US20030109676A1 - Human endothelin-bombesin receptor - Google Patents

Human endothelin-bombesin receptor Download PDF

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US20030109676A1
US20030109676A1 US10/292,525 US29252502A US2003109676A1 US 20030109676 A1 US20030109676 A1 US 20030109676A1 US 29252502 A US29252502 A US 29252502A US 2003109676 A1 US2003109676 A1 US 2003109676A1
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polypeptide
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US10/292,525
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Yi Li
Craig Rosen
Chandrika Kumar
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SmithKline Beecham Ltd
Human Genome Sciences Inc
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SmithKline Beecham Ltd
Human Genome Sciences Inc
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Priority to PCT/US1994/011843 priority Critical patent/WO1996011946A1/en
Priority to US08/465,687 priority patent/US5750370A/en
Priority to US09/030,970 priority patent/US6143519A/en
Priority to US09/520,210 priority patent/US6518404B1/en
Application filed by SmithKline Beecham Ltd, Human Genome Sciences Inc filed Critical SmithKline Beecham Ltd
Priority to US10/292,525 priority patent/US20030109676A1/en
Publication of US20030109676A1 publication Critical patent/US20030109676A1/en
Application status is Abandoned legal-status Critical

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72Receptors; Cell surface antigens; Cell surface determinants for hormones
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00Uses of viruses
    • C12N2799/02Uses of viruses as vector
    • C12N2799/021Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Abstract

A human endothelin-bombesin receptor polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for identifying agonists and antagonists to such polypeptide. Agonists to the endothelin-bombesin receptor polypeptide of the present invention may be used to treat asthma, Parkinson's Disease, acute heart failure, hypotension and osteoporosis. Antagonists against such polypeptides may be used therapeutically to treat hypertension, ulcerigenesis, subarachnoid hemorrhage, asthma, tumors, ciclosporin toxicity, cancer and septic shock. Also disclosed are diagnostic methods for detecting mutations in the polynucleotides of the present invention and for detecting levels of the soluble polypeptides in samples derived from a host.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a divisional of application Ser. No. 09/520,210, filed Mar. 7, 2000, which is a divisional of application Ser. No. 09/030,970, filed on Feb. 26, 1998, now U.S. Pat. No. 6,143,519, issued on Nov. 7, 2000, which is a divisional of application Ser. No. 08/465,687, filed on Jun. 6, 1995, now U.S. Pat. No. 5,750,370, issued on May 12, 1998, which is a continuation-in-part of International Application No. PCT/US94/11843, filed Oct. 17, 1994; priority to these applications is hereby claimed under 35 U.S.C. § 120, and each of these applications is hereby incorporated by reference in its entirety.[0001]
  • INTRODUCTION
  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a human 7-transmembrane receptor. The transmembrane receptor is a G-protein coupled receptor. More particularly, the 7-transmembrane receptor has been putatively identified as an endothelin-bombesin receptor, sometimes hereinafter referred to as “ETBR.” The invention also relates to inhibiting the action of such polypeptides. [0002]
  • BACKGROUND OF THE INVENTION
  • It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 351:353-354 (1991)). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins. Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B. K., et al., PNAS, 84:46-50 (1987); Kobilka, B. K., et al., Science, 238:650-656 (1987); Bunzow, J. R., et al., Nature, 336:783-787 (1988)), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M. I., et al., Science, 252:802-8 (1991)). [0003]
  • For example, in one form of signal transduction, the effect of hormone binding is activation of an enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP, and GTP also influences hormone binding. A G-protein connects the hormone receptors to adenylate cyclase. G-protein was shown to exchange GTP for bound GDP when activated by hormone receptors. The GTP-carrying form then binds to an activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal. [0004]
  • The peptide endothelin is a peptide of 21 amino acid residues and performs in vivo effects via endothelin receptors. Endothelin (ET) is a peptide present in various tissues in animals and is known as a strong vasoconstrictor. ET is one peptide of a family of at least 4 mammalian peptides characterized by 2 disulphide bridges and 6 conserved amino acid residues at the C-terminus. [0005]
  • Members of the family are called endothelin-[0006] 1 (ET-1), endothelin-2 (ET-2), and endothelin-3 (ET-3). A fourth peptide, vasointestinal contractor, is also sometimes described as the murine or rat form of ET-2. They differ mostly in the 29-membered ring system formed by the Cys-3-Cys-11 disulphide bond. Endothelins are produced by metabolism of a preproendothelin to a proendothelin, which is itself metabolized to the mature endothelin. The cleavage of proendothelin is thought to be due to the activity of a specific enzyme. ETs are distributed in a wide variety of vascular and non-vascular tissues (PNAS, USA, 86:2863-2867 (1989)).
  • It has previously been shown in vivo that ET-[0007] 1 and ET-2 are much stronger vasoconstrictors than ET-3, whereas the three ET isopeptides are roughly equipotent in producing the transient vasodilation. The analysis of nucleic acid sequences of ETs has revealed that various kinds of ET isopeptides exist. These ET isopeptides are also different in their properties. Therefore, it appears that various sub-types of ET-receptors exist. The existence of various sub-types of ET-receptors has been proven by the radioactive ligand binding studies of Watanabe, H., et al., Biochem-Biophys, Res. Commun., 161:1252-1259 (1989), and Martin, E. R., et al., J. Biol. Chem., 265:14044-14049 (1990). These studies indicate the existence of at least two kinds of ET-receptors. One of them has a higher affinity for ET-1 and ET-2 than for ET-3 and the other has an affinity for ET-1, ET-2 and ET-3 with no cell activity. The ETA receptors have a lower affinity for ET-3 and the ETB receptors are non-selective.
  • The receptors are homologous to other heptahelical receptors of the rhodopsin superfamily, having 7 hydrophobic regions predicted to form transmembrane helices. [0008]
  • The placenta has a very high expression of both receptors, as does the lung. In general the non-selective ET[0009] B receptor seems to be more widely expressed (e.g., in liver, kidney and uterus) and is probably the more prominent receptor in the CNS, a result that agrees with binding and functional studies. The heart is the only tissue about which there is a consensus that an ETA-type receptor predominates. The ETA receptors are associated with blood vessels and ETB receptors with glial, epithelial and ependymal cells, but few, if any, are associated with neurons. In the kidney, ETA receptors are located on blood vessel smooth-muscle cells, and ETB receptor expression occurs on a glomerular endothelium, vasa recti and the thin segments of Henle's loops.
  • Endothelins elicit biological responses by various signal transduction mechanisms, including the G-protein-coupled activation of phospholipase C and the activation of voltage-dependent Ca[0010] 2+ channels (Kasuya, Y., et al., Biochem. Biophys. Res. Commun., 61:1049-1055 (1989)). Thus, different sub-types of the endothelin receptor may use different signal-transduction mechanisms. Endothelin receptors have a relatively long N terminus preceding transmembrane segment I, and this portion may be involved in binding a relatively large endothelin peptide.
  • BRIEF SUMMARY OF THE INVENTION
  • Applicants have discovered a G-protein coupled receptor which has hydropathicity and amino acid homology which shows the existence of the 7 hydrophobic segments and a significant sequence similarity with other G-protein-coupled receptors. The 7 membrane-spanning domains and extra-cellular N-terminus and cytoplasmic C-terminus have also been identified. [0011]
  • The G-protein coupled receptor of the present invention has been putatively identified as an endothelin-bombesin receptor as a result of its homology to the known endothelin receptors ET[0012] A and ETB and as a result of its ability to bind endothelin and bombesin.
  • In accordance with one aspect of the present invention, there is provided a novel putative mature polypeptide which is a G-protein coupled receptor, as well as fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin. [0013]
  • In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides. [0014]
  • In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques. [0015]
  • In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides for therapeutic purposes, to measure the concentration of endothelin in vivo, or in soluble form as an antagonist. [0016]
  • In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides. [0017]
  • In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides. [0018]
  • In accordance with still another embodiment, there is provided a process for using the receptor to screen for receptor antagonists and/or receptor agonists and/or receptor ligands. [0019]
  • These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.[0020]
  • BRIEF DESCRIPTION OF THE FIGURES
  • The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims. [0021]
  • FIGS. [0022] 1A-H show the cDNA sequence and the corresponding deduced amino acid sequence of the G-protein coupled receptor of the present invention. The first 26 amino acids represent a putative signal sequence. The standard one-letter abbreviation for amino acids is used.
  • FIGS. [0023] 2A-B are an illustration of the secondary structural features of the G-protein coupled receptor. The first 7 illustrations set forth the regions of the amino acid sequence which are alpha helices, beta sheets, turn regions or coiled regions. The boxed areas are the areas which correspond to the region indicated. The second set of figures illustrate areas of the amino acid sequence which are exposed to intracellular, cytoplasmic or are membrane-spanning. The hydrophilicity part illustrates areas of the protein sequence which are the lipid bilayer of the membrane and are, therefore, hydrophobic, and areas outside the lipid bilayer membrane which are hydrophilic. The antigenic index corresponds to the hydrophilicity plot, since antigenic areas are areas outside the lipid bilayer membrane and are capable of binding antigens. The surface probability plot further corresponds to the antigenic index and the hydrophilicity plot. The amphipathic plots show those regions of the 13 sequences which are polar and non-polar. The flexible regions correspond to the second set of illustrations in the sense that flexible regions are those which are outside the membrane and inflexible regions are transmembrane regions.
  • FIGS. [0024] 3A-J, collectively, illustrate an amino acid alignment of the G-protein coupled receptor of the present invention and endothelin receptors from various species of animals. Faded areas are those areas which match with the other amino acid sequences in the figures. The comparative polypeptide sequences are represented by one-letter amino acid codes and each comparative row has six sets of lines representing the six amino acid sequences SEQ ID NO:2 and SEQ ID NOS:9-13, respectively.
  • FIG. 4 shows that ET[0025] 1, ET3 and Bombesin induced chloride currents in oocytes injected with pHHPEC49 derived RNA transcripts. The trace shows ET1 mediated chloride current (nanoamps). Arrow indicates ET1 addition. The inset shows the mean peak responses to 10 nM AII, Neuropeptide Y (NPY) and Bradykinin. The mean peak ±S.E. peak current response to ET1 is 150±50 (n=75), ET2 156±55 (n=75) and Bombesin 148±47 (n=75).
  • It should be pointed out that sequencing inaccuracies are a common problem which occurs in polynucleotide sequences. Accordingly, the sequence of the drawing is based on several sequencing runs and the sequencing accuracy is considered to be at least 97%. [0026]
  • DETAILED DESCRIPTION OF THE INVENTION
  • In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of FIGS. [0027] 1A-H (SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone deposited with the American Type Culture Collection (“ATCC”) on Jun. 24, 1994, and assigned ATCC Deposit No. 75823. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209.
  • A polynucleotide encoding a polypeptide of the present invention may be found in brain, liver and placenta. The polynucleotide of this invention was discovered in a cDNA library derived from a human brain. It is structurally related to the G protein-coupled receptor family. It contains an open reading frame encoding a protein of about 613 amino acid residues of which approximately the first 26 amino acids residues are the putative leader sequence such that the mature protein comprises 587 amino acids. The protein exhibits the highest degree of homology to a human ET[0028] A receptor with 30% identity and 55% similarity over a 420 amino acid stretch.
  • The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in FIGS. [0029] 1A-H (SEQ ID NO:1) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of FIGS. 1A-H (SEQ ID NO:1) or the deposited CDNA.
  • The polynucleotide which encodes for the mature polypeptide of FIGS. [0030] 1A-H (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.
  • Thus, the term “polynucleotide encoding a polypeptide” encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence. [0031]
  • The present invention further relates to variants of the hereinabove described polynucleotides which encoded for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of FIGS. [0032] 1A-H (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in FIGS. [0033] 1A-H (SEQ ID NO:2) or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of FIGS. 1A-H (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in FIGS. [0034] 1A-H (SEQ ID NO:1) or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternative form of a polynucleotide sequence which may have a substitution, deletion, or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein plus additional 5′ amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains. [0035]
  • Thus, for example, the polynucleotide of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence). [0036]
  • The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-[0037] 9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
  • The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). [0038]
  • Fragments of the full length gene of the present invention may be used as a hybridization probe for a CDNA library to isolate the full length CDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human CDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to. [0039]
  • The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term “stringent conditions” means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides with either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of FIGS. [0040] 1A-H (SEQ ID NO:1) or the deposited cDNA(s).
  • Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:[0041] 1, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
  • Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:[0042] 2 as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.
  • The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. § 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted. [0043]
  • The present invention further relates to a G-protein coupled receptor polypeptide which has the deduced amino acid sequence of FIGS. [0044] 1A-H (SEQ ID NO:2) or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
  • The terms “fragment,” “derivative” and “analog” when referring to the polypeptide of FIGS. [0045] 1A-H (SEQ ID NO:2) or that encoded by the deposited cDNA, means a polypeptide which either retains substantially the same biological function or activity as such polypeptide, i.e., functions as a G-protein coupled receptor, or retains the ability to bind the ligand or the receptor even though the polypeptide does not function as a G-protein coupled receptor, for example, a soluble form of the receptor. An analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide. [0046]
  • The fragment, derivative or analog of the polypeptide of FIGS. [0047] 1A-H (SEQ ID NO:2) or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide which are employed for purification of the mature polypeptide or a proprotein sequence or (v) one in which a fragment of the polypeptide is soluble, i.e., not membrane bound, yet still binds ligands to the membrane bound receptor. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
  • The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. [0048]
  • The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. [0049]
  • The polypeptides of the present invention include the polypeptide of SEQ ID NO:[0050] 2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
  • As known in the art “similarity” between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. [0051]
  • Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention. [0052]
  • The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. [0053]
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the ETBR genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. [0054]
  • The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV[0055] 40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.
  • The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art. [0056]
  • The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV[0057] 40 promoter, the E. coli. lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.
  • In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in [0058] E. coli.
  • The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. [0059]
  • As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as [0060] E. coli, Strentomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9 ; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE[0061] 70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK[0062] 232-8 and PCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)). [0063]
  • The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers. [0064]
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference. [0065]
  • Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV[0066] 40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of [0067] E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include [0068] E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
  • As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR[0069] 322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. [0070]
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. [0071]
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art. [0072]
  • Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-[0073] 7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • The G-protein coupled receptor polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing, configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. [0074]
  • The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue. [0075]
  • Bombesin, in addition to endothelin, has been found to bind to and stimulate the receptor of the present invention. Bombesin is a tetradecapeptide which has as a mammalian homolog the 27-amino acid peptide gastrin-releasing peptide (GRP). Bombesin is regarded as one of the most potent peptide to affect the central nervous system, since it has been reported as a thermoregulator in the rat (Brown, M. et al., Science, 196:998-1000 (1977)). Also, bombesin/gastrin releasing peptide is synthesized and secreted by small cell lung cancers (Davis, T. P. et al., Peptides, 13:401-17 (1992)). [0076]
  • The G-protein coupled receptor of the present invention may be employed in a process for screening for agonists and/or antagonists for the receptor. [0077]
  • In general, such screening procedures involve providing appropriate cells which express the receptor on the surface thereof. In particular, a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby express the G-protein coupled receptor. Such transfection may be accomplished by procedures as hereinabove described. [0078]
  • One such screening procedure involves the use of a melanophore which are transfected to express the G-protein coupled receptor of the present invention. Such a screening technique is described in PCT WO 92/01810 published Feb. 6, 1992. [0079]
  • Thus, for example, such assay may be employed for screening for a receptor antagonist by contacting the melanophore cells which encode the G-protein coupled receptor with both the receptor ligand and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor. [0080]
  • The screen may be employed for determining an agonist by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the receptor. [0081]
  • Other screening techniques include the use of cells which express the G-protein coupled receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, volume 246, pages 181-296 (October 1989). For example, potential agonists or antagonists may be contacted with a cell which expresses the G-protein coupled receptor and a second messenger response, e.g. signal transduction or pH changes, may be measured to determine whether the potential agonist or antagonist is effective. [0082]
  • Another such screening technique involves introducing RNA encoding the G-protein coupled receptor into Xenopus oocytes to transiently express the receptor. The receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition of a calcium signal. [0083]
  • Another screening technique involves expressing the G-protein coupled receptor in which the receptor is linked to a phospholipase C or D. As representative examples of such cells, there may be mentioned endothelial cells, smooth muscle cells, embryonic kidney cells, etc. The screening for an antagonist or agonist may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal. [0084]
  • Another method involves screening for antagonists by determining inhibition of binding of labeled ligand to cells which have the receptor on the surface thereof. Such a method involves transfecting a eukaryotic cell with DNA encoding the G-protein coupled receptor such that the cell expresses the receptor on its surface and contacting the cell with a potential antagonist in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the potential antagonist binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited. [0085]
  • In general, antagonists for G-protein coupled receptors which are determined by such screening procedures may be employed for a variety of therapeutic purposes. For example, such antagonists have been employed for treatment of hypertension, angina pectoris, myocardial infarction, ulcers, asthma, allergies, psychoses, depression, migraine, vomiting, and benign prostatic hypertrophy. [0086]
  • Agonists for G-protein coupled receptors are also useful for therapeutic purposes, such as the treatment of asthma, Parkinson's disease, acute heart failure, hypotension, urinary retention, and osteoporosis. [0087]
  • Examples of potential antagonists include an antibody, or in some cases an oligonucleotide, which binds to the G-protein coupled receptor but does not elicit a second messenger response such that the activity of the G-protein coupled receptor is prevented. Potential antagonists also include proteins which are closely related to the ligand of the G-protein coupled receptor, i.e. a fragment of the ligand, which have lost biological function and when binding to the G-protein coupled receptor, elicit no response. [0088]
  • A potential antagonist also includes an antisense construct prepared through the use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251:1360 (1991)), thereby preventing transcription and the production of G-protein coupled receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the G-protein coupled receptor (antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of G-protein coupled receptor. [0089]
  • Another potential antagonist is a small molecule which binds to the G-protein coupled receptor, making it inaccessible to ligands such that normal biological activity is prevented. Examples of small molecules include but are not limited to small peptides or peptide-like molecules. [0090]
  • Potential antagonists also include a soluble G-protein coupled receptor, e.g. a fragment of the receptor, which binds to the ligand and prevents the ligand from interacting with membrane bound G-protein coupled receptors. [0091]
  • An endothelin antagonist may be employed to offset the vasoconstrictive effects of endothelin and, therefore, may be employed to treat hypertension through vasodilation. These antagonists may also be used to treat the long-lasting vasospasms due to subarachnoid hemorrhages which cause increases in endothelin levels in cerebrospinal fluid and plasma. [0092]
  • Endothelin antagonists may also be employed to treat ulcerogenesis and gastric lesions. ET-[0093] 1 and ET-3 induce gastric lesions and enhance alcohol-induced lesions. Accordingly, inhibiting ET-1 and ET-3 from interacting with the ETBRs can prevent these conditions.
  • Endothelins potently contract pulmonary smooth muscle and levels of endothelins are increased in pulmonary lavage fluid during asthmatic attacks, therefore, antagonists for diminishing or preventing binding of endothelin may be employed to treat asthma. [0094]
  • Endothelin levels are increased in cancer tissue and a cancer-derived cell line can be stimulated to produce endothelin. ET-[0095] 1 itself stimulates growth of cancerous cells. Accordingly, endothelin antagonists may be employed to prevent the growth of cancer cells and tumors.
  • An increase in circulating endothelin levels is increased by ciclosporin, which may explain the toxic effects of ciclosporin. Accordingly, endothelin antagonists may be used to prevent and/or treat ciclosporin toxicity. [0096]
  • Endothelin antagonists may also be employed to treat septic shock which is caused by pathological levels of endothelins. Further, hypertension, congestive heart failure, coronary artery disease, atherosclerosis, restenosis, benign prostatic hypertrophy, renal failure and stroke may also be treated with the antagonist of ETBRs. [0097]
  • Bombesin antagonists may be employed to treat small cell lung cancers which synthesize and secret bombesin/gastrin releasing peptide. A bombesin antagonist will prevent bombesin from stimulating the ETBR of the present invention. The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described. [0098]
  • The present invention also provides a method for determining whether a ligand not known to be capable of binding to a G-protein coupled receptor can bind to such receptor which comprises contacting a mammalian cell which expresses a G-protein coupled receptor with the ligand under conditions permitting binding of ligands to the G-protein coupled receptor, detecting the presence of a ligand which binds to the receptor and thereby determining whether the ligand binds to the G-protein coupled receptor. The systems hereinabove described for determining agonists and/or antagonists may also be employed for determining ligands which bind to the receptor. [0099]
  • The ETBR polypeptides and antagonists or agonists may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. [0100]
  • The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptides and agonists and antagonists may be employed in conjunction with other therapeutic compounds. [0101]
  • The pharmaceutical compositions may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, the pharmaceutical compositions will be administered in an amount of at least about 10 μg/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc. [0102]
  • The ETBR polypeptides and antagonists or agonists which are polypeptides, may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as “gene therapy.”[0103]
  • Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention. [0104]
  • Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle. [0105]
  • Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus. [0106]
  • The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV[0107] 40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and b-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoA[0108] 1 promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the genes encoding the polypeptides.
  • The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE[0109] 501, PA317, y-2, y-AM, PA12, T19-14X, VT-19-17-H2, yCRE, yCRIP, GP+E−86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
  • The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells. [0110]
  • The present invention also provides a method for determining whether a ligand not known to be capable of binding to a G-protein coupled receptor can bind to such receptor which comprises contacting a mammalian cell which expresses a G-protein coupled receptor with the ligand under conditions permitting binding of ligands to the G-protein coupled receptor, detecting the presence of a ligand which binds to the receptor and thereby determining whether the ligand binds to the G-protein coupled receptor. The systems hereinabove described for determining agonists and/or antagonists may also be employed for determining ligands which bind to the receptor. [0111]
  • This invention also provides a method of detecting expression of a receptor polypeptide of the present invention on the surface of a cell by detecting the presence of mRNA coding for the receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 10 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the receptor by the cell. [0112]
  • The present invention also provides a method for identifying receptors related to the receptor polypeptides of the present invention. These related receptors may be identified by homology to a receptor polypeptide of the present invention, by low stringency cross hybridization, or by identifying receptors that interact with related natural or synthetic ligands and or elicit similar behaviors after genetic or pharmacological blockade of the receptor polypeptides of the present invention. [0113]
  • The present invention also contemplates the use of the genes of the present invention as a diagnostic, for example, some diseases result from inherited defective genes. These genes can be detected by comparing the sequences of the defective gene with that of a normal one. Subsequently, one can verify that a “mutant” gene is associated with abnormal receptor activity. In addition, one can insert mutant receptor genes into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, expression on MacConkey plates, complementation experiments, in a receptor deficient strain of HEK[0114] 293 cells) as yet another means to verify or identify mutations. Once “mutant” genes have been identified, one can then screen population for carriers of the “mutant” receptor gene.
  • Individuals carrying mutations in the gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis may be obtained from a patient's cells, including but not limited to such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki, et al., [0115] Nature, 324:163-166 1986) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complimentary to the nucleic acid of the instant invention can be used to identify and analyze mutations in the gene of the present invention. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radio labeled RNA of the invention or alternatively, radio labeled antisense DNA sequences of the invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Such a diagnostic would be particularly useful for prenatal or even neonatal testing. Sequence differences between the reference gene and “mutants” may be revealed by the direct DNA sequencing method. In addition, cloned DNA segments may be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequence primer is used with double stranded PCR product or a single stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radio labeled nucleotide or by an automatic sequencing procedure with fluorescent-tags.
  • Genetic testing based on DNA sequence differences may be achieved by detection of alterations in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Sequences changes at specific locations may also be revealed by nucleus protection assays, such RNase and S[0116] 1 protection or the chemical cleavage method (e.g. Cotton, et al., PNAS, USA, 85:4397-4401 1985). Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
  • Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease. [0117]
  • With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb). [0118]
  • The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments. [0119]
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide. [0120]
  • For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). [0121]
  • Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention. [0122]
  • The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight. [0123]
  • In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described. [0124]
  • “Plasmids” are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan. [0125]
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 microgram of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 microliter of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 micrograms of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment. [0126]
  • Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980). [0127]
  • “Oligonucleotides” refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5′ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated. [0128]
  • “Ligation” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T[0129] 4 DNA ligase (“ligase”) per 0.5 micrograms of approximately equimolar amounts of the DNA fragments to be ligated.
  • Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973). [0130]
  • Example 1
  • Bacterial Expression and Purification of ETBR [0131]
  • The DNA sequence encoding for ETBR, ATCC #75823, is initially amplified using PCR oligonucleotide primers corresponding to the 5′ and sequences of the processed ETBR protein (minus the signal peptide sequence) and the vector sequences 3′ to the ETBR gene. Additional nucleotides corresponding to ETBR were added to the 5′ and 3′ sequences respectively. The 5′ oligonucleotide primer has the sequence 5′ CACTAAGCTTAATGCGAGCCCCGGGCGCG 3′ (SEQ ID NO:[0132] 3) contains a HindIII restriction enzyme site followed by 18 nucleotides of ETBR coding sequence starting from the presumed terminal amino acid of the processed protein codon. The 3′ sequence 5′ GAACTTCTAGACCGTCAGCAATGAGTACCGAC 3′ (SEQ ID NO:4) contains complementary sequences to an XbaI site and is followed by 18 nucleotides of ETBR. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311). pQE-9 encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 was then digested with HindIII and XbaI. The amplified sequences were ligated into pQE-9 and were inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture was then used to transform E. coli HB101 by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired constructs were grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a final concentration of 1 mM. WYfG induces by inactivating the lacd repressor, clearing the P/O leading to increased gene expression. Cells were grown an extra 3 to 4 hours. Cells were then harvested by centrifugation. The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, solubilized ETBR was purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)). ETBR was eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.
  • Example 2
  • Expression of Recombinant ETBR in COS cells [0133]
  • The expression of plasmid, pETBR HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV[0134] 40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire ETBR protein and a HA tag fused in frame to its 3′ end was cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • The plasmid construction strategy is described as follows: [0135]
  • The DNA sequence encoding for ETBR, ATCC #75823, was constructed by PCR on the original EST cloned using two primers: the 5′ primer 5′ GTCCAAGCTTGCCACCATGCGAGCCCCGGGCGCG 3′ (SEQ ID NO:[0136] 5) contains a HindIII site followed by 18 nucleotides of ETBR coding sequence starting from the initiation codon; the 3′ sequence 5′ CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCAGCAATGAGTTC CGACAGA 3′ (SEQ ID NO:6) contains complementary sequences to an XhoI site, translation stop codon, HA tag and the last 18 nucleotides of the ETBR coding sequence (not including the stop codon). Therefore, the PCR product contains a HindIII site, ETBR coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an XhoI site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digested with HindIII and XhoI restriction enzyme and ligated. The ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037) the transformed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and examined by restriction analysis for the presence of the correct fragment. For expression of the recombinant ETBR, COS cells were transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the ETBR HA protein was detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with 35S-cysteine two days post transfection. Culture media were then collected and cells were lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media were precipitated with a HA specific monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.
  • Example 3
  • Cloning and expression of ETBR using the baculovirus expression system [0137]
  • The DNA sequence encoding the full length ETBR protein, ATCC #75823, amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ sequences of the gene: [0138]
  • The 5′ primer has the sequence 5′CGGGATCCGCCACC[0139] ATGCGAGCCCCGGGCGCG 3′ (SEQ ID NO:7) and contains a BamHI restriction enzyme site (in bold) followed by 6 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (J. Mol. Biol. 1987, 196, 947-950, Kozak, M.), and just behind, is the first 18 nucleotides of the ETBR gene (the initiation codon for translation “ATG” is underlined).
  • The 3′ primer has the sequence 5′CGGGATCCCGCTCAGCAATGAGTTCCGAC 3′ (SEQ ID NO:[0140] 8) and contains the cleavage site for the restriction endonuclease BamHI and 18 nucleotides complementary to the 3′ non-translated sequence of the ETBR gene. The amplified sequences were isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment was then digested with the endonucleases BamHI and then purified again on a 1% agarose gel. This fragment is designated F2.
  • The vector pRG[0141] 1 (modification of pVL941 vector, discussed below) is used for the expression of the ETBR protein using the baculovirus expression system (for review see: Summers, M. D. and Smith, G. E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction endonucleases Bamfll. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant viruses the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of cotransfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology, 170:31-39).
  • The plasmid was digested with the restriction enzymes BamHI and then dephosphorylated using calf intestinal phosphatase by procedures known in the air. The DNA was then isolated from a 1% agarose gel. This vector DNA is designated V[0142] 2.
  • Fragment F[0143] 2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E.coli HB101 cells were then transformed and bacteria identified that contained the plasmid (pBacETBR) with the ETBR gene using the enzymes BamHI. The sequence of the cloned fragment was confirmed by DNA sequencing.
  • 5 μg of the plasmid pBacETBR were cotransfected with 1.0 μg of a commercially available linearized baculovirus (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.) using the lipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)). [0144]
  • 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacETBR were mixed in a sterile well of a microtiter plate containing 50 μl of serum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture was added dropwise to the Sf[0145] 9 insect cells (ATCC CRL 171 1) seeded in a 35 mm tissue culture plate with 1 ml Grace' medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate was then incubated for 5 hours at 27° C. After 5 hours the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum was added. The plate was put back into an incubator and cultivation continued at 27° C. for four days.
  • After four days the supernatant was collected and a plaque assay performed similar as described by Summers and Smith (supra). As a modification an agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) was used which allows an easy isolation of blue stained plaques. (A detailed description of a “plaque assay” can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10). [0146]
  • Four days after the serial dilution, the viruses were added to the cells and blue stained plaques were picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculoviruses was used to infect Sf[0147] 9 cells seeded in 35 mm dishes. Four days later the supematants of these culture dishes were harvested and then stored at 4° C.
  • Sf[0148] 9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells were infected with the recombinant baculovirus V-ETBR at a multiplicity of infection (MOI) of 2. Six hours later the medium was removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 μCi of 35S-methionine and 5 μCi 35S cysteine (Amersham) were added. The cells were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.
  • Example 4
  • Xenopus Oocyte Assay to Identify Ligand [0149]
  • RNA was synthesized in vitro from linearized DNA, ATCC #75823, using an RNA transcription kit. This RNA was microinjected into Xenopus oocytes (10 ng of RNA/oocytes). The oocytes were manually defolliculated prior to microinjection to remove any endogenous receptors that might be present in the follicular membranes. The injected oocytes were maintained in modified Barth's medium at 18° C. for 48 hours to allow for receptor protein expression. Electrophysiology was performed using the voltage-clamp technique. Oocytes were clamped at −60 mV and the calcium activated chloride channel activity was recorded in Barth's medium at room temperature. Data were analyzed using Axotape software. [0150]
  • As shown in FIG. 4, oocytes injected with the synthetic RNA complementary to DNA from ATCC #75823, illicited fairly strong Cl[0151] currents upon addition of 10 nM ET1, ET3 as well as Bombesin. Addition of ET1, ET3 and Bombesin to uninjected oocytes on the other hand did not elicit any change in membrane potential (data not shown). The ET1 and ET3 mediated response was blocked by the ET receptor peptide antagonist BQ123. Addition of related peptide ligands like AII, Neuropeptide Y and Bradykinin did not illicit any response (FIG. 4). This indicates that the ETBR is functional and is capable of coupling to a second messenger system which leads to the mobilization of intercellular stores of calcium via production of inositol triphosphate. Since it responds to both ET and Bombesin it represents a novel endothelin-bombesin receptor.
  • Example 5
  • Expression via Gene Therapy [0152]
  • Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F[0153] 12 media, with 10% FBS, penicillin and streptomycin, is added. This is then incubated at 37° C. for approximately one week. At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
  • pMV-[0154] 7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
  • The CDNA encoding a polypeptide of the present invention is amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively. The 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T[0155] 4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is used to transform bacteria HB101, which are then plated onto agar-containing kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
  • The amphotropic pA[0156] 317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).
  • Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as [0157] neo or his.
  • The engineered fibroblasts are then injected into the-host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. [0158]
  • Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described. [0159]
  • 1 13 1 4156 DNA Human CDS (1779)..(3620) 1 cccactatgt tggccaggat ggtcttgatt tcttgacctc gtgttctgcc cgcctctacc 60 tcccaaagtg ccgggattac aggcgtgact gctgtgcccg gccccagcat cacttttata 120 gctttctgtg cctcttcctc tgggccttgg tgtatgaagc cacttgcctt tctctgttgg 180 gaagcgagca gaatcagatt gctactcatg atgcagtccg ggcagggcat actgtcacct 240 ttggctgtgg acacagttgt caggataggg gagaagccct ttaggtccgt cttcttgaca 300 cagccctcct acctggttac gctggtgctt tcgcttggtt tagacaacca agacacttga 360 gaattatgct gtcctcagaa tgtctgatga aaagaacaga ttcacttttt ggacacaatg 420 cccattagcc atctttggca gtgtttctga tcaaaggttc cccatgcctg ctctaggaaa 480 gtaaactttt ttcagaataa atcctcaaat ggattactga gtagtctttg caccattccc 540 atcagcctaa tcagactgaa tggtcacgct cagtgcaaaa agctgttttg ctgttaggat 600 gtttcagtgt ttcttgtctt tcctggaaca gttcagttgt ttaaatttag taattcaatc 660 ctgaccagtg taaacccact taattattgc agcctaaaga attcagctac ttctactctt 720 cataaatgtg cccaagtaaa tatgtgtttt taatattcaa ccctggaaaa ttagtaattc 780 agatgataaa agctcatgtt ttggtgtctt tgtactcaga ttgtgaacag gcatatttca 840 ctgatttaga cttagtatac ttgatgagaa tgctcaggtt gaagagatag ttctgtcagc 900 aatccaacat ctatagcaat gtggaaaaag taatcaactc atatttcacg aatttgatgt 960 atgttgtgat ttagagggca tgagataaag tttatatttg aactgtgtgg ggtaggggga 1020 agaagaggtt gcttaagcaa atgggggggt gattgaggaa caagatgtct ctaagatgag 1080 aagttatttt cttgcatcat agaagcactc tctccacccg ggagtgattg tgttaactat 1140 aaatcattta tatctgtaca ttaaagcaga ttccctcaat taggcaaatt tggttagcca 1200 agcccaagtt attgtttgta cttgaaagta ataaagctgc atttccttaa aaatatattc 1260 tgtagttaag actttgtctt gctttccgga attcctgttt ttcttttcct ctagagacct 1320 cggcttgcaa ctggatcaaa cgctgtcgaa aggatgtaaa taggcagagc aactgttacc 1380 aagaaggcca ccacccccac ccaaaggcag tgaggagtgt ggggcttcgt ctgggctccc 1440 ccgagtctca acagtaatca acagtcaggt gttgattgca acttttcaag gtcagccacc 1500 gggagtagcc tattccctct aggaaccttg gagggcatac cttgctggga ctcaacttgg 1560 ctgagaaatg cacaagatgc caaaggagga aggattatag ggggcgtgtg tgtgaccccc 1620 aagaccgatc ttccgctatc accctaatct ccggttcccc gctacccggg cgggggtgag 1680 tatgtgacat gtgcctaact ctcagcagca acttcggcag caggtgtcga tcctaactaa 1740 gcaggagctg cggctgccgg gtgtgccctc accaagcc atg cga gcc ccg ggc gcg 1796 Met Arg Ala Pro Gly Ala 1 5 ctt ctc gcc cgc atg tcg cgg cta ctg ctt ctg cta ctg ctc aag gtg 1844 Leu Leu Ala Arg Met Ser Arg Leu Leu Leu Leu Leu Leu Leu Lys Val 10 15 20 tct gcc tct tct gcc ctc ggg gtc gcc cct gcg tcc aga aac gaa act 1892 Ser Ala Ser Ser Ala Leu Gly Val Ala Pro Ala Ser Arg Asn Glu Thr 25 30 35 tgt ctg ggg gag agc tgt gca cct aca gtg atc cag cgc cgc ggc agg 1940 Cys Leu Gly Glu Ser Cys Ala Pro Thr Val Ile Gln Arg Arg Gly Arg 40 45 50 gac gcc tgg gga ccg gga aat tct gca aga gac gtt ctg cga gcc cga 1988 Asp Ala Trp Gly Pro Gly Asn Ser Ala Arg Asp Val Leu Arg Ala Arg 55 60 65 70 gca ccc agg gag gag cag ggg gca gcg ttt ctt gcg gga ccc tcc tgg 2036 Ala Pro Arg Glu Glu Gln Gly Ala Ala Phe Leu Ala Gly Pro Ser Trp 75 80 85 gac ctg ccg gcg gcc ccg gac cgt gac ccg gct gca ggc aga ggg gcg 2084 Asp Leu Pro Ala Ala Pro Asp Arg Asp Pro Ala Ala Gly Arg Gly Ala 90 95 100 gag gcg tcg aca gcc gga ccc ccg gga cct cca acc agg cca cct gtc 2132 Glu Ala Ser Thr Ala Gly Pro Pro Gly Pro Pro Thr Arg Pro Pro Val 105 110 115 ccc tgg agg tgg aaa ggt gct cgg ggt cag gag cct tct gaa act ttg 2180 Pro Trp Arg Trp Lys Gly Ala Arg Gly Gln Glu Pro Ser Glu Thr Leu 120 125 130 ggg aga ggg aac ccc acg gcc ctc cag ctc ttc ctt cag atc tca gag 2228 Gly Arg Gly Asn Pro Thr Ala Leu Gln Leu Phe Leu Gln Ile Ser Glu 135 140 145 150 gag gaa gag aag ggt ccc aga ggc gct gtc att tcc ggg cgt agc cag 2276 Glu Glu Glu Lys Gly Pro Arg Gly Ala Val Ile Ser Gly Arg Ser Gln 155 160 165 gag cag agt gtg aag aca gtc ccc gga gcc agc gat ctt ttt tac tgt 2324 Glu Gln Ser Val Lys Thr Val Pro Gly Ala Ser Asp Leu Phe Tyr Cys 170 175 180 cca agg aga gcc ggg aaa ctc cag ggt tcc cac cac aag ccc ctg tcc 2372 Pro Arg Arg Ala Gly Lys Leu Gln Gly Ser His His Lys Pro Leu Ser 185 190 195 aag acg gcc aat gga ctg gcg ggg cac gaa ggg tgg aca att gca ctc 2420 Lys Thr Ala Asn Gly Leu Ala Gly His Glu Gly Trp Thr Ile Ala Leu 200 205 210 ccg ggc cgg gcg ctg gcc cag aat gga tcc ttg ggt gaa gga atc cat 2468 Pro Gly Arg Ala Leu Ala Gln Asn Gly Ser Leu Gly Glu Gly Ile His 215 220 225 230 gat cct ggg ggt ccc cgc cgg gga aac agc acg aac cgg cgt gtg aga 2516 Asp Pro Gly Gly Pro Arg Arg Gly Asn Ser Thr Asn Arg Arg Val Arg 235 240 245 ctg aag aac ccc ttc tac ccg ctg acc cag gag tcc tat gga gcc tac 2564 Leu Lys Asn Pro Phe Tyr Pro Leu Thr Gln Glu Ser Tyr Gly Ala Tyr 250 255 260 gcg gtc atg tgt ctg tcc gtg gtg atc ttc ggg acc ggc atc att ggc 2612 Ala Val Met Cys Leu Ser Val Val Ile Phe Gly Thr Gly Ile Ile Gly 265 270 275 aac ctg gcg gtg atg tgc atc gtg tgc cac aac tac tac atg cgg agc 2660 Asn Leu Ala Val Met Cys Ile Val Cys His Asn Tyr Tyr Met Arg Ser 280 285 290 atc tcc aac tcc ctc ttg gcc aac ctg gtc ttc tgg gac ttt ctc atc 2708 Ile Ser Asn Ser Leu Leu Ala Asn Leu Val Phe Trp Asp Phe Leu Ile 295 300 305 310 atc ttc ttc tgc ctt ccg ctg gtc atc ttc cac gag ctg acc aag aag 2756 Ile Phe Phe Cys Leu Pro Leu Val Ile Phe His Glu Leu Thr Lys Lys 315 320 325 tgg ctg gtg gag gac ttc tcc tgc aag atc gtg ccc tat ata gag gtc 2804 Trp Leu Val Glu Asp Phe Ser Cys Lys Ile Val Pro Tyr Ile Glu Val 330 335 340 gct tct ctg gga gtc acc act ttc acc tta tgt gct ctg tgc ata gac 2852 Ala Ser Leu Gly Val Thr Thr Phe Thr Leu Cys Ala Leu Cys Ile Asp 345 350 355 cgc ttc cgt gct gcc acc aac gta cag atg tac tac gaa atg atc gaa 2900 Arg Phe Arg Ala Ala Thr Asn Val Gln Met Tyr Tyr Glu Met Ile Glu 360 365 370 aac tgt tcc tca aca act gcc aaa ctt gct gtt ata tgg gtg gga gct 2948 Asn Cys Ser Ser Thr Thr Ala Lys Leu Ala Val Ile Trp Val Gly Ala 375 380 385 390 cta ttg tta gca ctt cca gaa gtt gtt ctc cgc cag ctg agc aag gag 2996 Leu Leu Leu Ala Leu Pro Glu Val Val Leu Arg Gln Leu Ser Lys Glu 395 400 405 gat ttg ggg ttt agt ggc cga gct ccg gca gaa agg tgc att att aag 3044 Asp Leu Gly Phe Ser Gly Arg Ala Pro Ala Glu Arg Cys Ile Ile Lys 410 415 420 atc tct cct gat tta cca gac acc atc tat gtt cta gcc ctc acc tac 3092 Ile Ser Pro Asp Leu Pro Asp Thr Ile Tyr Val Leu Ala Leu Thr Tyr 425 430 435 gac agt gcg aga ctg tgg tgg tat ttt ggc tgt tac ttt tgt ttg ccc 3140 Asp Ser Ala Arg Leu Trp Trp Tyr Phe Gly Cys Tyr Phe Cys Leu Pro 440 445 450 acg ctt ttc acc atc acc tgc tct cta gtg act gcg agg aaa atc cgc 3188 Thr Leu Phe Thr Ile Thr Cys Ser Leu Val Thr Ala Arg Lys Ile Arg 455 460 465 470 aaa gca gag aaa gcc tgt acc cga ggg aat aaa cgg cag att caa cta 3236 Lys Ala Glu Lys Ala Cys Thr Arg Gly Asn Lys Arg Gln Ile Gln Leu 475 480 485 gag agt cag atg aac tgt aca gta gtg gca ctg acc att tta tat gga 3284 Glu Ser Gln Met Asn Cys Thr Val Val Ala Leu Thr Ile Leu Tyr Gly 490 495 500 ttg ggc att att cct gaa aat atc tgc aac att gtt act gcc tac atg 3332 Leu Gly Ile Ile Pro Glu Asn Ile Cys Asn Ile Val Thr Ala Tyr Met 505 510 515 gct aca ggg gtt tca cag cag aca atg gac ctc ctt aat atc atc agc 3380 Ala Thr Gly Val Ser Gln Gln Thr Met Asp Leu Leu Asn Ile Ile Ser 520 525 530 cag ttc ctt ttg ttc ttt aag tcc tgt gtc acc cca gtc ctc ctt ttc 3428 Gln Phe Leu Leu Phe Phe Lys Ser Cys Val Thr Pro Val Leu Leu Phe 535 540 545 550 tgt ctc tgc aaa ccc ttc agt cgg gcc ttc atg gag tgc tgc tgc tgt 3476 Cys Leu Cys Lys Pro Phe Ser Arg Ala Phe Met Glu Cys Cys Cys Cys 555 560 565 tgc tgt gag gaa tgc att cag aag tct tca acg gtg acc agt gat gac 3524 Cys Cys Glu Glu Cys Ile Gln Lys Ser Ser Thr Val Thr Ser Asp Asp 570 575 580 aat gac aac gag tac acc acg gaa ctc gaa ctc tcg cct ttc agt gcc 3572 Asn Asp Asn Glu Tyr Thr Thr Glu Leu Glu Leu Ser Pro Phe Ser Ala 585 590 595 ata cgc cgt gaa atg tcc act ttt gct tct gtc gga act cat tgc tga 3620 Ile Arg Arg Glu Met Ser Thr Phe Ala Ser Val Gly Thr His Cys 600 605 610 aggacagtac ttggttgggt cagatttatt tgtttgattt tcatatcccg tgaaagtttt 3680 taattcatat ttttccttat agggaaaaat gcaaaaaaga aacaataaag aaagaaatat 3740 taactactgt agaactgatt ttacaaatta atatttgtgc tttgaaaaaa agtttctatt 3800 tagttattta agaagaatga gaaggccaat agttttagat tattttatct ggtatggtgc 3860 taatatttta tttgaaaaaa gttactgcaa cttaacttaa aattgctaac gttttttctt 3920 cttttaaaaa tacaattatt gtatattaat tatagcaatg tgattttgta ggttatttta 3980 tatttgagtt gtgattgaaa gtatgttgta tatggtattg tgagatgatt tgtacttgga 4040 agcattcaca aagtagcacc aaataaatta cactttattc tttaatgtca ttgtcaatct 4100 acttttaacc aatattcaat aaatcttcta attgccttaa aaaaaaaaaa aaaaaa 4156 2 613 PRT Human 2 Met Arg Ala Pro Gly Ala Leu Leu Ala Arg Met Ser Arg Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Lys Val Ser Ala Ser Ser Ala Leu Gly Val Ala Pro 20 25 30 Ala Ser Arg Asn Glu Thr Cys Leu Gly Glu Ser Cys Ala Pro Thr Val 35 40 45 Ile Gln Arg Arg Gly Arg Asp Ala Trp Gly Pro Gly Asn Ser Ala Arg 50 55 60 Asp Val Leu Arg Ala Arg Ala Pro Arg Glu Glu Gln Gly Ala Ala Phe 65 70 75 80 Leu Ala Gly Pro Ser Trp Asp Leu Pro Ala Ala Pro Asp Arg Asp Pro 85 90 95 Ala Ala Gly Arg Gly Ala Glu Ala Ser Thr Ala Gly Pro Pro Gly Pro 100 105 110 Pro Thr Arg Pro Pro Val Pro Trp Arg Trp Lys Gly Ala Arg Gly Gln 115 120 125 Glu Pro Ser Glu Thr Leu Gly Arg Gly Asn Pro Thr Ala Leu Gln Leu 130 135 140 Phe Leu Gln Ile Ser Glu Glu Glu Glu Lys Gly Pro Arg Gly Ala Val 145 150 155 160 Ile Ser Gly Arg Ser Gln Glu Gln Ser Val Lys Thr Val Pro Gly Ala 165 170 175 Ser Asp Leu Phe Tyr Cys Pro Arg Arg Ala Gly Lys Leu Gln Gly Ser 180 185 190 His His Lys Pro Leu Ser Lys Thr Ala Asn Gly Leu Ala Gly His Glu 195 200 205 Gly Trp Thr Ile Ala Leu Pro Gly Arg Ala Leu Ala Gln Asn Gly Ser 210 215 220 Leu Gly Glu Gly Ile His Asp Pro Gly Gly Pro Arg Arg Gly Asn Ser 225 230 235 240 Thr Asn Arg Arg Val Arg Leu Lys Asn Pro Phe Tyr Pro Leu Thr Gln 245 250 255 Glu Ser Tyr Gly Ala Tyr Ala Val Met Cys Leu Ser Val Val Ile Phe 260 265 270 Gly Thr Gly Ile Ile Gly Asn Leu Ala Val Met Cys Ile Val Cys His 275 280 285 Asn Tyr Tyr Met Arg Ser Ile Ser Asn Ser Leu Leu Ala Asn Leu Val 290 295 300 Phe Trp Asp Phe Leu Ile Ile Phe Phe Cys Leu Pro Leu Val Ile Phe 305 310 315 320 His Glu Leu Thr Lys Lys Trp Leu Val Glu Asp Phe Ser Cys Lys Ile 325 330 335 Val Pro Tyr Ile Glu Val Ala Ser Leu Gly Val Thr Thr Phe Thr Leu 340 345 350 Cys Ala Leu Cys Ile Asp Arg Phe Arg Ala Ala Thr Asn Val Gln Met 355 360 365 Tyr Tyr Glu Met Ile Glu Asn Cys Ser Ser Thr Thr Ala Lys Leu Ala 370 375 380 Val Ile Trp Val Gly Ala Leu Leu Leu Ala Leu Pro Glu Val Val Leu 385 390 395 400 Arg Gln Leu Ser Lys Glu Asp Leu Gly Phe Ser Gly Arg Ala Pro Ala 405 410 415 Glu Arg Cys Ile Ile Lys Ile Ser Pro Asp Leu Pro Asp Thr Ile Tyr 420 425 430 Val Leu Ala Leu Thr Tyr Asp Ser Ala Arg Leu Trp Trp Tyr Phe Gly 435 440 445 Cys Tyr Phe Cys Leu Pro Thr Leu Phe Thr Ile Thr Cys Ser Leu Val 450 455 460 Thr Ala Arg Lys Ile Arg Lys Ala Glu Lys Ala Cys Thr Arg Gly Asn 465 470 475 480 Lys Arg Gln Ile Gln Leu Glu Ser Gln Met Asn Cys Thr Val Val Ala 485 490 495 Leu Thr Ile Leu Tyr Gly Leu Gly Ile Ile Pro Glu Asn Ile Cys Asn 500 505 510 Ile Val Thr Ala Tyr Met Ala Thr Gly Val Ser Gln Gln Thr Met Asp 515 520 525 Leu Leu Asn Ile Ile Ser Gln Phe Leu Leu Phe Phe Lys Ser Cys Val 530 535 540 Thr Pro Val Leu Leu Phe Cys Leu Cys Lys Pro Phe Ser Arg Ala Phe 545 550 555 560 Met Glu Cys Cys Cys Cys Cys Cys Glu Glu Cys Ile Gln Lys Ser Ser 565 570 575 Thr Val Thr Ser Asp Asp Asn Asp Asn Glu Tyr Thr Thr Glu Leu Glu 580 585 590 Leu Ser Pro Phe Ser Ala Ile Arg Arg Glu Met Ser Thr Phe Ala Ser 595 600 605 Val Gly Thr His Cys 610 3 29 DNA Artificial sequence Primer_Bind 3 cactaagctt aatgcgagcc ccgggcgcg 29 4 32 DNA Artificial sequence Primer_Bind 4 gaacttctag accgtcagca atgagtaccg ac 32 5 34 DNA Artificial sequence Primer_Bind 5 gtccaagctt gccaccatgc gagccccggg cgcg 34 6 61 DNA Artificial sequence Primer_Bind 6 ctagctcgag tcaagcgtag tctgggacgt cgtatgggta gcagcaatga gttccgacag 60 a 61 7 32 DNA Artificial sequence Primer_Bind 7 cgggatccgc caccatgcga gccccgggcg cg 32 8 29 DNA Artificial sequence Primer_Bind 8 cgggatcccg ctcagcaatg agttccgac 29 9 318 PRT Human protein 9 Met Glu Thr Leu Cys Leu Arg Ala Ser Phe Trp Leu Ala Leu Val Gly 1 5 10 15 Cys Val Ile Ser Asp Asn Pro Glu Arg Tyr Ser Thr Asn Leu Ser Asn 20 25 30 His Val Asp Asp Phe Thr Thr Phe Arg Gly Thr Glu Leu Ser Phe Leu 35 40 45 Val Thr Thr His Gln Pro Thr Asn Leu Val Leu Pro Ser Asn Gly Ser 50 55 60 Met His Asn Tyr Cys Pro Gln Gln Thr Lys Ile Thr Ser Ala Phe Lys 65 70 75 80 Tyr Ile Asn Thr Val Ile Ser Cys Thr Ile Phe Ile Val Gly Met Val 85 90 95 Gly Asn Ala Thr Leu Leu Arg Ile Ile Tyr Gln Asn Lys Cys Met Arg 100 105 110 Asn Gly Pro Asn Ala Leu Ile Ala Ser Leu Ala Leu Gly Asp Leu Ile 115 120 125 Tyr Val Val Ile Asp Leu Pro Ile Asn Val Phe Lys Phe Tyr Gln Asp 130 135 140 Val Lys Asp Trp Trp Leu Phe Gly Phe Tyr Phe Cys Met Pro Leu Val 145 150 155 160 Cys Thr Ala Ile Phe Tyr Thr Leu Met Thr Cys Glu Met Leu Asn Arg 165 170 175 Arg Asn Gly Ser Leu Arg Ile Ala Leu Ser Glu His Leu Lys Gln Arg 180 185 190 Arg Glu Val Ala Lys Thr Val Phe Cys Leu Val Val Ile Phe Ala Leu 195 200 205 Cys Trp Phe Pro Leu His Leu Ser Arg Ile Leu Lys Lys Thr Val Tyr 210 215 220 Asn Glu Met Asp Lys Asn Arg Cys Glu Leu Leu Ser Phe Leu Leu Leu 225 230 235 240 Met Asp Tyr Ile Gly Ile Asn Leu Ala Thr Met Asn Ser Cys Ile Asn 245 250 255 Pro Ile Ala Leu Tyr Phe Val Ser Lys Lys Phe Lys Asn Cys Phe Gln 260 265 270 Ser Cys Leu Cys Cys Cys Cys Tyr Gln Ser Lys Ser Leu Met Thr Ser 275 280 285 Val Pro Met Asn Gly Thr Ser Ile Gln Trp Lys Asn His Asp Gln Asn 290 295 300 Asn His Asn Thr Asp Arg Ser Ser His Lys Asp Ser Met Asn 305 310 315 10 442 PRT Homo sapiens 10 Met Gln Pro Pro Pro Ser Leu Cys Gly Pro Ala Leu Val Ala Leu Val 1 5 10 15 Leu Ala Cys Gly Leu Ser Arg Ile Trp Gly Glu Glu Arg Gly Phe Pro 20 25 30 Pro Asp Arg Ala Thr Pro Leu Leu Gln Thr Ala Glu Ile Met Thr Pro 35 40 45 Pro Thr Lys Thr Leu Trp Pro Lys Gly Ser Asn Ala Ser Leu Ala Arg 50 55 60 Ser Leu Ala Pro Ala Glu Val Pro Lys Gly Asp Arg Thr Ala Gly Ser 65 70 75 80 Pro Pro Arg Thr Ile Ser Pro Pro Pro Cys Gln Gly Pro Ile Glu Ile 85 90 95 Lys Glu Thr Phe Lys Tyr Ile Asn Thr Val Val Ser Cys Leu Val Phe 100 105 110 Val Leu Gly Ile Ile Gly Asn Ser Thr Leu Leu Arg Ile Ile Tyr Lys 115 120 125 Asn Lys Cys Met Arg Asn Gly Pro Asn Ile Leu Ile Ala Ser Leu Ala 130 135 140 Leu Gly Asp Leu Leu His Ile Val Ile Asp Ile Pro Ile Asn Val Tyr 145 150 155 160 Lys Leu Leu Ala Glu Asp Trp Pro Phe Gly Ala Glu Met Cys Lys Leu 165 170 175 Val Pro Phe Ile Gln Lys Ala Ser Val Gly Ile Thr Val Leu Ser Leu 180 185 190 Cys Ala Leu Ser Ile Asp Arg Tyr Arg Ala Val Ala Ser Trp Ser Arg 195 200 205 Ile Lys Gly Ile Gly Val Pro Lys Trp Thr Ala Val Glu Ile Val Leu 210 215 220 Ile Trp Val Val Ser Val Val Leu Ala Val Pro Glu Ala Ile Gly Phe 225 230 235 240 Asp Ile Ile Thr Met Asp Tyr Lys Gly Ser Tyr Leu Arg Ile Cys Leu 245 250 255 Leu His Pro Val Gln Lys Thr Ala Phe Met Gln Phe Tyr Lys Thr Ala 260 265 270 Lys Asp Trp Trp Leu Phe Ser Phe Tyr Phe Cys Leu Pro Leu Ala Ile 275 280 285 Thr Ala Phe Phe Tyr Thr Leu Met Thr Cys Glu Met Leu Arg Lys Lys 290 295 300 Ser Gly Met Gln Ile Ala Leu Asn Asp His Leu Lys Gln Arg Arg Glu 305 310 315 320 Val Ala Lys Thr Val Phe Cys Leu Val Leu Val Phe Ala Leu Cys Trp 325 330 335 Leu Pro Leu His Leu Ser Arg Ile Leu Lys Leu Thr Leu Tyr Asn Gln 340 345 350 Asn Asp Pro Asn Arg Cys Glu Leu Leu Ser Phe Leu Leu Val Leu Asp 355 360 365 Tyr Ile Gly Ile Asn Met Ala Ser Leu Asn Ser Cys Ile Asn Pro Ile 370 375 380 Ala Leu Tyr Leu Val Ser Lys Arg Phe Lys Asn Cys Phe Lys Ser Cys 385 390 395 400 Leu Cys Cys Trp Cys Gln Ser Phe Glu Glu Lys Gln Ser Leu Glu Glu 405 410 415 Lys Gln Ser Cys Leu Lys Phe Lys Ala Asn Asp His Gly Tyr Asp Asn 420 425 430 Phe Arg Ser Ser Asn Lys Tyr Ser Ser Ser 435 440 11 444 PRT Xenopus protein 11 Met Ala Thr Val Ile Leu Phe Val Ala Trp Met Ala Cys Leu Met Val 1 5 10 15 Gly Val Cys Tyr Gln Glu Phe Gln Thr Gln Gln Asn Phe Pro Asp Ile 20 25 30 Ser Asn Pro Ser Gln Glu Leu Asn Gln Glu Pro Ala His Arg Ile Val 35 40 45 Gln Leu Asp Ser Ile Gln Asn Asn Gly Ala Leu Asn Met Ser Thr Gly 50 55 60 Asn Val Leu Asn Met Ser Pro Pro Pro Pro Ser Pro Cys Leu Ser Arg 65 70 75 80 Ala Lys Ile Arg His Ala Phe Lys Tyr Val Thr Thr Ile Leu Ser Cys 85 90 95 Val Ile Phe Leu Val Gly Ile Val Gly Asn Ser Thr Leu Leu Arg Ile 100 105 110 Ile Tyr Lys Asn Lys Cys Met Arg Asn Gly Pro Asn Val Leu Ile Ala 115 120 125 Ser Leu Ala Leu Gly Asp Leu Phe Tyr Ile Leu Ile Ala Ile Pro Ile 130 135 140 Ile Ser Ile Ser Phe Trp Leu Ser Thr Gly His Ser Glu Tyr Ile Tyr 145 150 155 160 Gln Leu Val His Leu Tyr Arg Ala Arg Val Tyr Ser Leu Ser Leu Cys 165 170 175 Ala Leu Ser Ile Asp Arg Tyr Arg Ala Val Ala Ser Trp Asn Arg Ile 180 185 190 Arg Ser Ile Gly Ile Pro Val Arg Lys Ala Ile Glu Leu Thr Leu Ile 195 200 205 Trp Ala Val Ala Ile Ile Val Ala Val Pro Glu Ala Ile Ala Phe Asn 210 215 220 Leu Val Glu Leu Asp Phe Arg Gly Gln Thr Ile Leu Val Cys Met Leu 225 230 235 240 Pro Met Glu Gln Thr Ser Asp Phe Met Arg Phe Tyr Gln Glu Val Lys 245 250 255 Val Trp Trp Leu Phe Gly Phe Tyr Phe Cys Leu Pro Leu Ala Cys Thr 260 265 270 Gly Val Phe Tyr Thr Leu Met Ser Cys Glu Met Leu Ser Ile Lys Asn 275 280 285 Gly Met Arg Ile Ala Leu Asn Asp His Met Lys Gln Arg Arg Glu Val 290 295 300 Ala Lys Thr Val Phe Cys Leu Val Val Ile Phe Ala Leu Cys Trp Leu 305 310 315 320 Pro Leu His Val Ser Ser Ile Phe Val Arg Leu Ser Ala Thr Val Lys 325 330 335 Arg Ala Cys Ile Leu Lys Asn Lys Arg Ser Cys Ile Met Ala Glu Ile 340 345 350 Gln Thr Gly Val Asn Tyr Gln Leu Leu Met Val Met Asn Tyr Thr Gly 355 360 365 Ile Asn Met Ala Ser Leu Asn Ser Cys Ile Gly Pro Val Ala Leu Tyr 370 375 380 Phe Val Ser Arg Lys Phe Lys Asn Cys Phe Gln Ser Cys Leu Cys Cys 385 390 395 400 Trp Cys His Arg Pro Thr Leu Thr Ile Thr Pro Met Asp Glu Lys Gly 405 410 415 Ser Gly Gly Lys Trp Lys Ala Asn Gly His Asp Leu Asp Leu Asp Arg 420 425 430 Ser Ser Ser Arg Leu Ser Asn Lys Tyr Ser Ser Ser 435 440 12 384 PRT Human protein 12 Met Ala Leu Asn Asp Cys Phe Leu Leu Asn Leu Glu Val Asp His Phe 1 5 10 15 Met His Cys Asn Ile Ser Ser His Ser Ala Asp Leu Pro Val Asn Asp 20 25 30 Asp Trp Ser His Pro Gly Ile Leu Tyr Val Ile Pro Ala Val Tyr Gly 35 40 45 Val Ile Ile Leu Ile Gly Leu Ile Gly Asn Ile Thr Leu Ile Lys Ile 50 55 60 Phe Cys Thr Val Lys Ser Met Arg Asn Val Pro Asn Leu Phe Ile Ser 65 70 75 80 Ser Leu Ala Leu Gly Asp Leu Leu Leu Leu Ile Thr Cys Ala Pro Val 85 90 95 Asp Ala Ser Arg Tyr Leu Ala Asp Arg Trp Leu Phe Gly Arg Ile Gly 100 105 110 Cys Lys Leu Ile Pro Phe Ile Gln Leu Thr Ser Val Gly Val Ser Val 115 120 125 Phe Thr Leu Thr Ala Leu Ser Ala Asp Arg Tyr Lys Ala Ile Val Arg 130 135 140 Pro Met Asp Ile Gln Ala Ser His Ala Leu Met Lys Ile Cys Leu Lys 145 150 155 160 Ala Ala Phe Ile Trp Ile Ile Ser Met Leu Leu Ala Ile Pro Glu Ala 165 170 175 Val Phe Ser Asp Leu His Pro Phe His Glu Glu Ser Thr Asn Gln Thr 180 185 190 Phe Ile Ser Cys Ala Pro Tyr Pro His Ser Asn Glu Leu His Pro Lys 195 200 205 Ile His Ser Met Ala Ser Phe Leu Val Phe Tyr Val Ile Pro Leu Ser 210 215 220 Ile Ile Ser Val Tyr Tyr Tyr Phe Ile Ala Lys Asn Leu Ile Gln Ser 225 230 235 240 Ala Tyr Asn Leu Pro Val Glu Gly Asn Ile His Val Lys Lys Gln Ile 245 250 255 Glu Ser Arg Lys Arg Leu Ala Lys Thr Val Leu Val Phe Val Gly Leu 260 265 270 Phe Ala Phe Cys Trp Leu Pro Asn His Val Ile Tyr Leu Tyr Arg Ser 275 280 285 Tyr His Tyr Ser Glu Val Asp Thr Ser Met Leu His Phe Val Thr Ser 290 295 300 Ile Cys Ala Arg Leu Leu Ala Phe Thr Asn Ser Cys Val Asn Pro Phe 305 310 315 320 Ala Leu Tyr Leu Leu Ser Lys Ser Phe Arg Lys Gln Phe Asn Thr Gln 325 330 335 Leu Leu Cys Cys Gln Pro Gly Leu Ile Ile Arg Ser His Ser Thr Gly 340 345 350 Arg Ser Thr Thr Cys Met Thr Ser Leu Lys Ser Thr Asn Pro Ser Val 355 360 365 Ala Thr Phe Ser Leu Ile Asn Gly Asn Ile Cys His Glu Arg Tyr Val 370 375 380 13 390 PRT Rattus protein 13 Met Pro Pro Arg Ser Leu Pro Asn Leu Ser Leu Pro Thr Glu Ala Ser 1 5 10 15 Glu Ser Glu Leu Glu Pro Glu Val Trp Glu Asn Asp Phe Leu Pro Asp 20 25 30 Ser Asp Gly Thr Thr Ala Glu Leu Val Ile Arg Cys Val Ile Pro Ser 35 40 45 Leu Tyr Leu Ile Ile Ile Ser Val Gly Leu Leu Gly Asn Ile Met Leu 50 55 60 Val Lys Ile Phe Leu Thr Asn Ser Thr Met Arg Ser Val Pro Asn Ile 65 70 75 80 Phe Ile Ser Asn Leu Ala Ala Gly Asp Leu Leu Leu Leu Leu Thr Cys 85 90 95 Val Pro Val Asp Ala Ser Arg Tyr Phe Phe Asp Glu Trp Val Phe Gly 100 105 110 Lys Leu Gly Cys Lys Leu Ile Pro Ala Ile Gln Leu Thr Ser Val Gly 115 120 125 Val Ser Val Phe Thr Leu Thr Ala Leu Ser Ala Asp Arg Tyr Arg Ala 130 135 140 Ile Val Asn Pro Met Asp Met Gln Thr Ser Gly Val Val Leu Trp Thr 145 150 155 160 Ser Leu Lys Ala Val Gly Ile Trp Val Val Ser Val Leu Leu Ala Val 165 170 175 Pro Glu Ala Val Phe Ser Glu Val Ala Arg Ile Gly Ser Ser Asp Asn 180 185 190 Ser Ser Phe Thr Ala Cys Ile Pro Tyr Pro Gln Thr Asp Glu Leu His 195 200 205 Pro Lys Ile His Ser Val Leu Ile Phe Leu Val Tyr Phe Leu Ile Pro 210 215 220 Leu Val Ile Ile Ser Ile Tyr Tyr Tyr His Ile Ala Lys Thr Leu Ile 225 230 235 240 Arg Ser Ala His Asn Leu Pro Gly Glu Tyr Asn Glu His Thr Lys Lys 245 250 255 Gln Met Glu Thr Arg Lys Arg Leu Ala Lys Ile Val Leu Val Phe Val 260 265 270 Gly Cys Phe Val Phe Cys Trp Phe Pro Asn His Ile Leu Tyr Leu Tyr 275 280 285 Arg Ser Phe Asn Tyr Lys Glu Ile Asp Pro Ser Leu Gly His Met Ile 290 295 300 Val Thr Leu Val Ala Arg Val Leu Ser Phe Ser Asn Ser Cys Val Asn 305 310 315 320 Pro Phe Ala Leu Tyr Leu Leu Ser Glu Ser Phe Arg Lys His Phe Asn 325 330 335 Ser Gln Leu Cys Cys Gly Gln Lys Ser Tyr Pro Glu Arg Ser Thr Ser 340 345 350 Tyr Leu Leu Ser Ser Ser Ala Val Arg Met Thr Ser Leu Lys Ser Asn 355 360 365 Ala Lys Asn Val Val Thr Asn Ser Val Leu Leu Asn Gly His Ser Thr 370 375 380 Lys Gln Glu Ile Ala Leu 385 390

Claims (20)

What is claimed is:
1. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide encoding the polypeptide comprising amino acid-27 to 613 as set forth in SEQ ID NO:2;
(b) a polynucleotide encoding the polypeptide comprising amino acid 1 to 613 as set forth in SEQ ID NO:2;
(c) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a) or (b); and
(d) a polynucleotide fragment of the polynucleotide of (a), (b) or (c).
2. The polynucleotide of claim 1 wherein the polynucleotide is DNA.
3. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide encoding a mature polypeptide encoded by the DNA contained in ATCC Deposit No. 75823;
(b) a polynucleotide encoding a polypeptide expressed by the DNA contained in ATCC Deposit No. 75823;
(c) a polynucleotide capable of hybridizing to and which is at least 70% identical to the polynucleotide of (a) or (b); and
(d) a polynucleotide fragment of the polynucleotide of (a), (b) or (c).
4. A vector containing the DNA of claim 2.
5. A host cell transformed or transfected with the vector of claim 4.
6. A process for producing a polypeptide comprising: expressing from the host cell of claim 5 the polypeptide encoded by said DNA.
7. A process for producing cells capable of expressing a polypeptide comprising transforming or transfecting the cells with the vector of claim 4.
8. A receptor polypeptide comprising a member selected from the group consisting of:
(a) a polypeptide having the deduced amino acid sequence of SEQ ID NO:2 and fragments, analogs and derivatives thereof; and
(b) a polypeptide encoded by the cDNA of ATCC Deposit No. 75823 and fragments, analogs and derivatives of said polypeptide.
9. An antibody against the polypeptide of claim 8.
10. A compound which activates the polypeptide of claim 8.
11. A compound which inhibits activation the polypeptide of claim 8.
12. A method for the treatment of a patient having need to activate an ETBR receptor comprising administering to the patient a therapeutically effective amount of the compound of claim 10.
13. A method for the treatment of a patient having need to inhibit an ETBR receptor comprising administering to the patient a therapeutically effective amount of the compound of claim 11.
14. The method of claim 12 wherein said compound is a polypeptide and a therapeutically effective amount of the compound is administered by providing to the patient DNA encoding said agonist and expressing said agonist in vivo.
15. The method of claim 13 wherein said compound is a polypeptide and a therapeutically effective amount of the compound is administered by providing to the patient DNA encoding said antagonist and expressing said antagonist in vivo.
16. A method for identifying compounds which bind to and activate the receptor polypeptide of claim 8 comprising:
(a) contacting a cell expressing on the surface thereof the receptor polypeptide, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor polypeptide, with a compound under conditions sufficient to permit binding of the compound to the receptor polypeptide; and
(b) identifying if the compound is capable of receptor binding by detecting the signal produced by said second component.
17. A method for identifying compounds which bind to and inhibit activation of the polypeptide of claim 8 comprising:
(a) contacting a cell expressing on the surface thereof the receptor polypeptide, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor polypeptide, with an analytically detectable ligand known to bind to the receptor polypeptide and a compound to be screened under conditions to permit binding to the receptor polypeptide; and
(b) determining whether the compound inhibits activation of the polypeptide by detecting the absence of a signal generated from the interaction of the ligand with the polypeptide.
18. A process for diagnosing a disease or a susceptibility to a disease related to an under-expression of the polypeptide of claim 8 comprising determining a mutation in the nucleic acid sequence encoding said polypeptide.
19. The polypeptide of claim 8 wherein the polypeptide is a soluble fragment of the polypeptide and is capable of binding a ligand for the receptor.
20. A diagnostic process comprising analyzing for the presence of the polypeptide of claim 19 in a sample derived from a host.
US10/292,525 1994-10-17 2002-11-13 Human endothelin-bombesin receptor Abandoned US20030109676A1 (en)

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US08/465,687 US5750370A (en) 1995-06-06 1995-06-06 Nucleic acid encoding human endothlein-bombesin receptor and method of producing the receptor
US09/030,970 US6143519A (en) 1994-10-17 1998-02-26 Human endothelin-bombesin receptor
US09/520,210 US6518404B1 (en) 1994-10-17 2000-03-07 Human endothelin-bombesin receptor antibodies
US10/292,525 US20030109676A1 (en) 1994-10-17 2002-11-13 Human endothelin-bombesin receptor

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US09/520,210 Expired - Lifetime US6518404B1 (en) 1994-10-17 2000-03-07 Human endothelin-bombesin receptor antibodies
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US09/520,210 Expired - Lifetime US6518404B1 (en) 1994-10-17 2000-03-07 Human endothelin-bombesin receptor antibodies

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