc cggcctcgcc tgcccggggg caaggcggcc 720 ctggccccag cgctggccgt gggccaattt gcagcctgct ggctgcctta tggctgcgcg 780 tgcctggcgc ccgcagcgcg ggccgcggaa gccgaagcgg ctgtcacctg ggtcgcctac 840 tcggccttcg cggctcaccc cttcctgtac gggctgctgc agcgccccgt gcgcttggca 900 ctgggccgcc tctctcgccg tgcactgcct ggacctgtgc gggcctgcac tccgcaagcc 960 tggcacccgc gggcactctt gcaatgcctc cagagacccc cagagggccc tgccgtaggc 1020 ccttctgagg ctccagaaca gacccccgag ttggcaggag ggcggagccc cgcataccag 1080 gggccacctg agagttctct ctcctga 1107 6 368 PRT Homo sapiens 6 Met Ala Asn Ser Thr Gly Leu Asn Ala Ser Glu Val Ala Gly Ser Leu 1 5 10 15 Gly Leu Ile Leu Ala Ala Val Val Glu Val Gly Ala Leu Leu Gly Asn 20 25 30 Gly Ala Leu Leu Val Val Val Leu Arg Thr Pro Gly Leu Arg Asp Ala 35 40 45 Leu Tyr Leu Ala His Leu Cys Val Val Asp Leu Leu Ala Ala Ala Ser 50 55 60 Ile Met Pro Leu Gly Leu Leu Ala Ala Pro Pro Pro Gly Leu Gly Arg 65 70 75 80 Val Arg Leu Gly Pro Ala Pro Cys Arg Ala Ala Arg Phe Leu Ser Ala 85 90 95 Ala Leu Leu Pro Ala Cys Thr Leu Gly Val Ala Ala Leu Gly Leu Ala 100 105 110 Arg Tyr Arg Leu Ile Val His Pro Leu Arg Pro Gly Ser Arg Pro Pro 115 120 125 Pro Val Leu Val Leu Thr Ala Val Trp Ala Ala Ala Gly Leu Leu Gly 130 135 140 Ala Leu Ser Leu Leu Gly Pro Pro Pro Ala Pro Pro Pro Ala Pro Ala 145 150 155 160 Arg Cys Ser Val Leu Ala Gly Gly Leu Gly Pro Phe Arg Pro Leu Trp 165 170 175 Ala Leu Leu Ala Phe Ala Leu Pro Ala Leu Leu Leu Leu Gly Ala Tyr 180 185 190 Gly Gly Ile Phe Val Val Ala Arg Arg Ala Ala Leu Arg Pro Pro Arg 195 200 205 Pro Ala Arg Gly Ser Arg Leu Arg Ser Asp Ser Leu Asp Ser Arg Leu 210 215 220 Ser Ile Leu Pro Pro Leu Arg Pro Arg Leu Pro Gly Gly Lys Ala Ala 225 230 235 240 Leu Ala Pro Ala Leu Ala Val Gly Gln Phe Ala Ala Cys Trp Leu Pro 245 250 255 Tyr Gly Cys Ala Cys Leu Ala Pro Ala Ala Arg Ala Ala Glu Ala Glu 260 265 270 Ala Ala Val Thr Trp Val Ala Tyr Ser Ala Phe Ala Ala His Pro Phe 275 280 285 Leu Tyr Gly Leu Leu Gln Arg Pro Val Arg Leu Ala Leu Gly Arg Leu 290 295 300 Ser Arg Arg Ala Leu Pro Gly Pro Val Arg Ala Cys Thr Pro Gln Ala 305 310 315 320 Trp His Pro Arg Ala Leu Leu Gln Cys Leu Gln Arg Pro Pro Glu Gly 325 330 335 Pro Ala Val Gly Pro Ser Glu Ala Pro Glu Gln Thr Pro Glu Leu Ala 340 345 350 Gly Gly Arg Ser Pro Ala Tyr Gln Gly Pro Pro Glu Ser Ser Leu Ser 355 360 365 7 1008 DNA Homo sapiens 7 atggaatcat ctttctcatt tggagtgatc cttgctgtcc tggcctccct catcattgct 60 actaacacac tagtggctgt ggctgtgctg ctgttgatcc acaagaatga tggtgtcagt 120 ctctgcttca ccttgaatct ggctgtggct gacaccttga ttggtgtggc catctctggc 180 ctactcacag accagctctc cagcccttct cggcccacac agaagaccct gtgcagcctg 240 cggatggcat ttgtcacttc ctccgcagct gcctctgtcc tcacggtcat gctgatcacc 300 tttgacaggt accttgccat caagcagccc ttccgctact tgaagatcat gagtgggttc 360 gtggccgggg cctgcattgc cgggctgtgg ttagtgtctt acctcattgg cttcctccca 420 ctcggaatcc ccatgttcca gcagactgcc tacaaagggc agtgcagctt ctttgctgta 480 tttcaccctc acttcgtgct gaccctctcc tgcgttggct tcttcccagc catgctcctc 540 tttgtcttct tctactgcga catgctcaag attgcctcca tgcacagcca gcagattcga 600 aagatggaac atgcaggagc catggctgga ggttatcgat ccccacggac tcccagcgac 660 ttcaaagctc tccgtactgt gtctgttctc attgggagct ttgctctatc ctggaccccc 720 ttccttatca ctggcattgt gcaggtggcc tgccaggagt gtcacctcta cctagtgctg 780 gaacggtacc tgtggctgct cggcgtgggc aactccctgc tcaacccact catctatgcc 840 tattggcaga aggaggtgcg actgcagctc taccacatgg ccctaggagt gaagaaggtg 900 ctcacctcat tcctcctctt tctctcggcc aggaattgtg gcccagagag gcccagggaa 960 agttcctgtc acatcgtcac tatctccagc tcagagtttg atggctaa 1008 8 335 PRT Homo sapiens 8 Met Glu Ser Ser Phe Ser Phe Gly Val Ile Leu Ala Val Leu Ala Ser 1 5 10 15 Leu Ile Ile Ala Thr Asn Thr Leu Val Ala Val Ala Val Leu Leu Leu 20 25 30 Ile His Lys Asn Asp Gly Val Ser Leu Cys Phe Thr Leu Asn Leu Ala 35 40 45 Val Ala Asp Thr Leu Ile Gly Val Ala Ile Ser Gly Leu Leu Thr Asp 50 55 60 Gln Leu Ser Ser Pro Ser Arg Pro Thr Gln Lys Thr Leu Cys Ser Leu 65 70 75 80 Arg Met Ala Phe Val Thr Ser Ser Ala Ala Ala Ser Val Leu Thr Val 85 90 95 Met Leu Ile Thr Phe Asp Arg Tyr Leu Ala Ile Lys Gln Pro Phe Arg 100 105 110 Tyr Leu Lys Ile Met Ser Gly Phe Val Ala Gly Ala Cys Ile Ala Gly 115 120 125 Leu Trp Leu Val Ser Tyr Leu Ile Gly Phe Leu Pro Leu Gly Ile Pro 130 135 140 Met Phe Gln Gln Thr Ala Tyr Lys Gly Gln Cys Ser Phe Phe Ala Val 145 150 155 160 Phe His Pro His Phe Val Leu Thr Leu Ser Cys Val Gly Phe Phe Pro 165 170 175 Ala Met Leu Leu Phe Val Phe Phe Tyr Cys Asp Met Leu Lys Ile Ala 180 185 190 Ser Met His Ser Gln Gln Ile Arg Lys Met Glu His Ala Gly Ala Met 195 200 205 Ala Gly Gly Tyr Arg Ser Pro Arg Thr Pro Ser Asp Phe Lys Ala Leu 210 215 220 Arg Thr Val Ser Val Leu Ile Gly Ser Phe Ala Leu Ser Trp Thr Pro 225 230 235 240 Phe Leu Ile Thr Gly Ile Val Gln Val Ala Cys Gln Glu Cys His Leu 245 250 255 Tyr Leu Val Leu Glu Arg Tyr Leu Trp Leu Leu Gly Val Gly Asn Ser 260 265 270 Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp Gln Lys Glu Val Arg Leu 275 280 285 Gln Leu Tyr His Met Ala Leu Gly Val Lys Lys Val Leu Thr Ser Phe 290 295 300 Leu Leu Phe Leu Ser Ala Arg Asn Cys Gly Pro Glu Arg Pro Arg Glu 305 310 315 320 Ser Ser Cys His Ile Val Thr Ile Ser Ser Ser Glu Phe Asp Gly 325 330 335 9 1413 DNA Homo sapiens 9 atggacacta ccatggaagc tgacctgggt gccactggcc acaggccccg cacagagctt 60 gatgatgagg actcctaccc ccaaggtggc tgggacacgg tcttcctggt ggccctgctg 120 ctccttgggc tgccagccaa tgggttgatg gcgtggctgg ccggctccca ggcccggcat 180 ggagctggca cgcgtctggc gctgctcctg ctcagcctgg ccctctctga cttcttgttc 240 ctggcagcag cggccttcca gatcctagag atccggcatg ggggacactg gccgctgggg 300 acagctgcct gccgcttcta ctacttccta tggggcgtgt cctactcctc cggcctcttc 360 ctgctggccg ccctcagcct cgaccgctgc ctgctggcgc tgtgcccaca ctggtaccct 420 gggcaccgcc cagtccgcct gcccctctgg gtctgcgccg gtgtctgggt gctggccaca 480 ctcttcagcg tgccctggct ggtcttcccc gaggctgccg tctggtggta cgacctggtc 540 atctgcctgg acttctggga cagcgaggag ctgtcgctga ggatgctgga ggtcctgggg 600 ggcttcctgc ctttcctcct gctgctcgtc tgccacgtgc tcacccaggc cacagcctgt 660 cgcacctgcc accgccaaca gcagcccgca gcctgccggg gcttcgcccg tgtggccagg 720 accattctgt cagcctatgt ggtcctgagg ctgccctacc agctggccca gctgctctac 780 ctggccttcc tgtgggacgt ctactctggc tacctgctct gggaggccct ggtctactcc 840 gactacctga tcctactcaa cagctgcctc agccccttcc tctgcctcat ggccagtgcc 900 gacctccgga ccctgctgcg ctccgtgctc tcgtccttcg cggcagctct ctgcgaggag 960 cggccgggca gcttcacgcc cactgagcca cagacccagc tagattctga gggtccaact 1020 ctgccagagc cgatggcaga ggcccagtca cagatggatc ctgtggccca gcctcaggtg 1080 aaccccacac tccagccacg atcggatccc acagctcagc cacagctgaa ccctacggcc 1140 cagccacagt cggatcccac agcccagcca cagctgaacc tcatggccca gccacagtca 1200 gattctgtgg cccagccaca ggcagacact aacgtccaga cccctgcacc tgctgccagt 1260 tctgtgccca gtccctgtga tgaagcttcc ccaaccccat cctcgcatcc taccccaggg 1320 gcccttgagg acccagccac acctcctgcc tctgaaggag aaagccccag cagcaccccg 1380 ccagaggcgg ccccgggcgc aggccccacg tga 1413 10 468 PRT Homo sapiens 10 Met Asp Thr Thr Met Glu Ala Asp Leu Gly Ala Thr Gly His Arg Pro 1 5 10 15 Arg Thr Glu Leu Asp Asp Glu Asp Ser Tyr Pro Gln Gly Gly Trp Asp 20 25 30 Thr Val Phe Leu Val Ala Leu Leu Leu Leu Gly Leu Pro Ala Asn Gly 35 40 45 Leu Met Ala Trp Leu Ala Gly Ser Gln Ala Arg His Gly Ala Gly Thr 50 55 60 Arg Leu Ala Leu Leu Leu Leu Ser Leu Ala Leu Ser Asp Phe Leu Phe 65 70 75 80 Leu Ala Ala Ala Ala Phe Gln Ile Leu Glu Ile Arg His Gly Gly His 85 90 95 Trp Pro Leu Gly Thr Ala Ala Cys Arg Phe Tyr Tyr Phe Leu Trp Gly 100 105 110 Val Ser Tyr Ser Ser Gly Leu Phe Leu Leu Ala Ala Leu Ser Leu Asp 115 120 125 Arg Cys Leu Leu Ala Leu Cys Pro His Trp Tyr Pro Gly His Arg Pro 130 135 140 Val Arg Leu Pro Leu Trp Val Cys Ala Gly Val Trp Val Leu Ala Thr 145 150 155 160 Leu Phe Ser Val Pro Trp Leu Val Phe Pro Glu Ala Ala Val Trp Trp 165 170 175 Tyr Asp Leu Val Ile Cys Leu Asp Phe Trp Asp Ser Glu Glu Leu Ser 180 185 190 Leu Arg Met Leu Glu Val Leu Gly Gly Phe Leu Pro Phe Leu Leu Leu 195 200 205 Leu Val Cys His Val Leu Thr Gln Ala Thr Arg Thr Cys His Arg Gln 210 215 220 Gln Gln Pro Ala Ala Cys Arg Gly Phe Ala Arg Val Ala Arg Thr Ile 225 230 235 240 Leu Ser Ala Tyr Val Val Leu Arg Leu Pro Tyr Gln Leu Ala Gln Leu 245 250 255 Leu Tyr Leu Ala Phe Leu Trp Asp Val Tyr Ser Gly Tyr Leu Leu Trp 260 265 270 Glu Ala Leu Val Tyr Ser Asp Tyr Leu Ile Leu Leu Asn Ser Cys Leu 275 280 285 Ser Pro Phe Leu Cys Leu Met Ala Ser Ala Asp Leu Arg Thr Leu Leu 290 295 300 Arg Ser Val Leu Ser Ser Phe Ala Ala Ala Leu Cys Glu Glu Arg Pro 305 310 315 320 Gly Ser Phe Thr Pro Thr Glu Pro Gln Thr Gln Leu Asp Ser Glu Gly 325 330 335 Pro Thr Leu Pro Glu Pro Met Ala Glu Ala Gln Ser Gln Met Asp Pro 340 345 350 Val Ala Gln Pro Gln Val Asn Pro Thr Leu Gln Pro Arg Ser Asp Pro 355 360 365 Thr Ala Gln Pro Gln Leu Asn Pro Thr Ala Gln Pro Gln Ser Asp Pro 370 375 380 Thr Ala Gln Pro Gln Leu Asn Leu Met Ala Gln Pro Gln Ser Asp Ser 385 390 395 400 Val Ala Gln Pro Gln Ala Asp Thr Asn Val Gln Thr Pro Ala Pro Ala 405 410 415 Ala Ser Ser Val Pro Ser Pro Cys Asp Glu Ala Ser Pro Thr Pro Ser 420 425 430 Ser His Pro Thr Pro Gly Ala Leu Glu Asp Pro Ala Thr Pro Pro Ala 435 440 445 Ser Glu Gly Glu Ser Pro Ser Ser Thr Pro Pro Glu Ala Ala Pro Gly 450 455 460 Ala Gly Pro Thr 465 11 1248 DNA Homo sapiens 11 atgtcaggga tggaaaaact tcagaatgct tcctggatct accagcagaa actagaagat 60 ccattccaga aacacctgaa cagcaccgag gagtatctgg ccttcctctg cggacctcgg 120 cgcagccact tcttcctccc cgtgtctgtg gtgtatgtgc caatttttgt ggtgggggtc 180 attggcaatg tcctggtgtg cctggtgatt ctgcagcacc aggctatgaa gacgcccacc 240 aactactacc tcttcagcct ggcggtctct gacctcctgg tcctgctcct tggaatgccc 300 ctggaggtct atgagatgtg gcgcaactac cctttcttgt tcgggcccgt gggctgctac 360 ttcaagacgg ccctctttga gaccgtgtgc ttcgcctcca tcctcagcat caccaccgtc 420 agcgtggagc gctacgtggc catcctacac ccgttccgcg ccaaactgca gagcacccgg 480 cgccgggccc tcaggatcct cggcatcgtc tggggcttct ccgtgctctt ctccctgccc 540 aacaccagca tccatggcat caagttccac tacttcccca atgggtccct ggtcccaggt 600 tcggccacct gtacggtcat caagcccatg tggatctaca atttcatcat ccaggtcacc 660 tccttcctat tctacctcct ccccatgact gtcatcagtg tcctctacta cctcatggca 720 ctcagactaa agaaagacaa atctcttgag gcagatgaag ggaatgcaaa tattcaaaga 780 ccctgcagaa aatcagtcaa caagatgctg tttgtcttgg tcttagtgtt tgctatctgt 840 tgggccccgt tccacattga ccgactcttc ttcagctttg tggaggagtg gagtgaatcc 900 ctggctgctg tgttcaacct cgtccatgtg gtgtcaggtg tcttcttcta cctgagctca 960 gctgtcaacc ccattatcta taacctactg tctcgccgct tccaggcagc attccagaat 1020 gtgatctctt ctttccacaa acagtggcac tcccagcatg acccacagtt gccacctgcc 1080 cagcggaaca tcttcctgac agaatgccac tttgtggagc tgaccgaaga tataggtccc 1140 caattcccat gtcagtcatc catgcacaac tctcacctcc caacagccct ctctagtgaa 1200 cagatgtcaa gaacaaacta tcaaagcttc cactttaaca aaacctga 1248 12 415 PRT Homo sapiens 12 Met Ser Gly Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln 1 5 10 15 Lys Leu Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr 20 25 30 Leu Ala Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val 35 40 45 Ser Val Val Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val 50 55 60 Leu Val Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr 65 70 75 80 Asn Tyr Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu 85 90 95 Leu Gly Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe 100 105 110 Leu Phe Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr 115 120 125 Val Cys Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg 130 135 140 Tyr Val Ala Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg 145 150 155 160 Arg Arg Ala Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu 165 170 175 Phe Ser Leu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe 180 185 190 Pro Asn Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys 195 200 205 Pro Met Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe 210 215 220 Tyr Leu Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala 225 230 235 240 Leu Arg Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala 245 250 255 Asn Ile Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Phe Val 260 265 270 Leu Val Leu Val Phe Ala Ile Cys Trp Ala Pro Phe His Ile Asp Arg 275 280 285 Leu Phe Phe Ser Phe Val Glu Glu Trp Ser Glu Ser Leu Ala Ala Val 290 295 300 Phe Asn Leu Val His Val Val Ser Gly Val Phe Phe Tyr Leu Ser Ser 305 310 315 320 Ala Val Asn Pro Ile Ile Tyr Asn Leu Leu Ser Arg Arg Phe Gln Ala 325 330 335 Ala Phe Gln Asn Val Ile Ser Ser Phe His Lys Gln Trp His Ser Gln 340 345 350 His Asp Pro Gln Leu Pro Pro Ala Gln Arg Asn Ile Phe Leu Thr Glu 355 360 365 Cys His Phe Val Glu Leu Thr Glu Asp Ile Gly Pro Gln Phe Pro Cys 370 375 380 Gln Ser Ser Met His Asn Ser His Leu Pro Thr Ala Leu Ser Ser Glu 385 390 395 400 Gln Met Ser Arg Thr Asn Tyr Gln Ser Phe His Phe Asn Lys Thr 405 410 415 13 1173 DNA Homo sapiens 13 atgccagata ctaatagcac aatcaattta tcactaagca ctcgtgttac tttagcattt 60 tttatgtcct tagtagcttt tgctataatg ctaggaaatg ctttggtcat tttagctttt 120 gtggtggaca aaaaccttag acatcgaagt agttattttt ttcttaactt ggccatctct 180 gacttctttg tgggtgtgat ctccattcct ttgtacatcc ctcacacgct gttcgaatgg 240 gattttggaa aggaaatctg tgtattttgg ctcactactg actatctgtt atgtacagca 300 tctgtatata acattgtcct catcagctat gatcgatacc tgtcagtctc aaatgctgtg 360 tcttatagaa ctcaacatac tggggtcttg aagattgtta ctctgatggt ggccgtttgg 420 gtgctggcct tcttagtgaa tgggccaatg attctagttt cagagtcttg gaaggatgaa 480 ggtagtgaat gtgaacctgg atttttttcg gaatggtaca tccttgccat cacatcattc 540 ttggaattcg tgatcccagt catcttagtc gcttatttca acatgaatat ttattggagc 600 ctgtggaagc gtgatcatct cagtaggtgc caaagccatc ctggactgac tgctgtctct 660 tccaacatct gtggacactc attcagaggt agactatctt caaggagatc tctttctgca 720 tcgacagaag ttcctgcatc ctttcattca gagagacaga ggagaaagag tagtctcatg 780 ttttcctcaa gaaccaagat gaatagcaat acaattgctt ccaaaatggg ttccttctcc 840 caatcagatt ctgtagctct tcaccaaagg gaacatgttg aactgcttag agccaggaga 900 ttagccaagt cactggccat tctcttaggg gtttttgctg tttgctgggc tccatattct 960 ctgttcacaa ttgtcctttc attttattcc tcagcaacag gtcctaaatc agtttggtat 1020 agaattgcat tttggcttca gtggttcaat tcctttgtca atcctctttt gtatccattg 1080 tgtcacaagc gctttcaaaa ggctttcttg aaaatatttt gtataaaaaa gcaacctcta 1140 ccatcacaac acagtcggtc agtatcttct taa 1173 14 390 PRT Homo sapiens 14 Met Pro Asp Thr Asn Ser Thr Ile Asn Leu Ser Leu Ser Thr Arg Val 1 5 10 15 Thr Leu Ala Phe Phe Met Ser Leu Val Ala Phe Ala Ile Met Leu Gly 20 25 30 Asn Ala Leu Val Ile Leu Ala Phe Val Val Asp Lys Asn Leu Arg His 35 40 45 Arg Ser Ser Tyr Phe Phe Leu Asn Leu Ala Ile Ser Asp Phe Phe Val 50 55 60 Gly Val Ile Ser Ile Pro Leu Tyr Ile Pro His Thr Leu Phe Glu Trp 65 70 75 80 Asp Phe Gly Lys Glu Ile Cys Val Phe Trp Leu Thr Thr Asp Tyr Leu 85 90 95 Leu Cys Thr Ala Ser Val Tyr Asn Ile Val Leu Ile Ser Tyr Asp Arg 100 105 110 Tyr Leu Ser Val Ser Asn Ala Val Ser Tyr Arg Thr Gln His Thr Gly 115 120 125 Val Leu Lys Ile Val Thr Leu Met Val Ala Val Trp Val Leu Ala Phe 130 135 140 Leu Val Asn Gly Pro Met Ile Leu Val Ser Glu Ser Trp Lys Asp Glu 145 150 155 160 Gly Ser Glu Cys Glu Pro Gly Phe Phe Ser Glu Trp Tyr Ile Leu Ala 165 170 175 Ile Thr Ser Phe Leu Glu Phe Val Ile Pro Val Ile Leu Val Ala Tyr 180 185 190 Phe Asn Met Asn Ile Tyr Trp Ser Leu Trp Lys Arg Asp His Leu Ser 195 200 205 Arg Cys Gln Ser His Pro Gly Leu Thr Ala Val Ser Ser Asn Ile Cys 210 215 220 Gly His Ser Phe Arg Gly Arg Leu Ser Ser Arg Arg Ser Leu Ser Ala 225 230 235 240 Ser Thr Glu Val Pro Ala Ser Phe His Ser Glu Arg Gln Arg Arg Lys 245 250 255 Ser Ser Leu Met Phe Ser Ser Arg Thr Lys Met Asn Ser Asn Thr Ile 260 265 270 Ala Ser Lys Met Gly Ser Phe Ser Gln Ser Asp Ser Val Ala Leu His 275 280 285 Gln Arg Glu His Val Glu Leu Leu Arg Ala Arg Arg Leu Ala Lys Ser 290 295 300 Leu Ala Ile Leu Leu Gly Val Phe Ala Val Cys Trp Ala Pro Tyr Ser 305 310 315 320 Leu Phe Thr Ile Val Leu Ser Phe Tyr Ser Ser Ala Thr Gly Pro Lys 325 330 335 Ser Val Trp Tyr Arg Ile Ala Phe Trp Leu Gln Trp Phe Asn Ser Phe 340 345 350 Val Asn Pro Leu Leu Tyr Pro Leu Cys His Lys Arg Phe Gln Lys Ala 355 360 365 Phe Leu Lys Ile Phe Cys Ile Lys Lys Gln Pro Leu Pro Ser Gln His 370 375 380 Ser Arg Ser Val Ser Ser 385 390 15 1128 DNA Homo sapiens 15 atggcgaacg cgagcgagcc gggtggcagc ggcggcggcg aggcggccgc cctgggcctc 60 aagctggcca cgctcagcct gctgctgtgc gtgagcctag cgggcaacgt gctgttcgcg 120 ctgctgatcg tgcgggagcg cagcctgcac cgcgccccgt actacctgct gctcgacctg 180 tgcctggccg acgggctgcg cgcgctcgcc tgcctcccgg ccgtcatgct ggcggcgcgg 240 cgtgcggcgg ccgcggcggg ggcgccgccg ggcgcgctgg gctgcaagct gctcgccttc 300 ctggccgcgc tcttctgctt ccacgccgcc ttcctgctgc tgggcgtggg cgtcacccgc 360 tacctggcca tcgcgcacca ccgcttctat gcagagcgcc tggccggctg gccgtgcgcc 420 gccatgctgg tgtgcgccgc ctgggcgctg gcgctggccg cggccttccc gccagtgctg 480 gacggcggtg gcgacgacga ggacgcgccg tgcgccctgg agcagcggcc cgacggcgcc 540 cccggcgcgc tgggcttcct gctgctgctg gccgtggtgg tgggcgccac gcacctcgtc 600 tacctccgcc tgctcttctt catccacgac cgccgcaaga tgcggcccgc gcgcctggtg 660 cccgccgtca gccacgactg gaccttccac ggcccgggcg ccaccggcca ggcggccgcc 720 aactggacgg cgggcttcgg ccgcgggccc acgccgcccg cgcttgtggg catccggccc 780 gcagggccgg gccgcggcgc gcgccgcctc ctcgtgctgg aagaattcaa gacggagaag 840 aggctgtgca agatgttcta cgccgtcacg ctgctcttcc tgctcctctg ggggccctac 900 gtcgtggcca gctacctgcg ggtcctggtg cggcccggcg ccgtccccca ggcctacctg 960 acggcctccg tgtggctgac cttcgcgcag gccggcatca accccgtcgt gtgcttcctc 1020 ttcaacaggg agctgaggga ctgcttcagg gcccagttcc cctgctgcca gagcccccgg 1080 accacccagg cgacccatcc ctgcgacctg aaaggcattg gtttatga 1128 16 375 PRT Homo sapiens 16 Met Ala Asn Ala Ser Glu Pro Gly Gly Ser Gly Gly Gly Glu Ala Ala 1 5 10 15 Ala Leu Gly Leu Lys Leu Ala Thr Leu Ser Leu Leu Leu Cys Val Ser 20 25 30 Leu Ala Gly Asn Val Leu Phe Ala Leu Leu Ile Val Arg Glu Arg Ser 35 40 45 Leu His Arg Ala Pro Tyr Tyr Leu Leu Leu Asp Leu Cys Leu Ala Asp 50 55 60 Gly Leu Arg Ala Leu Ala Cys Leu Pro Ala Val Met Leu Ala Ala Arg 65 70 75 80 Arg Ala Ala Ala Ala Ala Gly Ala Pro Pro Gly Ala Leu Gly Cys Lys 85 90 95 Leu Leu Ala Phe Leu Ala Ala Leu Phe Cys Phe His Ala Ala Phe Leu 100 105 110 Leu Leu Gly Val Gly Val Thr Arg Tyr Leu Ala Ile Ala His His Arg 115 120 125 Phe Tyr Ala Glu Arg Leu Ala Gly Trp Pro Cys Ala Ala Met Leu Val 130 135 140 Cys Ala Ala Trp Ala Leu Ala Leu Ala Ala Ala Phe Pro Pro Val Leu 145 150 155 160 Asp Gly Gly Gly Asp Asp Glu Asp Ala Pro Cys Ala Leu Glu Gln Arg 165 170 175 Pro Asp Gly Ala Pro Gly Ala Leu Gly Phe Leu Leu Leu Leu Ala Val 180 185 190 Val Val Gly Ala Thr His Leu Val Tyr Leu Arg Leu Leu Phe Phe Ile 195 200 205 His Asp Arg Arg Lys Met Arg Pro Ala Arg Leu Val Pro Ala Val Ser 210 215 220 His Asp Trp Thr Phe His Gly Pro Gly Ala Thr Gly Gln Ala Ala Ala 225 230 235 240 Asn Trp Thr Ala Gly Phe Gly Arg Gly Pro Thr Pro Pro Ala Leu Val 245 250 255 Gly Ile Arg Pro Ala Gly Pro Gly Arg Gly Ala Arg Arg Leu Leu Val 260 265 270 Leu Glu Glu Phe Lys Thr Glu Lys Arg Leu Cys Lys Met Phe Tyr Ala 275 280 285 Val Thr Leu Leu Phe Leu Leu Leu Trp Gly Pro Tyr Val Val Ala Ser 290 295 300 Tyr Leu Arg Val Leu Val Arg Pro Gly Ala Val Pro Gln Ala Tyr Leu 305 310 315 320 Thr Ala Ser Val Trp Leu Thr Phe Ala Gln Ala Gly Ile Asn Pro Val 325 330 335 Val Cys Phe Leu Phe Asn Arg Glu Leu Arg Asp Cys Phe Arg Ala Gln 340 345 350 Phe Pro Cys Cys Gln Ser Pro Arg Thr Thr Gln Ala Thr His Pro Cys 355 360 365 Asp Leu Lys Gly Ile Gly Leu 370 375 17 1002 DNA Homo sapiens 17 atgaacacca cagtgatgca aggcttcaac agatctgagc ggtgccccag agacactcgg 60 atagtacagc tggtattccc agccctctac acagtggttt tcttgaccgg catcctgctg 120 aatactttgg ctctgtgggt gtttgttcac atccccagct cctccacctt catcatctac 180 ctcaaaaaca ctttggtggc cgacttgata atgacactca tgcttccttt caaaatcctc 240 tctgactcac acctggcacc ctggcagctc agagcttttg tgtgtcgttt ttcttcggtg 300 atattttatg agaccatgta tgtgggcatc gtgctgttag ggctcatagc ctttgacaga 360 ttcctcaaga tcatcagacc tttgagaaat atttttctaa aaaaacctgt ttttgcaaaa 420 acggtctcaa tcttcatctg gttctttttg ttcttcatct ccctgccaaa tacgatcttg 480 agcaacaagg aagcaacacc atcgtctgtg aaaaagtgtg cttccttaaa ggggcctctg 540 gggctgaaat ggcatcaaat ggtaaataac atatgccagt ttattttctg gactgttttt 600 atcctaatgc ttgtgtttta tgtggttatt gcaaaaaaag tatatgattc ttatagaaag 660 tccaaaagta aggacagaaa aaacaacaaa aagctggaag gcaaagtatt tgttgtcgtg 720 gctgtcttct ttgtgtgttt tgctccattt cattttgcca gagttccata tactcacagt 780 caaaccaaca ataagactga ctgtagactg caaaatcaac tgtttattgc taaagaaaca 840 actctctttt tggcagcaac taacatttgt atggatccct taatatacat attcttatgt 900 aaaaaattca cagaaaagct accatgtatg caagggagaa agaccacagc atcaagccaa 960 gaaaatcata gcagtcagac agacaacata accttaggct ga 1002 18 333 PRT Homo sapiens 18 Met Asn Thr Thr Val Met Gln Gly Phe Asn Arg Ser Glu Arg Cys Pro 1 5 10 15 Arg Asp Thr Arg Ile Val Gln Leu Val Phe Pro Ala Leu Tyr Thr Val 20 25 30 Val Phe Leu Thr Gly Ile Leu Leu Asn Thr Leu Ala Leu Trp Val Phe 35 40 45 Val His Ile Pro Ser Ser Ser Thr Phe Ile Ile Tyr Leu Lys Asn Thr 50 55 60 Leu Val Ala Asp Leu Ile Met Thr Leu Met Leu Pro Phe Lys Ile Leu 65 70 75 80 Ser Asp Ser His Leu Ala Pro Trp Gln Leu Arg Ala Phe Val Cys Arg 85 90 95 Phe Ser Ser Val Ile Phe Tyr Glu Thr Met Tyr Val Gly Ile Val Leu 100 105 110 Leu Gly Leu Ile Ala Phe Asp Arg Phe Leu Lys Ile Ile Arg Pro Leu 115 120 125 Arg Asn Ile Phe Leu Lys Lys Pro Val Phe Ala Lys Thr Val Ser Ile 130 135 140 Phe Ile Trp Phe Phe Leu Phe Phe Ile Ser Leu Pro Asn Thr Ile Leu 145 150 155 160 Ser Asn Lys Glu Ala Thr Pro Ser Ser Val Lys Lys Cys Ala Ser Leu 165 170 175 Lys Gly Pro Leu Gly Leu Lys Trp His Gln Met Val Asn Asn Ile Cys 180 185 190 Gln Phe Ile Phe Trp Thr Val Phe Ile Leu Met Leu Val Phe Tyr Val 195 200 205 Val Ile Ala Lys Lys Val Tyr Asp Ser Tyr Arg Lys Ser Lys Ser Lys 210 215 220 Asp Arg Lys Asn Asn Lys Lys Leu Glu Gly Lys Val Phe Val Val Val 225 230 235 240 Ala Val Phe Phe Val Cys Phe Ala Pro Phe His Phe Ala Arg Val Pro 245 250 255 Tyr Thr His Ser Gln Thr Asn Asn Lys Thr Asp Cys Arg Leu Gln Asn 260 265 270 Gln Leu Phe Ile Ala Lys Glu Thr Thr Leu Phe Leu Ala Ala Thr Asn 275 280 285 Ile Cys Met Asp Pro Leu Ile Tyr Ile Phe Leu Cys Lys Lys Phe Thr 290 295 300 Glu Lys Leu Pro Cys Met Gln Gly Arg Lys Thr Thr Ala Ser Ser Gln 305 310 315 320 Glu Asn His Ser Ser Gln Thr Asp Asn Ile Thr Leu Gly 325 330 19 1122 DNA Homo sapiens 19 atggccaaca ctaccggaga gcctgaggag gtgagcggcg ctctgtcccc accgtccgca 60 tcagcttatg tgaagctggt actgctggga ctgattatgt gcgtgagcct ggcgggtaac 120 gccatcttgt ccctgctggt gctcaaggag cgtgccctgc acaaggctcc ttactacttc 180 ctgctggacc tgtgcctggc cgatggcata cgctctgccg tctgcttccc ctttgtgctg 240 gcttctgtgc gccacggctc ttcatggacc ttcagtgcac tcagctgcaa gattgtggcc 300 tttatggccg tgctcttttg cttccatgcg gccttcatgc tgttctgcat cagcgtcacc 360 cgctacatgg ccatcgccca ccaccgcttc tacgccaagc gcatgacact ctggacatgc 420 gcggctgtca tctgcatggc ctggaccctg tctgtggcca tggccttccc acctgtcttt 480 gacgtgggca cctacaagtt tattcgggag gaggaccagt gcatctttga gcatcgctac 540 ttcaaggcca atgacacgct gggcttcatg cttatgttgg ctgtgctcat ggcagctacc 600 catgctgtct acggcaagct gctcctcttc gagtatcgtc accgcaagat gaagccagtg 660 cagatggtgc cagccatcag ccagaactgg acattccatg gtcccggggc caccggccag 720 gctgctgcca actggatcgc cggctttggc cgtgggccca tgccaccaac cctgctgggt 780 atccggcaga atgggcatgc agccagccgg cggctactgg gcatggacga ggtcaagggt 840 gaaaagcagc tgggccgcat gttctacgcg atcacactgc tctttctgct cctctggtca 900 ccctacatcg tggcctgcta ctggcgagtg tttgtgaaag cctgtgctgt gccccaccgc 960 tacctggcca ctgctgtttg gatgagcttc gcccaggctg ccgtcaaccc aattgtctgc 1020 ttcctgctca acaaggacct caagaagtgc ctgaccactc acgccccctg ctggggcaca 1080 ggaggtgccc cggctcccag agaaccctac tgtgtcatgt ga 1122 20 373 PRT Homo sapiens 20 Met Ala Asn Thr Thr Gly Glu Pro Glu Glu Val Ser Gly Ala Leu Ser 1 5 10 15 Pro Pro Ser Ala Ser Ala Tyr Val Lys Leu Val Leu Leu Gly Leu Ile 20 25 30 Met Cys Val Ser Leu Ala Gly Asn Ala Ile Leu Ser Leu Leu Val Leu 35 40 45 Lys Glu Arg Ala Leu His Lys Ala Pro Tyr Tyr Phe Leu Leu Asp Leu 50 55 60 Cys Leu Ala Asp Gly Ile Arg Ser Ala Val Cys Phe Pro Phe Val Leu 65 70 75 80 Ala Ser Val Arg His Gly Ser Ser Trp Thr Phe Ser Ala Leu Ser Cys 85 90 95 Lys Ile Val Ala Phe Met Ala Val Leu Phe Cys Phe His Ala Ala Phe 100 105 110 Met Leu Phe Cys Ile Ser Val Thr Arg Tyr Met Ala Ile Ala His His 115 120 125 Arg Phe Tyr Ala Lys Arg Met Thr Leu Trp Thr Cys Ala Ala Val Ile 130 135 140 Cys Met Ala Trp Thr Leu Ser Val Ala Met Ala Phe Pro Pro Val Phe 145 150 155 160 Asp Val Gly Thr Tyr Lys Phe Ile Arg Glu Glu Asp Gln Cys Ile Phe 165 170 175 Glu His Arg Tyr Phe Lys Ala Asn Asp Thr Leu Gly Phe Met Leu Met 180 185 190 Leu Ala Val Leu Met Ala Ala Thr His Ala Val Tyr Gly Lys Leu Leu 195 200 205 Leu Phe Glu Tyr Arg His Arg Lys Met Lys Pro Val Gln Met Val Pro 210 215 220 Ala Ile Ser Gln Asn Trp Thr Phe His Gly Pro Gly Ala Thr Gly Gln 225 230 235 240 Ala Ala Ala Asn Trp Ile Ala Gly Phe Gly Arg Gly Pro Met Pro Pro 245 250 255 Thr Leu Leu Gly Ile Arg Gln Asn Gly His Ala Ala Ser Arg Arg Leu 260 265 270 Leu Gly Met Asp Glu Val Lys Gly Glu Lys Gln Leu Gly Arg Met Phe 275 280 285 Tyr Ala Ile Thr Leu Leu Phe Leu Leu Leu Trp Ser Pro Tyr Ile Val 290 295 300 Ala Cys Tyr Trp Arg Val Phe Val Lys Ala Cys Ala Val Pro His Arg 305 310 315 320 Tyr Leu Ala Thr Ala Val Trp Met Ser Phe Ala Gln Ala Ala Val Asn 325 330 335 Pro Ile Val Cys Phe Leu Leu Asn Lys Asp Leu Lys Lys Cys Leu Thr 340 345 350 Thr His Ala Pro Cys Trp Gly Thr Gly Gly Ala Pro Ala Pro Arg Glu 355 360 365 Pro Tyr Cys Val Met 370 21 1053 DNA Homo sapiens 21 atggctttgg aacagaacca gtcaacagat tattattatg aggaaaatga aatgaatggc 60 acttatgact acagtcaata tgaattgatc tgtatcaaag aagatgtcag agaatttgca 120 aaagttttcc tccctgtatt cctcacaata gctttcgtca ttggacttgc aggcaattcc 180 atggtagtgg caatttatgc ctattacaag aaacagagaa ccaaaacaga tgtgtacatc 240 ctgaatttgg ctgtagcaga tttactcctt ctattcactc tgcctttttg ggctgttaat 300 gcagttcatg ggtgggtttt agggaaaata atgtgcaaaa taacttcagc cttgtacaca 360 ctaaactttg tctctggaat gcagtttctg gcttgcatca gcatagacag atatgtggca 420 gtaactaatg tccccagcca atcaggagtg ggaaaaccat gctggatcat ctgtttctgt 480 gtctggatgg ctgccatctt gctgagcata ccccagctgg ttttttatac agtaaatgac 540 aatgctaggt gcattcccat tttcccccgc tacctaggaa catcaatgaa agcattgatt 600 caaatgctag agatctgcat tggatttgta gtaccctttc ttattatggg ggtgtgctac 660 tttatcacgg caaggacact catgaagatg ccaaacatta aaatatctcg acccctaaaa 720 gttctgctca cagtcgttat agttttcatt gtcactcaac tgccttataa cattgtcaag 780 ttctgccgag ccatagacat catctactcc ctgatcacca gctgcaacat gagcaaacgc 840 atggacatcg ccatccaagt cacagaaagc attgcactct ttcacagctg cctcaaccca 900 atcctttatg tttttatggg agcatctttc aaaaactacg ttatgaaagt ggccaagaaa 960 tatgggtcct ggagaagaca gagacaaagt gtggaggagt ttccttttga ttctgagggt 1020 cctacagagc caaccagtac ttttagcatt taa 1053 22 350 PRT Homo sapiens 22 Met Ala Leu Glu Gln Asn Gln Ser Thr Asp Tyr Tyr Tyr Glu Glu Asn 1 5 10 15 Glu Met Asn Gly Thr Tyr Asp Tyr Ser Gln Tyr Glu Leu Ile Cys Ile 20 25 30 Lys Glu Asp Val Arg Glu Phe Ala Lys Val Phe Leu Pro Val Phe Leu 35 40 45 Thr Ile Ala Phe Val Ile Gly Leu Ala Gly Asn Ser Met Val Val Ala 50 55 60 Ile Tyr Ala Tyr Tyr Lys Lys Gln Arg Thr Lys Thr Asp Val Tyr Ile 65 70 75 80 Leu Asn Leu Ala Val Ala Asp Leu Leu Leu Leu Phe Thr Leu Pro Phe 85 90 95 Trp Ala Val Asn Ala Val His Gly Trp Val Leu Gly Lys Ile Met Cys 100 105 110 Lys Ile Thr Ser Ala Leu Tyr Thr Leu Asn Phe Val Ser Gly Met Gln 115 120 125 Phe Leu Ala Cys Ile Ser Ile Asp Arg Tyr Val Ala Val Thr Asn Val 130 135 140 Pro Ser Gln Ser Gly Val Gly Lys Pro Cys Trp Ile Ile Cys Phe Cys 145 150 155 160 Val Trp Met Ala Ala Ile Leu Leu Ser Ile Pro Gln Leu Val Phe Tyr 165 170 175 Thr Val Asn Asp Asn Ala Arg Cys Ile Pro Ile Phe Pro Arg Tyr Leu 180 185 190 Gly Thr Ser Met Lys Ala Leu Ile Gln Met Leu Glu Ile Cys Ile Gly 195 200 205 Phe Val Val Pro Phe Leu Ile Met Gly Val Cys Tyr Phe Ile Thr Ala 210 215 220 Arg Thr Leu Met Lys Met Pro Asn Ile Lys Ile Ser Arg Pro Leu Lys 225 230 235 240 Val Leu Leu Thr Val Val Ile Val Phe Ile Val Thr Gln Leu Pro Tyr 245 250 255 Asn Ile Val Lys Phe Cys Arg Ala Ile Asp Ile Ile Tyr Ser Leu Ile 260 265 270 Thr Ser Cys Asn Met Ser Lys Arg Met Asp Ile Ala Ile Gln Val Thr 275 280 285 Glu Ser Ile Ala Leu Phe His Ser Cys Leu Asn Pro Ile Leu Tyr Val 290 295 300 Phe Met Gly Ala Ser Phe Lys Asn Tyr Val Met Lys Val Ala Lys Lys 305 310 315 320 Tyr Gly Ser Trp Arg Arg Gln Arg Gln Ser Val Glu Glu Phe Pro Phe 325 330 335 Asp Ser Glu Gly Pro Thr Glu Pro Thr Ser Thr Phe Ser Ile 340 345 350 23 1116 DNA Homo sapiens 23 atgccaggaa acgccacccc agtgaccacc actgccccgt gggcctccct gggcctctcc 60 gccaagacct gcaacaacgt gtccttcgaa gagagcagga tagtcctggt cgtggtgtac 120 agcgcggtgt gcacgctggg ggtgccggcc aactgcctga ctgcgtggct ggcgctgctg 180 caggtactgc agggcaacgt gctggccgtc tacctgctct gcctggcact ctgcgaactg 240 ctgtacacag gcacgctgcc actctgggtc atctatatcc gcaaccagca ccgctggacc 300 ctaggcctgc tggcctcgaa ggtgaccgcc tacatcttct tctgcaacat ctacgtcagc 360 atcctcttcc tgtgctgcat ctcctgcgac cgcttcgtgg ccgtggtgta cgcgctggag 420 agtcggggcc gccgccgccg gaggaccgcc atcctcatct ccgcctgcat cttcatcctc 480 gtcgggatcg ttcactaccc ggtgttccag acggaagaca aggagacctg ctttgacatg 540 ctgcagatgg acagcaggat tgccgggtac tactacgcca ggttcaccgt tggctttgcc 600 atccctctct ccatcatcgc cttcaccaac caccggattt tcaggagcat caagcagagc 660 atgggcttaa gcgctgccca gaaggccaag gtgaagcact cggccatcgc ggtggttgtc 720 atcttcctag tctgcttcgc cccgtaccac ctggttctcc tcgtcaaagc cgctgccttt 780 tcctactaca gaggagacag gaacgccatg tgcggcttgg aggaaaggct gtacacagcc 840 tctgtggtgt ttctgtgcct gtccacggtg aacggcgtgg ctgaccccat tatctacgtg 900 ctggccacgg accattcccg ccaagaagtg tccagaatcc ataaggggtg gaaagagtgg 960 tccatgaaga cagacgtcac caggctcacc cacagcaggg acaccgagga gctgcagtcg 1020 cccgtggccc ttgcagacca ctacaccttc tccaggcccg tgcacccacc agggtcacca 1080 tgccctgcaa agaggctgat tgaggagtcc tgctga 1116 24 371 PRT Homo sapiens 24 Met Pro Gly Asn Ala Thr Pro Val Thr Thr Thr Ala Pro Trp Ala Ser 1 5 10 15 Leu Gly Leu Ser Ala Lys Thr Cys Asn Asn Val Ser Phe Glu Glu Ser 20 25 30 Arg Ile Val Leu Val Val Val Tyr Ser Ala Val Cys Thr Leu Gly Val 35 40 45 Pro Ala Asn Cys Leu Thr Ala Trp Leu Ala Leu Leu Gln Val Leu Gln 50 55 60 Gly Asn Val Leu Ala Val Tyr Leu Leu Cys Leu Ala Leu Cys Glu Leu 65 70 75 80 Leu Tyr Thr Gly Thr Leu Pro Leu Trp Val Ile Tyr Ile Arg Asn Gln 85 90 95 His Arg Trp Thr Leu Gly Leu Leu Ala Ser Lys Val Thr Ala Tyr Ile 100 105 110 Phe Phe Cys Asn Ile Tyr Val Ser Ile Leu Phe Leu Cys Cys Ile Ser 115 120 125 Cys Asp Arg Phe Val Ala Val Val Tyr Ala Leu Glu Ser Arg Gly Arg 130 135 140 Arg Arg Arg Arg Thr Ala Ile Leu Ile Ser Ala Cys Ile Phe Ile Leu 145 150 155 160 Val Gly Ile Val His Tyr Pro Val Phe Gln Thr Glu Asp Lys Glu Thr 165 170 175 Cys Phe Asp Met Leu Gln Met Asp Ser Arg Ile Ala Gly Tyr Tyr Tyr 180 185 190 Ala Arg Phe Thr Val Gly Phe Ala Ile Pro Leu Ser Ile Ile Ala Phe 195 200 205 Thr Asn His Arg Ile Phe Arg Ser Ile Lys Gln Ser Met Gly Leu Ser 210 215 220 Ala Ala Gln Lys Ala Lys Val Lys His Ser Ala Ile Ala Val Val Val 225 230 235 240 Ile Phe Leu Val Cys Phe Ala Pro Tyr His Leu Val Leu Leu Val Lys 245 250 255 Ala Ala Ala Phe Ser Tyr Tyr Arg Gly Asp Arg Asn Ala Met Cys Gly 260 265 270 Leu Glu Glu Arg Leu Tyr Thr Ala Ser Val Val Phe Leu Cys Leu Ser 275 280 285 Thr Val Asn Gly Val Ala Asp Pro Ile Ile Tyr Val Leu Ala Thr Asp 290 295 300 His Ser Arg Gln Glu Val Ser Arg Ile His Lys Gly Trp Lys Glu Trp 305 310 315 320 Ser Met Lys Thr Asp Val Thr Arg Leu Thr His Ser Arg Asp Thr Glu 325 330 335 Glu Leu Gln Ser Pro Val Ala Leu Ala Asp His Tyr Thr Phe Ser Arg 340 345 350 Pro Val His Pro Pro Gly Ser Pro Cys Pro Ala Lys Arg Leu Ile Glu 355 360 365 Glu Ser Cys 370 25 1113 DNA Homo sapiens 25 atggcgaact atagccatgc agctgacaac attttgcaaa atctctcgcc tctaacagcc 60 tttctgaaac tgacttcctt gggtttcata ataggagtca gcgtggtggg caacctcctg 120 atctccattt tgctagtgaa agataagacc ttgcatagag caccttacta cttcctgttg 180 gatctttgct gttcagatat cctcagatct gcaatttgtt tcccatttgt gttcaactct 240 gtcaaaaatg gctctacctg gacttatggg actctgactt gcaaagtgat tgcctttctg 300 ggggttttgt cctgtttcca cactgctttc atgctcttct gcatcagtgt caccagatac 360 ttagctatcg cccatcaccg cttctataca aagaggctga ccttttggac gtgtctggct 420 gtgatctgta tggtgtggac tctgtctgtg gccatggcat ttcccccggt tttagacgtg 480 ggcacttact cattcattag ggaggaagat caatgcacct tccaacaccg ctccttcagg 540 gctaatgatt ccttaggatt tatgctgctt cttgctctca tcctcctagc cacacagctt 600 gtctacctca agctgatatt tttcgtccac gatcgaagaa aaatgaagcc agtccagttt 660 gtagcagcag tcagccagaa ctggactttt catggtcctg gagccagtgg ccaggcagct 720 gccaattggc tagcaggatt tggaaggggt cccacaccac ccaccttgct gggcatcagg 780 caaaatgcaa acaccacagg cagaagaagg ctattggtct tagacgagtt caaaatggag 840 aaaagaatca gcagaatgtt ctatataatg acttttctgt ttctaacctt gtggggcccc 900 tacctggtgg cctgttattg gagagttttt gcaagagggc ctgtagtacc agggggattt 960 ctaacagctg ctgtctggat gagttttgcc caagcaggaa tcaatccttt tgtctgcatt 1020 ttctcaaaca gggagctgag gcgctgtttc agcacaaccc ttctttactg cagaaaatcc 1080 aggttaccaa gggaacctta ctgtgttata tga 1113 26 370 PRT Homo sapiens 26 Met Ala Asn Tyr Ser His Ala Ala Asp Asn Ile Leu Gln Asn Leu Ser 1 5 10 15 Pro Leu Thr Ala Phe Leu Lys Leu Thr Ser Leu Gly Phe Ile Ile Gly 20 25 30 Val Ser Val Val Gly Asn Leu Leu Ile Ser Ile Leu Leu Val Lys Asp 35 40 45 Lys Thr Leu His Arg Ala Pro Tyr Tyr Phe Leu Leu Asp Leu Cys Cys 50 55 60 Ser Asp Ile Leu Arg Ser Ala Ile Cys Phe Pro Phe Val Phe Asn Ser 65 70 75 80 Val Lys Asn Gly Ser Thr Trp Thr Tyr Gly Thr Leu Thr Cys Lys Val 85 90 95 Ile Ala Phe Leu Gly Val Leu Ser Cys Phe His Thr Ala Phe Met Leu 100 105 110 Phe Cys Ile Ser Val Thr Arg Tyr Leu Ala Ile Ala His His Arg Phe 115 120 125 Tyr Thr Lys Arg Leu Thr Phe Trp Thr Cys Leu Ala Val Ile Cys Met 130 135 140 Val Trp Thr Leu Ser Val Ala Met Ala Phe Pro Pro Val Leu Asp Val 145 150 155 160 Gly Thr Tyr Ser Phe Ile Arg Glu Glu Asp Gln Cys Thr Phe Gln His 165 170 175 Arg Ser Phe Arg Ala Asn Asp Ser Leu Gly Phe Met Leu Leu Leu Ala 180 185 190 Leu Ile Leu Leu Ala Thr Gln Leu Val Tyr Leu Lys Leu Ile Phe Phe 195 200 205 Val His Asp Arg Arg Lys Met Lys Pro Val Gln Phe Val Ala Ala Val 210 215 220 Ser Gln Asn Trp Thr Phe His Gly Pro Gly Ala Ser Gly Gln Ala Ala 225 230 235 240 Ala Asn Trp Leu Ala Gly Phe Gly Arg Gly Pro Thr Pro Pro Thr Leu 245 250 255 Leu Gly Ile Arg Gln Asn Ala Asn Thr Thr Gly Arg Arg Arg Leu Leu 260 265 270 Val Leu Asp Glu Phe Lys Met Glu Lys Arg Ile Ser Arg Met Phe Tyr 275 280 285 Ile Met Thr Phe Leu Phe Leu Thr Leu Trp Gly Pro Tyr Leu Val Ala 290 295 300 Cys Tyr Trp Arg Val Phe Ala Arg Gly Pro Val Val Pro Gly Gly Phe 305 310 315 320 Leu Thr Ala Ala Val Trp Met Ser Phe Ala Gln Ala Gly Ile Asn Pro 325 330 335 Phe Val Cys Ile Phe Ser Asn Arg Glu Leu Arg Arg Cys Phe Ser Thr 340 345 350 Thr Leu Leu Tyr Cys Arg Lys Ser Arg Leu Pro Arg Glu Pro Tyr Cys 355 360 365 Val Ile 370 27 1080 DNA Homo sapiens 27 atgcaggtcc cgaacagcac cggcccggac aacgcgacgc tgcagatgct gcggaacccg 60 gcgatcgcgg tggccctgcc cgtggtgtac tcgctggtgg cggcggtcag catcccgggc 120 aacctcttct ctctgtgggt gctgtgccgg cgcatggggc ccagatcccc gtcggtcatc 180 ttcatgatca acctgagcgt cacggacctg atgctggcca gcgtgttgcc tttccaaatc 240 tactaccatt gcaaccgcca ccactgggta ttcggggtgc tgctttgcaa cgtggtgacc 300 gtggcctttt acgcaaacat gtattccagc atcctcacca tgacctgtat cagcgtggag 360 cgcttcctgg gggtcctgta cccgctcagc tccaagcgct ggcgccgccg tcgttacgcg 420 gtggccgcgt gtgcagggac ctggctgctg ctcctgaccg ccctgtgccc gctggcgcgc 480 accgatctca cctacccggt gcacgccctg ggcatcatca cctgcttcga cgtcctcaag 540 tggacgatgc tccccagcgt ggccatgtgg gccgtgttcc tcttcaccat cttcatcctg 600 ctgttcctca tcccgttcgt gatcaccgtg gcttgttaca cggccaccat cctcaagctg 660 ttgcgcacgg aggaggcgca cggccgggag cagcggaggc gcgcggtggg cctggccgcg 720 gtggtcttgc tggcctttgt cacctgcttc gcccccaaca acttcgtgct cctggcgcac 780 atcgtgagcc gcctgttcta cggcaagagc tactaccacg tgtacaagct cacgctgtgt 840 ctcagctgcc tcaacaactg tctggacccg tttgtttatt actttgcgtc ccgggaattc 900 cagctgcgcc tgcgggaata tttgggctgc cgccgggtgc ccagagacac cctggacacg 960 cgccgcgaga gcctcttctc cgccaggacc acgtccgtgc gctccgaggc cggtgcgcac 1020 cctgaaggga tggagggagc caccaggccc ggcctccaga ggcaggagag tgtgttctga 1080 28 359 PRT Homo sapiens 28 Met Gln Val Pro Asn Ser Thr Gly Pro Asp Asn Ala Thr Leu Gln Met 1 5 10 15 Leu Arg Asn Pro Ala Ile Ala Val Ala Leu Pro Val Val Tyr Ser Leu 20 25 30 Val Ala Ala Val Ser Ile Pro Gly Asn Leu Phe Ser Leu Trp Val Leu 35 40 45 Cys Arg Arg Met Gly Pro Arg Ser Pro Ser Val Ile Phe Met Ile Asn 50 55 60 Leu Ser Val Thr Asp Leu Met Leu Ala Ser Val Leu Pro Phe Gln Ile 65 70 75 80 Tyr Tyr His Cys Asn Arg His His Trp Val Phe Gly Val Leu Leu Cys 85 90 95 Asn Val Val Thr Val Ala Phe Tyr Ala Asn Met Tyr Ser Ser Ile Leu 100 105 110 Thr Met Thr Cys Ile Ser Val Glu Arg Phe Leu Gly Val Leu Tyr Pro 115 120 125 Leu Ser Ser Lys Arg Trp Arg Arg Arg Arg Tyr Ala Val Ala Ala Cys 130 135 140 Ala Gly Thr Trp Leu Leu Leu Leu Thr Ala Leu Cys Pro Leu Ala Arg 145 150 155 160 Thr Asp Leu Thr Tyr Pro Val His Ala Leu Gly Ile Ile Thr Cys Phe 165 170 175 Asp Val Leu Lys Trp Thr Met Leu Pro Ser Val Ala Met Trp Ala Val 180 185 190 Phe Leu Phe Thr Ile Phe Ile Leu Leu Phe Leu Ile Pro Phe Val Ile 195 200 205 Thr Val Ala Cys Tyr Thr Ala Thr Ile Leu Lys Leu Leu Arg Thr Glu 210 215 220 Glu Ala His Gly Arg Glu Gln Arg Arg Arg Ala Val Gly Leu Ala Ala 225 230 235 240 Val Val Leu Leu Ala Phe Val Thr Cys Phe Ala Pro Asn Asn Phe Val 245 250 255 Leu Leu Ala His Ile Val Ser Arg Leu Phe Tyr Gly Lys Ser Tyr Tyr 260 265 270 His Val Tyr Lys Leu Thr Leu Cys Leu Ser Cys Leu Asn Asn Cys Leu 275 280 285 Asp Pro Phe Val Tyr Tyr Phe Ala Ser Arg Glu Phe Gln Leu Arg Leu 290 295 300 Arg Glu Tyr Leu Gly Cys Arg Arg Val Pro Arg Asp Thr Leu Asp Thr 305 310 315 320 Arg Arg Glu Ser Leu Phe Ser Ala Arg Thr Thr Ser Val Arg Ser Glu 325 330 335 Ala Gly Ala His Pro Glu Gly Met Glu Gly Ala Thr Arg Pro Gly Leu 340 345 350 Gln Arg Gln Glu Ser Val Phe 355 29 1503 DNA Homo sapiens 29 atggagcgtc cctgggagga cagcccaggc ccggaggggg cagctgaggg ctcgcctgtg 60 ccagtcgccg ccggggcgcg ctccggtgcc gcggcgagtg gcacaggctg gcagccatgg 120 gctgagtgcc cgggacccaa ggggaggggg caactgctgg cgaccgccgg ccctttgcgt 180 cgctggcccg ccccctcgcc tgccagctcc agccccgccc ccggagcggc gtccgctcac 240 tcggttcaag gcagcgcgac tgcgggtggc gcacgaccag ggcgcagacc ttggggcgcg 300 cggcccatgg agtcggggct gctgcggccg gcgccggtga gcgaggtcat cgtcctgcat 360 tacaactaca ccggcaagct ccgcggtgcg agctaccagc cgggtgccgg cctgcgcgcc 420 gacgccgtgg tgtgcctggc ggtgtgcgcc ttcatcgtgc tagagaatct agccgtgttg 480 ttggtgctcg gacgccaccc gcgcttccac gctcccatgt tcctgctcct gggcagcctc 540 acgttgtcgg atctgctggc aggcgccgcc tacgccgcca acatcctact gtcggggccg 600 ctcacgctga aactgtcccc cgcgctctgg ttcgcacggg agggaggcgt cttcgtggca 660 ctcactgcgt ccgtgctgag cctcctggcc atcgcgctgg agcgcagcct caccatggcg 720 cgcagggggc ccgcgcccgt ctccagtcgg gggcgcacgc tggcgatggc agccgcggcc 780 tggggcgtgt cgctgctcct cgggctcctg ccagcgctgg gctggaattg cctgggtcgc 840 ctggacgctt gctccactgt cttgccgctc tacgccaagg cctacgtgct cttctgcgtg 900 ctcgccttcg tgggcatcct ggccgcgatc tgtgcactct acgcgcgcat ctactgccag 960 gtacgcgcca acgcgcggcg cctgccggca cggcccggga ctgcggggac cacctcgacc 1020 cgggcgcgtc gcaagccgcg ctctctggcc ttgctgcgca cgctcagcgt ggtgctcctg 1080 gcctttgtgg catgttgggg ccccctcttc ctgctgctgt tgctcgacgt ggcgtgcccg 1140 gcgcgcacct gtcctgtact cctgcaggcc gatcccttcc tgggactggc catggccaac 1200 tcacttctga accccatcat ctacacgctc accaaccgcg acctgcgcca cgcgctcctg 1260 cgcctggtct gctgcggacg ccactcctgc ggcagagacc cgagtggctc ccagcagtcg 1320 gcgagcgcgg ctgaggcttc cgggggcctg cgccgctgcc tgcccccggg ccttgatggg 1380 agcttcagcg gctcggagcg ctcatcgccc cagcgcgacg ggctggacac cagcggctcc 1440 acaggcagcc ccggtgcacc cacagccgcc cggactctgg tatcagaacc ggctgcagac 1500 tga 1503 30 500 PRT Homo sapiens 30 Met Glu Arg Pro Trp Glu Asp Ser Pro Gly Pro Glu Gly Ala Ala Glu 1 5 10 15 Gly Ser Pro Val Pro Val Ala Ala Gly Ala Arg Ser Gly Ala Ala Ala 20 25 30 Ser Gly Thr Gly Trp Gln Pro Trp Ala Glu Cys Pro Gly Pro Lys Gly 35 40 45 Arg Gly Gln Leu Leu Ala Thr Ala Gly Pro Leu Arg Arg Trp Pro Ala 50 55 60 Pro Ser Pro Ala Ser Ser Ser Pro Ala Pro Gly Ala Ala Ser Ala His 65 70 75 80 Ser Val Gln Gly Ser Ala Thr Ala Gly Gly Ala Arg Pro Gly Arg Arg 85 90 95 Pro Trp Gly Ala Arg Pro Met Glu Ser Gly Leu Leu Arg Pro Ala Pro 100 105 110 Val Ser Glu Val Ile Val Leu His Tyr Asn Tyr Thr Gly Lys Leu Arg 115 120 125 Gly Ala Ser Tyr Gln Pro Gly Ala Gly Leu Arg Ala Asp Ala Val Val 130 135 140 Cys Leu Ala Val Cys Ala Phe Ile Val Leu Glu Asn Leu Ala Val Leu 145 150 155 160 Leu Val Leu Gly Arg His Pro Arg Phe His Ala Pro Met Phe Leu Leu 165 170 175 Leu Gly Ser Leu Thr Leu Ser Asp Leu Leu Ala Gly Ala Ala Tyr Ala 180 185 190 Ala Asn Ile Leu Leu Ser Gly Pro Leu Thr Leu Lys Leu Ser Pro Ala 195 200 205 Leu Trp Phe Ala Arg Glu Gly Gly Val Phe Val Ala Leu Thr Ala Ser 210 215 220 Val Leu Ser Leu Leu Ala Ile Ala Leu Glu Arg Ser Leu Thr Met Ala 225 230 235 240 Arg Arg Gly Pro Ala Pro Val Ser Ser Arg Gly Arg Thr Leu Ala Met 245 250 255 Ala Ala Ala Ala Trp Gly Val Ser Leu Leu Leu Gly Leu Leu Pro Ala 260 265 270 Leu Gly Trp Asn Cys Leu Gly Arg Leu Asp Ala Cys Ser Thr Val Leu 275 280 285 Pro Leu Tyr Ala Lys Ala Tyr Val Leu Phe Cys Val Leu Ala Phe Val 290 295 300 Gly Ile Leu Ala Ala Ile Cys Ala Leu Tyr Ala Arg Ile Tyr Cys Gln 305 310 315 320 Val Arg Ala Asn Ala Arg Arg Leu Pro Ala Arg Pro Gly Thr Ala Gly 325 330 335 Thr Thr Ser Thr Arg Ala Arg Arg Lys Pro Arg Ser Leu Ala Leu Leu 340 345 350 Arg Thr Leu Ser Val Val Leu Leu Ala Phe Val Ala Cys Trp Gly Pro 355 360 365 Leu Phe Leu Leu Leu Leu Leu Asp Val Ala Cys Pro Ala Arg Thr Cys 370 375 380 Pro Val Leu Leu Gln Ala Asp Pro Phe Leu Gly Leu Ala Met Ala Asn 385 390 395 400 Ser Leu Leu Asn Pro Ile Ile Tyr Thr Leu Thr Asn Arg Asp Leu Arg 405 410 415 His Ala Leu Leu Arg Leu Val Cys Cys Gly Arg His Ser Cys Gly Arg 420 425 430 Asp Pro Ser Gly Ser Gln Gln Ser Ala Ser Ala Ala Glu Ala Ser Gly 435 440 445 Gly Leu Arg Arg Cys Leu Pro Pro Gly Leu Asp Gly Ser Phe Ser Gly 450 455 460 Ser Glu Arg Ser Ser Pro Gln Arg Asp Gly Leu Asp Thr Ser Gly Ser 465 470 475 480 Thr Gly Ser Pro Gly Ala Pro Thr Ala Ala Arg Thr Leu Val Ser Glu 485 490 495 Pro Ala Ala Asp 500 31 1029 DNA Homo sapiens 31 atgcaagccg tcgacaatct cacctctgcg cctgggaaca ccagtctgtg caccagagac 60 tacaaaatca cccaggtcct cttcccactg ctctacactg tcctgttttt tgttggactt 120 atcacaaatg gcctggcgat gaggattttc tttcaaatcc ggagtaaatc aaactttatt 180 atttttctta agaacacagt catttctgat cttctcatga ttctgacttt tccattcaaa 240 attcttagtg atgccaaact gggaacagga ccactgagaa cttttgtgtg tcaagttacc 300 tccgtcatat tttatttcac aatgtatatc agtatttcat tcctgggact gataactatc 360 gatcgctacc agaagaccac caggccattt aaaacatcca accccaaaaa tctcttgggg 420 gctaagattc tctctgttgt catctgggca ttcatgttct tactctcttt gcctaacatg 480 attctgacca acaggcagcc gagagacaag aatgtgaaga aatgctcttt ccttaaatca 540 gagttcggtc tagtctggca tgaaatagta aattacatct gtcaagtcat tttctggatt 600 aatttcttaa ttgttattgt atgttataca ctcattacaa aagaactgta ccggtcatac 660 gtaagaacga ggggtgtagg taaagtcccc aggaaaaagg tgaacgtcaa agttttcatt 720 atcattgctg tattctttat ttgttttgtt cctttccatt ttgcccgaat tccttacacc 780 ctgagccaaa cccgggatgt ctttgactgc actgctgaaa atactctgtt ctatgtgaaa 840 gagagcactc tgtggttaac ttccttaaat gcatgcctgg atccgttcat ctattttttc 900 ctttgcaagt ccttcagaaa ttccttgata agtatgctga agtgccccaa ttctgcaaca 960 tctctgtccc aggacaatag gaaaaaagaa caggatggtg gtgacccaaa tgaagagact 1020 ccaatgtaa 1029 32 342 PRT Homo sapiens 32 Met Gln Ala Val Asp Asn Leu Thr Ser Ala Pro Gly Asn Thr Ser Leu 1 5 10 15 Cys Thr Arg Asp Tyr Lys Ile Thr Gln Val Leu Phe Pro Leu Leu Tyr 20 25 30 Thr Val Leu Phe Phe Val Gly Leu Ile Thr Asn Gly Leu Ala Met Arg 35 40 45 Ile Phe Phe Gln Ile Arg Ser Lys Ser Asn Phe Ile Ile Phe Leu Lys 50 55 60 Asn Thr Val Ile Ser Asp Leu Leu Met Ile Leu Thr Phe Pro Phe Lys 65 70 75 80 Ile Leu Ser Asp Ala Lys Leu Gly Thr Gly Pro Leu Arg Thr Phe Val 85 90 95 Cys Gln Val Thr Ser Val Ile Phe Tyr Phe Thr Met Tyr Ile Ser Ile 100 105 110 Ser Phe Leu Gly Leu Ile Thr Ile Asp Arg Tyr Gln Lys Thr Thr Arg 115 120 125 Pro Phe Lys Thr Ser Asn Pro Lys Asn Leu Leu Gly Ala Lys Ile Leu 130 135 140 Ser Val Val Ile Trp Ala Phe Met Phe Leu Leu Ser Leu Pro Asn Met 145 150 155 160 Ile Leu Thr Asn Arg Gln Pro Arg Asp Lys Asn Val Lys Lys Cys Ser 165 170 175 Phe Leu Lys Ser Glu Phe Gly Leu Val Trp His Glu Ile Val Asn Tyr 180 185 190 Ile Cys Gln Val Ile Phe Trp Ile Asn Phe Leu Ile Val Ile Val Cys 195 200 205 Tyr Thr Leu Ile Thr Lys Glu Leu Tyr Arg Ser Tyr Val Arg Thr Arg 210 215 220 Gly Val Gly Lys Val Pro Arg Lys Lys Val Asn Val Lys Val Phe Ile 225 230 235 240 Ile Ile Ala Val Phe Phe Ile Cys Phe Val Pro Phe His Phe Ala Arg 245 250 255 Ile Pro Tyr Thr Leu Ser Gln Thr Arg Asp Val Phe Asp Cys Thr Ala 260 265 270 Glu Asn Thr Leu Phe Tyr Val Lys Glu Ser Thr Leu Trp Leu Thr Ser 275 280 285 Leu Asn Ala Cys Leu Asp Pro Phe Ile Tyr Phe Phe Leu Cys Lys Ser 290 295 300 Phe Arg Asn Ser Leu Ile Ser Met Leu Lys Cys Pro Asn Ser Ala Thr 305 310 315 320 Ser Leu Ser Gln Asp Asn Arg Lys Lys Glu Gln Asp Gly Gly Asp Pro 325 330 335 Asn Glu Glu Thr Pro Met 340 33 1077 DNA Homo sapiens 33 atgtcggtct gctaccgtcc cccagggaac gagacactgc tgagctggaa gacttcgcgg 60 gccacaggca cagccttcct gctgctggcg gcgctgctgg ggctgcctgg caacggcttc 120 gtggtgtgga gcttggcggg ctggcggcct gcacgggggc gaccgctggc ggccacgctt 180 gtgctgcacc tggcgctggc cgacggcgcg gtgctgctgc tcacgccgct ctttgtggcc 240 ttcctgaccc ggcaggcctg gccgctgggc caggcgggct gcaaggcggt gtactacgtg 300 tgcgcgctca gcatgtacgc cagcgtgctg ctcaccggcc tgctcagcct gcagcgctgc 360 ctcgcagtca cccgcccctt cctggcgcct cggctgcgca gcccggccct ggcccgccgc 420 ctgctgctgg cggtctggct ggccgccctg ttgctcgccg tcccggccgc cgtctaccgc 480 cacctgtgga gggaccgcgt atgccagctg tgccacccgt cgccggtcca cgccgccgcc 540 cacctgagcc tggagactct gaccgctttc gtgcttcctt tcgggctgat gctcggctgc 600 tacagcgtga cgctggcacg gctgcggggc gcccgctggg gctccgggcg gcacggggcg 660 cgggtgggcc ggctggtgag cgccatcgtg cttgccttcg gcttgctctg ggccccctac 720 cacgcagtca accttctgca ggcggtcgca gcgctggctc caccggaagg ggccttggcg 780 aagctgggcg gagccggcca ggcggcgcga gcgggaacta cggccttggc cttcttcagt 840 tctagcgtca acccggtgct ctacgtcttc accgctggag atctgctgcc ccgggcaggt 900 ccccgtttcc tcacgcggct cttcgaaggc tctggggagg cccgaggggg cggccgctct 960 agggaaggga ccatggagct ccgaactacc cctcagctga aagtggtggg gcagggccgc 1020 ggcaatggag acccgggggg tgggatggag aaggacggtc cggaatggga cctttga 1077 34 358 PRT Homo sapiens 34 Met Ser Val Cys Tyr Arg Pro Pro Gly Asn Glu Thr Leu Leu Ser Trp 1 5 10 15 Lys Thr Ser Arg Ala Thr Gly Thr Ala Phe Leu Leu Leu Ala Ala Leu 20 25 30 Leu Gly Leu Pro Gly Asn Gly Phe Val Val Trp Ser Leu Ala Gly Trp 35 40 45 Arg Pro Ala Arg Gly Arg Pro Leu Ala Ala Thr Leu Val Leu His Leu 50 55 60 Ala Leu Ala Asp Gly Ala Val Leu Leu Leu Thr Pro Leu Phe Val Ala 65 70 75 80 Phe Leu Thr Arg Gln Ala Trp Pro Leu Gly Gln Ala Gly Cys Lys Ala 85 90 95 Val Tyr Tyr Val Cys Ala Leu Ser Met Tyr Ala Ser Val Leu Leu Thr 100 105 110 Gly Leu Leu Ser Leu Gln Arg Cys Leu Ala Val Thr Arg Pro Phe Leu 115 120 125 Ala Pro Arg Leu Arg Ser Pro Ala Leu Ala Arg Arg Leu Leu Leu Ala 130 135 140 Val Trp Leu Ala Ala Leu Leu Leu Ala Val Pro Ala Ala Val Tyr Arg 145 150 155 160 His Leu Trp Arg Asp Arg Val Cys Gln Leu Cys His Pro Ser Pro Val 165 170 175 His Ala Ala Ala His Leu Ser Leu Glu Thr Leu Thr Ala Phe Val Leu 180 185 190 Pro Phe Gly Leu Met Leu Gly Cys Tyr Ser Val Thr Leu Ala Arg Leu 195 200 205 Arg Gly Ala Arg Trp Gly Ser Gly Arg His Gly Ala Arg Val Gly Arg 210 215 220 Leu Val Ser Ala Ile Val Leu Ala Phe Gly Leu Leu Trp Ala Pro Tyr 225 230 235 240 His Ala Val Asn Leu Leu Gln Ala Val Ala Ala Leu Ala Pro Pro Glu 245 250 255 Gly Ala Leu Ala Lys Leu Gly Gly Ala Gly Gln Ala Ala Arg Ala Gly 260 265 270 Thr Thr Ala Leu Ala Phe Phe Ser Ser Ser Val Asn Pro Val Leu Tyr 275 280 285 Val Phe Thr Ala Gly Asp Leu Leu Pro Arg Ala Gly Pro Arg Phe Leu 290 295 300 Thr Arg Leu Phe Glu Gly Ser Gly Glu Ala Arg Gly Gly Gly Arg Ser 305 310 315 320 Arg Glu Gly Thr Met Glu Leu Arg Thr Thr Pro Gln Leu Lys Val Val 325 330 335 Gly Gln Gly Arg Gly Asn Gly Asp Pro Gly Gly Gly Met Glu Lys Asp 340 345 350 Gly Pro Glu Trp Asp Leu 355 35 1005 DNA Homo sapiens 35 atgctgggga tcatggcatg gaatgcaact tgcaaaaact ggctggcagc agaggctgcc 60 ctggaaaagt actacctttc cattttttat gggattgagt tcgttgtggg agtccttgga 120 aataccattg ttgtttacgg ctacatcttc tctctgaaga actggaacag cagtaatatt 180 tatctcttta acctctctgt ctctgactta gcttttctgt gcaccctccc catgctgata 240 aggagttatg ccaatggaaa ctggatatat ggagacgtgc tctgcataag caaccgatat 300 gtgcttcatg ccaacctcta taccagcatt ctctttctca cttttatcag catagatcga 360 tacttgataa ttaagtatcc tttccgagaa caccttctgc aaaagaaaga gtttgctatt 420 ttaatctcct tggccatttg ggttttagta accttagagt tactacccat acttcccctt 480 ataaatcctg ttataactga caatggcacc acctgtaatg attttgcaag ttctggagac 540 cccaactaca acctcattta cagcatgtgt ctaacactgt tggggttcct tattcctctt 600 tttgtgatgt gtttctttta ttacaagatt gctctcttcc taaagcagag gaataggcag 660 gttgctactg ctctgcccct tgaaaagcct ctcaacttgg tcatcatggc agtggtaatc 720 ttctctgtgc tttttacacc ctatcacgtc atgcggaatg tgaggatcgc ttcacgcctg 780 gggagttgga agcagtatca gtgcactcag gtcgtcatca actcctttta cattgtgaca 840 cggcctttgg cctttctgaa cagtgtcatc aaccctgtct tctattttct tttgggagat 900 cacttcaggg acatgctgat gaatcaactg agacacaact tcaaatccct tacatccttt 960 agcagatggg ctcatgaact cctactttca ttcagagaaa agtga 1005 36 334 PRT Homo sapiens 36 Met Leu Gly Ile Met Ala Trp Asn Ala Thr Cys Lys Asn Trp Leu Ala 1 5 10 15 Ala Glu Ala Ala Leu Glu Lys Tyr Tyr Leu Ser Ile Phe Tyr Gly Ile 20 25 30 Glu Phe Val Val Gly Val Leu Gly Asn Thr Ile Val Val Tyr Gly Tyr 35 40 45 Ile Phe Ser Leu Lys Asn Trp Asn Ser Ser Asn Ile Tyr Leu Phe Asn 50 55 60 Leu Ser Val Ser Asp Leu Ala Phe Leu Cys Thr Leu Pro Met Leu Ile 65 70 75 80 Arg Ser Tyr Ala Asn Gly Asn Trp Ile Tyr Gly Asp Val Leu Cys Ile 85 90 95 Ser Asn Arg Tyr Val Leu His Ala Asn Leu Tyr Thr Ser Ile Leu Phe 100 105 110 Leu Thr Phe Ile Ser Ile Asp Arg Tyr Leu Ile Ile Lys Tyr Pro Phe 115 120 125 Arg Glu His Leu Leu Gln Lys Lys Glu Phe Ala Ile Leu Ile Ser Leu 130 135 140 Ala Ile Trp Val Leu Val Thr Leu Glu Leu Leu Pro Ile Leu Pro Leu 145 150 155 160 Ile Asn Pro Val Ile Thr Asp Asn Gly Thr Thr Cys Asn Asp Phe Ala 165 170 175 Ser Ser Gly Asp Pro Asn Tyr Asn Leu Ile Tyr Ser Met Cys Leu Thr 180 185 190 Leu Leu Gly Phe Leu Ile Pro Leu Phe Val Met Cys Phe Phe Tyr Tyr 195 200 205 Lys Ile Ala Leu Phe Leu Lys Gln Arg Asn Arg Gln Val Ala Thr Ala 210 215 220 Leu Pro Leu Glu Lys Pro Leu Asn Leu Val Ile Met Ala Val Val Ile 225 230 235 240 Phe Ser Val Leu Phe Thr Pro Tyr His Val Met Arg Asn Val Arg Ile 245 250 255 Ala Ser Arg Leu Gly Ser Trp Lys Gln Tyr Gln Cys Thr Gln Val Val 260 265 270 Ile Asn Ser Phe Tyr Ile Val Thr Arg Pro Leu Ala Phe Leu Asn Ser 275 280 285 Val Ile Asn Pro Val Phe Tyr Phe Leu Leu Gly Asp His Phe Arg Asp 290 295 300 Met Leu Met Asn Gln Leu Arg His Asn Phe Lys Ser Leu Thr Ser Phe 305 310 315 320 Ser Arg Trp Ala His Glu Leu Leu Leu Ser Phe Arg Glu Lys 325 330 37 1296 DNA Homo sapiens 37 atgcaggcgc ttaacattac cccggagcag ttctctcggc tgctgcggga ccacaacctg 60 acgcgggagc agttcatcgc tctgtaccgg ctgcgaccgc tcgtctacac cccagagctg 120 ccgggacgcg ccaagctggc cctcgtgctc accggcgtgc tcatcttcgc cctggcgctc 180 tttggcaatg ctctggtgtt ctacgtggtg acccgcagca aggccatgcg caccgtcacc 240 aacatcttta tctgctcctt ggcgctcagt gacctgctca tcaccttctt ctgcattccc 300 gtcaccatgc tccagaacat ttccgacaac tggctggggg gtgctttcat ttgcaagatg 360 gtgccatttg tccagtctac cgctgttgtg acagaaatgc tcactatgac ctgcattgct 420 gtggaaaggc accagggact tgtgcatcct tttaaaatga agtggcaata caccaaccga 480 agggctttca caatgctagg tgtggtctgg ctggtggcag tcatcgtagg atcacccatg 540 tggcacgtgc aacaacttga gatcaaatat gacttcctat atgaaaagga acacatctgc 600 tgcttagaag agtggaccag ccctgtgcac cagaagatct acaccacctt catccttgtc 660 atcctcttcc tcctgcctct tatggtgatg cttattctgt acagtaaaat tggttatgaa 720 ctttggataa agaaaagagt tggggatggt tcagtgcttc gaactattca tggaaaagaa 780 atgtccaaaa tagccaggaa gaagaaacga gctgtcatta tgatggtgac agtggtggct 840 ctctttgctg tgtgctgggc accattccat gttgtccata tgatgattga atacagtaat 900 tttgaaaagg aatatgatga tgtcacaatc aagatgattt ttgctatcgt gcaaattatt 960 ggattttcca actccatctg taatcccatt gtctatgcat ttatgaatga aaacttcaaa 1020 aaaaatgttt tgtctgcagt ttgttattgc atagtaaata aaaccttctc tccagcacaa 1080 aggcatggaa attcaggaat tacaatgatg cggaagaaag caaagttttc cctcagagag 1140 aatccagtgg aggaaaccaa aggagaagca ttcagtgatg gcaacattga agtcaaattg 1200 tgtgaacaga cagaggagaa gaaaaagctc aaacgacatc ttgctctctt taggtctgaa 1260 ctggctgaga attctccttt agacagtggg cattaa 1296 38 431 PRT Homo sapiens 38 Met Gln Ala Leu Asn Ile Thr Pro Glu Gln Phe Ser Arg Leu Leu Arg 1 5 10 15 Asp His Asn Leu Thr Arg Glu Gln Phe Ile Ala Leu Tyr Arg Leu Arg 20 25 30 Pro Leu Val Tyr Thr Pro Glu Leu Pro Gly Arg Ala Lys Leu Ala Leu 35 40 45 Val Leu Thr Gly Val Leu Ile Phe Ala Leu Ala Leu Phe Gly Asn Ala 50 55 60 Leu Val Phe Tyr Val Val Thr Arg Ser Lys Ala Met Arg Thr Val Thr 65 70 75 80 Asn Ile Phe Ile Cys Ser Leu Ala Leu Ser Asp Leu Leu Ile Thr Phe 85 90 95 Phe Cys Ile Pro Val Thr Met Leu Gln Asn Ile Ser Asp Asn Trp Leu 100 105 110 Gly Gly Ala Phe Ile Cys Lys Met Val Pro Phe Val Gln Ser Thr Ala 115 120 125 Val Val Thr Glu Met Leu Thr Met Thr Cys Ile Ala Val Glu Arg His 130 135 140 Gln Gly Leu Val His Pro Phe Lys Met Lys Trp Gln Tyr Thr Asn Arg 145 150 155 160 Arg Ala Phe Thr Met Leu Gly Val Val Trp Leu Val Ala Val Ile Val 165 170 175 Gly Ser Pro Met Trp His Val Gln Gln Leu Glu Ile Lys Tyr Asp Phe 180 185 190 Leu Tyr Glu Lys Glu His Ile Cys Cys Leu Glu Glu Trp Thr Ser Pro 195 200 205 Val His Gln Lys Ile Tyr Thr Thr Phe Ile Leu Val Ile Leu Phe Leu 210 215 220 Leu Pro Leu Met Val Met Leu Ile Leu Tyr Ser Lys Ile Gly Tyr Glu 225 230 235 240 Leu Trp Ile Lys Lys Arg Val Gly Asp Gly Ser Val Leu Arg Thr Ile 245 250 255 His Gly Lys Glu Met Ser Lys Ile Ala Arg Lys Lys Lys Arg Ala Val 260 265 270 Ile Met Met Val Thr Val Val Ala Leu Phe Ala Val Cys Trp Ala Pro 275 280 285 Phe His Val Val His Met Met Ile Glu Tyr Ser Asn Phe Glu Lys Glu 290 295 300 Tyr Asp Asp Val Thr Ile Lys Met Ile Phe Ala Ile Val Gln Ile Ile 305 310 315 320 Gly Phe Ser Asn Ser Ile Cys Asn Pro Ile Val Tyr Ala Phe Met Asn 325 330 335 Glu Asn Phe Lys Lys Asn Val Leu Ser Ala Val Cys Tyr Cys Ile Val 340 345 350 Asn Lys Thr Phe Ser Pro Ala Gln Arg His Gly Asn Ser Gly Ile Thr 355 360 365 Met Met Arg Lys Lys Ala Lys Phe Ser Leu Arg Glu Asn Pro Val Glu 370 375 380 Glu Thr Lys Gly Glu Ala Phe Ser Asp Gly Asn Ile Glu Val Lys Leu 385 390 395 400 Cys Glu Gln Thr Glu Glu Lys Lys Lys Leu Lys Arg His Leu Ala Leu 405 410 415 Phe Arg Ser Glu Leu Ala Glu Asn Ser Pro Leu Asp Ser Gly His 420 425 430 39 24 DNA Homo sapiens 39 ctgtgtacag cagttcgcag agtg 24 40 24 DNA Homo sapiens 40 gagtgccagg cagagcaggt agac 24 41 31 DNA Homo sapiens 41 cccgaattcc tgcttgctcc cagcttggcc c 31 42 32 DNA Homo sapiens 42 tgtggatcct gctgtcaaag gtcccattcc gg 32 43 20 DNA Homo sapiens 43 tcacaatgct aggtgtggtc 20 44 22 DNA Homo sapiens 44 tgcatagaca atgggattac ag 22 45 511 DNA Homo sapiens 45 tcacaatgct aggtgtggtc tggctggtgg cagtcatcgt aggatcaccc atgtggcacg 60 tgcaacaact tgagatcaaa tatgacttcc tatatgaaaa ggaacacatc tgctgcttag 120 aagagtggac cagccctgtg caccagaaga tctacaccac cttcatcctt gtcatcctct 180 tcctcctgcc tcttatggtg atgcttattc tgtacgtaaa attggttatg aactttggat 240 aaagaaaaga gttggggatg gttcagtgct tcgaactatt catggaaaag aaatgtccaa 300 aatagccagg aagaagaaac gagctgtcat tatgatggtg acagtggtgg ctctctttgc 360 tgtgtgctgg gcaccattcc atgttgtcca tatgatgatt gaatacagta attttgaaaa 420 ggaatatgat gatgtcacaa tcaagatgat ttttgctatc gtgcaaatta ttggattttc 480 caactccatc tgtaatccca ttgtctatgc a 511 46 21 DNA Homo sapiens 46 ctgcttagaa gagtggacca g 21 47 22 DNA Homo sapiens 47 ctgtgcacca gaagatctac ac 22 48 21 DNA Homo sapiens 48 caaggatgaa ggtggtgtag a 21 49 23 DNA Homo sapiens 49 gtgtagatct tctggtgcac agg 23 50 21 DNA Homo sapiens 50 gcaatgcagg tcatagtgag c 21 51 27 DNA Homo sapiens 51 tggagcatgg tgacgggaat gcagaag 27 52 27 DNA Homo sapiens 52 gtgatgagca ggtcactgag cgccaag 27 53 23 DNA Homo sapiens 53 gcaatgcagg cgcttaacat tac 23 54 22 DNA Homo sapiens 54 ttgggttaca atctgaaggg ca 22 55 23 DNA Homo sapiens 55 actccgtgtc cagcaggact ctg 23 56 24 DNA Homo sapiens 56 tgcgtgttcc tggaccctca cgtg 24 57 29 DNA Homo sapiens 57 caggccttgg attttaatgt cagggatgg 29 58 27 DNA Homo sapiens 58 ggagagtcag ctctgaaaga attcagg 27 59 27 DNA Homo sapiens 59 tgatgtgatg ccagatacta atagcac 27 60 27 DNA Homo sapiens 60 cctgattcat ttaggtgaga ttgagac 27 61 21 DNA Homo sapiens 61 gacaggtacc ttgccatcaa g 21 62 22 DNA Homo sapiens 62 ctgcacaatg ccagtgataa gg 22 63 27 DNA Homo sapiens 63 ctgacttctt gttcctggca gcagcgg 27 64 27 DNA Homo sapiens 64 agaccagcca gggcacgctg aagagtg 27 65 32 DNA Homo sapiens 65 gatcaagctt ccatcctact gaaaccatgg tc 32 66 35 DNA Homo sapiens 66 gatcagatct cagttccaat attcacacca ccgtc 35 67 22 DNA Homo sapiens 67 ctggtgtgct ccatggcatc cc 22 68 22 DNA Homo sapiens 68 gtaagcctcc cagaacgaga gg 22 69 24 DNA Homo sapiens 69 cagcgcaggg tgaagcctga gagc 24 70 24 DNA Homo sapiens 70 ggcacctgct gtgacctgtg cagg 24 71 22 DNA Homo sapiens 71 gtcctgccac ttcgagacat gg 22 72 23 DNA Homo sapiens 72 gaaacttctc tgcccttacc gtc 23 73 26 DNA Homo sapiens 73 ccaacaccag catccatggc atcaag 26 74 27 DNA Homo sapiens 74 ggagagtcag ctctgaaaga attcagg 27

Claims (76)

What is claimed is:
1. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 1.
2. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 1 comprising SEQ.ID.NO.: 2.
3. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:1.
4. A Host Cell comprising the Plasmid of claim 3.
5. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 3.
6. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 3 comprising SEQ.ID.NO.: 4.
7. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:3.
8. A Host Cell comprising the Plasmid of claim 7.
9. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 5.
10. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 5 comprising SEQ.ID.NO.: 6.
11. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:5.
12. A Host Cell comprising the Plasmid of claim 11.
13. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 7.
14. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 7 comprising SEQ.ID.NO.: 8.
15. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:7.
16. A Host Cell comprising the Plasmid of claim 15.
17. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 9.
18. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 9 comprising SEQ.ID.NO.: 10.
19. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:9.
20. A Host Cell comprising the Plasmid of claim 19.
21. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 11.
22. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 11 comprising SEQ.ID.NO.:12.
23. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:11.
24. A Host Cell comprising the Plasmid of claim 23.
25. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 13.
26. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 13 comprising SEQ.ID.NO.: 14.
27. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.: 13.
28. A Host Cell comprising the Plasmid of claim 27.
29. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 15.
30. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 15 comprising SEQ.ID.NO.: 16.
31. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:15.
32. A Host Cell comprising the Plasmid of claim 31.
33. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 17.
34. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 17 comprising SEQ.ID.NO.: 18.
35. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.: 17.
36. A Host Cell comprising the Plasmid of claim 35.
37. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 19.
38. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 19 comprising SEQ.ID.NO.: 20.
39. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.: 19.
40. A Host Cell comprising the Plasmid of claim 39.
41. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 21.
42. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 21 comprising SEQ.ID.NO.: 22.
43. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:21.
44. A Host Cell comprising the Plasmid of claim 43.
45. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 23.
46. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 23 comprising SEQ.ID.NO.: 24.
47. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.: 23.
48. A Host Cell comprising the Plasmid of claim 47.
49. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 25.
50. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 25 comprising SEQ.ID.NO.: 26.
51. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:25.
52. A Host Cell comprising the Plasmid of claim 51.
53. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 27.
54. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 27 comprising SEQ.ID.NO.: 28.
55. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:27.
56. A Host Cell comprising the Plasmid of claim 55.
57. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 29.
58. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 29 comprising SEQ.ID.NO.: 30.
59. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:29.
60. A Host Cell comprising the Plasmid of claim 59.
61. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 31.
62. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 31 comprising SEQ.ID.NO.: 32.
63. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:31.
64. A Host Cell comprising the Plasmid of claim 63.
65. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 33.
66. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 33 comprising SEQ.ID.NO.: 34.
67. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:33.
68. A Host Cell comprising the Plasmid of claim 67.
69. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 35.
70. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 35 comprising SEQ.ID.NO.: 36.
71. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:35.
72. A Host Cell comprising the Plasmid of claim 71.
73. A cDNA encoding a human G protein-coupled receptor comprising SEQ.ID.NO.: 37.
74. A human G protein-coupled receptor encoded by the cDNA of SEQ.ID.NO.: 37 comprising SEQ.ID.NO.: 38.
75. A Plasmid comprising a Vector and the cDNA of SEQ.ID.NO.:37.
76. A Host Cell comprising the Plasmid of claim 75.
US09/875,076 1998-11-20 2001-06-06 Human orphan G protein-coupled receptors Abandoned US20030017528A1 (en)

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US10921398P 1998-11-20 1998-11-20
US12041699P 1999-02-16 1999-02-16
US12185299P 1999-02-26 1999-02-26
US12394999P 1999-03-12 1999-03-12
US12394699P 1999-03-12 1999-03-12
US13643999P 1999-05-28 1999-05-28
US13643799P 1999-05-28 1999-05-28
US13656799P 1999-05-28 1999-05-28
US13713199P 1999-05-28 1999-05-28
US13712799P 1999-05-28 1999-05-28
US13643699P 1999-05-28 1999-05-28
US14144899P 1999-06-29 1999-06-29
US15633399P 1999-09-28 1999-09-28
US15655599P 1999-09-29 1999-09-29
US15663499P 1999-09-29 1999-09-29
US41704499A 1999-10-12 1999-10-12
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