AU763711B2 - Human neuropeptide receptor - Google Patents

Human neuropeptide receptor Download PDF

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AU763711B2
AU763711B2 AU27737/00A AU2773700A AU763711B2 AU 763711 B2 AU763711 B2 AU 763711B2 AU 27737/00 A AU27737/00 A AU 27737/00A AU 2773700 A AU2773700 A AU 2773700A AU 763711 B2 AU763711 B2 AU 763711B2
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antibody
seq
amino acid
protein
leu
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Yi Li
Craig A Rosen
Daniel R Soppet
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Human Genome Sciences Inc
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Human Genome Sciences Inc
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Description

-1-
AUSTRALIA
PATENTS ACT 1990
ORIGINAL
COMPLETE SPECIFICATION Name of Applicant: Address of Applicant: Actual Inventor(s): Human Genome Sciences, Inc.
9410 Key West Avenue, Rockville, Maryland 20850- 3338, United States of America SOPPET, Daniel R.
LI, Yi ROSEN, Craig A, Address for Service: DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.
Complete Specification for the invention entitled: Human neuropeptide receptor r The following statement is a full description of this invention, including the best method of performing it known to us: G:\oper\mro\282192 divO98.doc- 13/4/00 P:opcn'ro2282192 spc.doc-II April -1A- HUMAN NEUROPEPTIDE RECEPTOR This invention relates to newly identified polynucleotides, polypeptide encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptides of the present invention are human 7-transmembrane G-protein coupled receptors. More particularly, the polypeptides of the present invention are neuropeptide receptor polypeptides, sometimes hereinafter referred to as neuropeptide receptor polypeptides. The invention also relates to inhibiting the action of such polypeptides.
Obesity is the commonest nutritional disorder in Western societies. More than three ii ten adult Americans weight at least 20% in excess of their ideal body weight (Burroa, The New York Times, 17 July 1994). Increased body weight is an important public health problem because it is associated with Type II diabetes, hypertension, hyperlipidemia and certain cancers (Grundy, S. and Barnett, J. Disease-a-Month, 36:645-696 (1990)).
Five single-gene mutations in the mouse obesity gene (ob) which result in an obese phenotype have been described (Friedman, J. M. Leibel, R. Cell, 66: 217- 220(1990)). The cloning and sequencing of the mouse ob gene and its human homologue have been reported (Zhang, Y, e: al., Nature, 372:425-431 (1994)). The ob gene encodes a 4.5-kb adipose tissue mRNA with a highly conserved 167-amino-acid open reading frame. T:.e predicted amino-acid sequence is 84% identical between human and mouse and has features of a secreted protein. The ob gene product may function as part of a signalling pathway from adipose tissue that acts to regulate the size of the body fat depot (id. 425).
Of the brain regions implicated in the regulation of feeding behavior, the ventromedial nucleus of the hypothalamus (VMH) is considered to be the most important satiety center in the central nervous system (CNS). The energy balance in mammals is therefore postulated to be controlled by a feedback -loop in which the amount of stored energy is sensed by the hypothalamus, which adjusts food intake and energy expenditure to maintain a constant body weight (Ombeck, Yale J. Biol. Med., 20:545-552 (1948) and Kennedy, Proc. R. Soc.148:57 8 592 (1953)). In the lipostasis theory, the size of the body fat depot is regulated by the CNS, with a product of body fat metabolism affecting energy balance by interacting with the hypothalamus (Kennedy, Proc. R. Soc.148:57 8 592 (1953)).
The inability to identify the putative signal from fat has hindered the validation of the lipostasis theory. The possibility that at least one component of the signalling system circulates in the bloodstream was first suggested by Hervey (Dietrich, et al., Genetics, 131:423-447 (1992)), who showed that the transfer of blood from an animal with a VMH lesion across a vascular graft to an untreated animal (a oarabiosis experiment) resulted in a reduction of food intake in the intact animal. It is now significant that there is evidence that the ob gene product is secreted, suggesting that ob may encode .his circulating factor.
The ob signal may act directly or indirectly on the CNS to inhibit food intake and/or regulate energy expenditure as part of a homeostatic mechanism to maintain constancy or :ne adipose mass (Zhang, et al., Nature, 372:425-431, 431 (1994)) The ob gene apparently encodes a protein secreted by fat, and mutations apparently prevent translation or b y k T Nature, 372:406-407 expression of the gene (Rink, Nature, 372:406-407 (1994)).
Parabiosis experiments suggest that the ob receptor is encoded by the mouse db (diabetes) gene (Coleman,
D.L.,
DiabetolcTia, 14:141-148 (1978)). Mice having a mutation in the db gene are also obese, with the defect possibly being a receptor defect. (Id. at 406) Neuropeptide Y is similar to the ob gene product in that it mediates the feeding response. Neuropeptide Y acts on at least four types of neuropeptide Y receptors called Y, Y 3 and an atypical Y, receptor, which mediates the feeding response stimulated by neuropeptide
Y.
Neuropeptide Y has a wide range of biological functions.
Neuropeptide Y is found to be widely distributed in the central nervous system (CNS) and the peripheral nervous system (PNS). In the PNS, neuropeptide Y is found in the noradrenergic sympathetic innervation of blood vessels and other smooth muscle tissues and in neurons within the enteric nervous system. Neuropeptide Y immunoreactive fibers also occur in the non-vascular smooth muscle, surrounding exocrine glands and surface epithelia Neuropeptide Y also occurs in subpopulations of neurons and is generally co-localized with other neurotran tters, particular noradrenaline.
In the CNS, neuropepride Y is contained in GABAergic interneurons in higher centers and in predominantly catecholaminergic cells that project further caudally For example, neuropeptide Y is contained in interneurons in the cortex, hippocampus, amygdala, basal forebrain and striatum, whereas in the brain stem, neuropeptide Y is contained in noradrenergic neurons of the A, and A. groups in the medulla, and the locus coeruleus (LC) .the hypothala WO96/34877 WO 96/34877 PCT/US95/05616 neuropeptide Y is found predominantly in the arcuate nucleus and lateral hypothalamus.
Within the peripheral nervous system, neuropeptide Y is present in postganglionic sympathetic nerves, and is colocalized as stated above with other neurotransmitters, including catecholamines. When used pharmacologically, neuropeptide Y has been shown to have a potent vasoconstrictor activity as well as dramatically potentiating the vasoconstriction caused by many other pressor agents.
Particularly high concentrations of n-.uropeptide Y are found in the sympathetic nerves supplying the coronary, cerebral and renal vasculature and when infused into these vascular beds, neuropeptide Y causes prolonged vasoconstriction that is not reversed by adrenergic blocking agents. These observations have lead to the proposal that neuropeptide Y is the candidate transmitter for pathological vasospasm, a major cause of morbidity and mortality when involving the coronary and cerebral vessels.
Neuropeptide Y also appears to be involved in interaction with the renin angiotensin system. Neuropeptide Y containing sympathetic nerve terminals are found on the juxta-glomerular apparatus of the renal cortex and neuropeptide y influences renin release. These data, together with the demonstration of all durations in neuropeptide Y concentrations in hypertensive animal models the pressor response to infusion of the peptide, have .*:::resulted in implications of this peptide in hypertension.
Within the central nervous system neuropeptide Y is .''".located predominantly within interneurons where it appears to a regulatory role. It therefore has widespread and diverse effects including effects on memory and a possible role in Alzheimer's disease. Neuropeptide Y is the most potent known substance to cause an increase in feeding and may play a role in the genetic basis of Type II Diabetes Mellitus. Neuropeptide Y may also play a role as a regulaorY agen-- and iuizar f:nction as wel as ac:_n:la egulaory agen.t ant :1a neuromodulatory function in stress responses an reproductive function.
In accordance with one aspect of the presen: invention, there are provided novel ma-ure receptor polyeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The receptor polypeptides of the present invention are of human origin.
origIn accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the receptor polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with a further aspect o the present invention, there are provided processes for producing such receptor polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells containing nucleic acid sequences encoding the receptor polpeptides of the present i nvn n, under receptor polYPeptides o f said polyeptides and conditions promoting expression of said polypeptides and subsequent recovery of said polypeptides.
In accordance with vet a further aspect of the present invention, there are provided antibodies against such receptor polypeptides. e rese In accordance with another aspect Of the presert invention there are provided methods of screening for compounds which bind to and activate or i2hibit acivatio n of the receptor polypeptides of the present invention.
In accordance with still another embodiment of the present inventior- there are provided processes of ministerineg compounds to a host which bind to and activate administering compounds Co r invention which are the receptor polypeptide of the resent invention which are useful in the prevention and/or treatment of obesity, r r S.
S
hvderipidemia, certa.n cancers, to s auae ne growth, to regulate neurocrans-ssi o n, r.hance ac:ivitv levels and utilization of ingested foods.
In accordance with another aspect of the present invention there is provided a method of administerin the receptor oolypeptides of the present invention via gene therapy to treat conditions related to underexpression of the polypeptides or underexpression of a ligand to the receptor polypeptide.
In accordance with still another embodiment of the oresent invention there are provided processes of administering compounds to a host which bind to and inhibit activation of the receptor polypeptides of the present invention which are useful in the prevention and/or treatment of Alzheimer's disease, Type II Diabetes Mellitus, epilepsy, stress, anxiety, hypertension, cardiovascular disease, psychotic conditions and obesity caused by neuropeptide
Y:
In accordance with yet another aspect of the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the polynucleotide sequences of the present invention.
In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences encoding such polypeptides and for detecting an altered level of the soluble form of the receptor polypeptides.
In accordance with yet a further aspect of the present invention, there are provided processes for utilizing such receptor polypeptides, or polyucleotides encoding sucn polypeptides, for in vitro purposes related to scientific esearch, synthesis of DNA and manufacture of DNA vectors.
r These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
The folowing drawings are illuscra 1e zf eociCens of the invention and are not meant to limit the sccoe of tne invention as encompassed by the claims.
Figure 1 shows the cDNA s-quence and the corresponding deduced amino acid sequence of th neuropepOtde receptor polypeptide of the present invention. The standard oneletter abbreviation for amino acids is used. Seuenng was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.) Figure 2 shows the cDNA sequence and the corresponding deduced amino acid sequence of the neuropeptide receptor solice variant 1 polypeptide of the present inventio- The standard one-ltter abbreviation for amino acids is used.
Figure 3 shows the cDNA sequence and the corresponding deduced amino acid serquence of the neuropeotide receptor splice variant 2 polypeptide of the present invention. The standard one-letter abbreviation for amino acids is used.
Figure 4 illustrates the amino acid sequence and seven transmembrane regions of the neuropeptide receptor. The transmembrane regions are underlined and denoted with a TM.
Fig-ure 5 illustrates the amino acid sequence and seven trnmerane regions of the neuropeptide receptor splice variant The transebrane regions are underlined and denoted with a TM.
igure 6 illustrates the amino acid sequence and seven transemrane regions of the neuropeptide receptor splice ransmembrane regionare uderlined and variant 2. The transmembrane regions are derlined a denoted with a TM.
F Figure 7 shows the amino acid homology between the humn Figu r e h p sent invention neuropeptide receptor polypeptide of the resent nvention (and the human neuropeotide Y receptor) The receptor polypeptides of the present invention are S receptors for ligands, both known and unknown, which modulate the activity of cells in both the central nervous system and Speripheral tissues regulated by the cenral nrvus system oe Examples of such ligands are neuropeptide Y, substance P, the human ob gene product and neurokinin B. Accordingly, modulation of the activity of receptor polypeptides of the present invention will have a broad range of therapeutic and diagnostic applications, particularly with respect to the treatment of obesity.
The present inventors have isolated a full-length cDNA clone encoding a human neuropeptide receptor polypeptide.
The present full-length cDNA has been mapped to a location on human chromosome 1 position p31-3 4 which corresponds to a location on the mouse chromosome 4 where the db gene is found. The mouse db gene is thought to encode the receptor for the obesity gene product.
In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature polypeptide having the deduced amino acid sequence of Figures 2 (SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of the clone(s) deposited as ATCC Deposit No. 97128 on April 28, 1995.
The polynucleotide of this invention was discovered in a cDNA library derived from human adult hypothalamus. It is structurally related to the G protein-coupled receptor family. The neuropeptide receptor polypeptide contains an open reading frame encoding a protein of 402 amino acid ,residues. The neuropeptide receptor protein exhibits the highest degree of homology to human neuropeptide YI receptor protein with 52 similarity and 26 identity over the entire amino acid sequence.
The polynucleotides 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 doublestranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequences which encode the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID or ha of e deposied izlone(s) cr z e eZL:en NO:1) or Ona f odingr :e acoding sequence which coding sequence, as a resut of tne redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure (SEQ
NO:)
or the deposited cDNA(s) The polynucleotides which encode for the mature polypeptide of Figure 2 (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited cDNA(s) may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding secuence, 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 polypepcide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequence of Figure 2 (SEQ ID NOC:2 or the polypeptide encoded by the cDNA of the deposited clone The variants of the polynucleotide may be naturally occurring allelic variants of the polynucleotides or non-naturally occurring variants of the polynucleotides.
Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 2 S (SEQ D NO:2) or the same mature polypeptide encoded by the cDNA of the deposited clone(s) as well as variants of such polyucleotide which variants encode for a fragment, derivative or analog of the polypeptide of Figure 2 (SEQ ID NO:2) or the polypept-de encoded by the cDNA of the deposited clone Such nucleotide variants inclde deletion variants, substitution variants and addition or insertion variants. Specific examples of such variants include the oDolvuclctide sCeqences as se: forth in SEQ ID NOS:3 and which encode for splice variant 1 and 2, respectively, of the polypeptide of the present invention.
As hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SEQ ID NO:1) or of the coding sequence of the deposited clone(s) As known in the art, an allelic variant is an alternate form of polynucleotide sequences which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptides.
The polynucleotides may also encode for a soluble form of the neuropeptide receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved from the TM and intracellular domain of the fulllength polypeptide of the present invention.
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 hexahistidine tag supplied by a pQE-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 S sequence may be a hemagglutinin (KA) tag when a mammalian 9 host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, et al., Cell, 37:767 (1984)).
The present invention further relates to polynucleotides which hybridize to the hereinabov -described seauences if there is at least 70%, preferably at least and more preferably at ]east 95% identity between the
S:
sequences. The present i.nvention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, t I the term "stringent conditions" means nybcrdation -wi occur only if there is at least 95% and preferably at lease 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID NO:1) or the deposited cDNA(s), i.e. function as a soluble neuropeptide receptor by retaining the ability to bind the ligands for the receptor even though the polypeptide does not function as a membrane bound neuropeptide receptor, for example, by eliciting a second messenger response.
Alternatively, the- polynucleotides may be polynucleotides which have at least 20 bases, preferably bases and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which have an identity thereto, as hereinabove described, and which does not retain activity. Such polynucleotides may be employed as orobes for the polynucleotide of SEQ ID NO: 1, or for variants thereof, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International S Recognition of the Deposit of Micro-organisms for purposes of P atent 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. ยง112.
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 S* required to make, use or sell the deposited materials, and no such license is hereby granted.
-11- The oresent invention further relates to a polypeptae which has the deduced amino acid secuence of Figure 2 (SEQ ID NO:2) or which has the amino acid sequence encoded by the deposited cDNA(s), as well as fragments, analogs and derivatives of such polypeptide.
The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 2 (SEQ ID NO:2) or that encoded by the deposited cDNA(s), means polypeptides which either retain substantially the same biological function or activity as such polypeptides, function as a soluble neuropeptide receptor by retaining the ability to bind the ligands of the receptors even though the polypeptides do not function as membrane bound neuropeptide receptors. An analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. Specific examples are splice variant 1 and 2 of Figures 2 and 3 (SEQ ID NO:4 and 6), respectively.
The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.
A fragment, derivative or analog of the polypeptide of Figure 2 (SEQ ID NO:2) or that encoded by the deposited cDNA(s) may be one in which one or more ofthe 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 no" be one encoded by the genetic code, (ii) one in which one S or more of the amino acid residues includes a substituent group, (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), (iv) one in which the additional amino acids are fused to the mature polypeptide, such as sequence which is employed for purification of the mature polypeptide sequence -12- 0V-!20or (iv) splice variants of the mature polypete w=n ma have one or more amino acids deleted from the mature polypeptide yet still retain activity corresponding to the mature polvyeptide. 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.
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).
The term "isolated" means that the material is removed from its original environment the natural environment if it is naturally occurring). For example, a naturallyoccurring 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 comosition is not part of its natural environment.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the i: nvention and the production of polypeptides of the invention by r-combinant techniques.
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 -13engineered hosc cells ca- be cu e nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the human neuropeptide receptor genes. The culture cbnditions, 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.
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 SV40; 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 reolicable 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.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence (s) S (promoter) to direct mRNA synthesis. As representative examles of such promoters, there may be mentioned: LTR or promoter, the E. coli lac or tr, the phage lambda
PL
promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
S The exoression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
**14 -14- The veccr may also include appropriate se--ence3 for amplifying expression.
In addition, the expression vec:ors preferably contain one or more selectabie 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 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.
As reoresentative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomvces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Soodootera 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: pQE70, pQE60, pQE-9 (Qiagen), pbs, pDl0, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pr16a, pNHlSA, pNH46A (Stratagene) pTRC99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, oSV2CAT, pOG-4, oXTI, oSG ':ratcaene; SVX3, s-p,
MSG-,
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 PKK232-8 and PCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PC 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, Battey, Basic Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by he reco nant sequ Alternatively, the polypeptides of the recombinant sequence t.he invention can be synthetically produced by conventional eptide synthesizers.
Fragments of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments S may be employed as intermedi--es for producing the fulllength polypeptides. Fragments of the polynucleotides of the S present invention may be used in a similar manner to -16synthesize the full-length polynucleotides of e present invention.
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, (1989), the disclosure of which is hereby incorporated by reference.
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 SV40 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, the ampicillin Sresistance gene of E. coli and S. cerevisiae TRP1 gene, and S 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 te-mination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic spade or extracellular medium. Optionally, the heterologous sequence *1 -17e can encode a fusion rocen incl ud e an N-:er-nal identification pep-ide imparting desired characteristics, 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 E. coli Bacillus subtilis, Salmonella tvphimurium 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 pBR322
(ATCC
37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, 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 temperature shift -or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting S crude extract retained for further purification.
-18- Microbial cells employed in expression of protcens car 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.
Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-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 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The neuropeptide receptor polypeptide of the present invention 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 comoleting configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The neuropeptide receptor polypeptide 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 -19a* mammalian cells In cul-.re- Deoenc' Do employed in a recombinan 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.
The oolynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease.
The human neuropeptide receptor polypeptides of the present invention may be employed in a process for screening compounds which bind to and activate the receptor polypeptide and for compounds which bind to and inhibit activation of the receptor polypeptides of the present invention.
In general, the neuropeptide receptor in isolated, immobilized or cell bound form is contacted with a plurality of compounds and those compounds are selected which bind to and interact with the receptor. The binding or interaction can be measured directly by using radioactively labeled compounds of interest or by the second messenger effect resulting from the interaction or binding of the candidate compound. Alternatively, the candidate compounds can be subjected to competition screening assays, in which a known ligand, preferably labeled with an analytically detectable reagent, most preferably radioactivity, is .introduced with the compound to be tested and the compound's capacity to inhibit or enhance the binding of the labeled ligand is measured. Compounds are screened for their increased afffinity and selectivity to the receptor polypeptide of the present invention.
One such screening procedure involves the use of melanophores which are transfected to express the neuropeptide receptor of the present invention Such a screening technique is described in PCT WO 92/01810 published February 6, 1992.
For example, to screen for compounds which iLnbit activation of the receptor polypeptide of the present invention, the compound and a ligand known to bind to the receptor are both contacted with the melanophor2 cells.
Inhibition of the signal generated by the ligand indicates that the compound inhibits activation of the receptor.
The screen may be employed for determining a compound which binds to and activates the receptor polypeptide of the present invention by contacting such cells with cc-pounds to be screened and determining whether such compound generates a signal, activates the receptor.
Other examples include the use of cells which express a neuropeptide receptor of the present invention (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, compounds may be contacted with a cell which expresses an neuropeptide receptor polypeptide of the present invention and a second messenger response, e.g. signal transduction or pH changes, may be measured to determine whether the potential compound is effective as an activator or inhibitor.
Another example involves introducing RNA encoding a neuropeptide receptor of the present invention into Xenopus oocytes to transiently express the receptor. The oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition of or an increase in intracellular calcium.
Another example involves expressing a neuropeptide S receptor polypeptide of the present invention on the surface of a cell wherein 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, embrryonic kidney cells, etc. The screening may be accomplished as hereinabove described by detecting activation -21e of the receptor or inhliL:!ic of ac:iva:ion or recep.rfrom the phospholipase second signal.
Another method involves determining inhibition of binding of labeled ligand to cells which have a neuropeptide receotor on the surface thereof. Such a method involves cransfecting a eukaryotic cell with DNA encoding an neuropeptide receptor polypeptide of the present invention such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand. The ligand can be labeled, by radioactivity. The amount of labeled ligand bound to the receptors is measured, by measuring radioactivity of the receptors. If the compound 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.
Another screening technique involves expressing a neuropeptide receptor polypeptide on the surface of a cell wherein the receptor is linked to a second messenger to increase cytosolic calcium levels in transfected CHO cells.
An example of such a method comprises transfecting CHO cells with a nucleic acid sequence encoding a receptor of the present invention such that the receptor is expressed on the surface thereof. The transfected cell is then incubated in a reaction mixture with labeled calcium in the presence of a compound to be screened. The ability of the compound to increase calcium up-take or inhibit calcium up-take can then be determined by measuring the amount of labeled calcium transoorted into the cells by taking advantage of the label, e.g, radioactivity.
Compounds may also be identified by the above methods which bind to specific subregions within the CNS that are omportant for specific behaviors through indirect interactions with a neuropeptide receptor polypeptide of the presnt invention.
-22- &V 0: To measure intraceilular cyclic AMP levels, cyclic
AMP
is assayed in whole cells treated for 15 minutes at 37 0 C with 100 micromolar isobutylmethylxanthine (IBMX; Sigma).
Transfected cells (1 x 106 0.5 ml reaction) are incubated with 10 micromolar forskolin and various concentrations of known or unknown ligands to the receptor. Reactions are terminated with the addition of HC1 to 0.1M, incubation at room temperature for 15 minutes, neutralization and sample dilution in 50 mM sodium acetate, pH 6.2. Cyclic AMP is quantified by using a radioimmunoa.ssay (Dupont/NEN) To measure levels of intracellular calcium, transfected cells are suspended in loading medium (modified RPMI 1640 medium/1 0 mM Hepes/l% newborn calf serum) and incubated in a spinner flask at 37 0 C for 2.5 hour at 1 x 106 cells per ml.
Cells are then treated with 1 micromolar Fura-2 acetoxymethyl ester (fura-2 AM; Molecular Probes) for 30 minutes at 37 0
C,
washed twice with loading medium, and resuspended at 5 x 106 cells/ml. Immediately before fluorescence spectroscopy, cells are recovered by centrifugation at 1000 rpm and resuspended at 1 x 10 cells/m- in a modified Krebs buffer (135 mM NaC1/4.
7 mM KCI/1.
2 mM MgSO 4 /1.
2 mM KH,PO 4 /5 mM NaHC03/1 mM CaC12/ 2 8 mM glucose/1 0 mM hepes, pH 7.4) containing sulfinpyrazone. Bombesin is purchased from Sigma and Auspep. Fluorescence recordings are made on a Hitachi fluorescence spectrometer (F4010) at 340 nm (excitation) and 505 nm (emission) over 10 minutes with slit widths of 5 nm and response time of 2 seconds. Intracellular calcium is quantified by using equations described by Grkiewicz, ec al., J. Bio. Chem. 260:3440-3450, 1985.
The invention also provides a method of treating and/or preventing obesity by administering to a host a compound which binds to and activates the receptor polypeptides of the present invention. Such a compound is ot.eur than the ob gene product disclosed in Zhang, et al., Nature, 372:425-431 (1994). The receptor polypeptide of the present invention -23maps to a human chromosome which corresponds to the position of the mouse chromosome which encodes for the receptor of the ob gene product. The human ob gene encodes a "satiety" factor which binds to and activates the receptor polypeptide of the present invention. Accordingly, a compound which activates the receptor of the present invention will decrease appetite and prevent obesity.
The compounds described above may also be employed to enhance activity level, modify eating behavior, enhance utilization of ingested foods and regulate deposition of fat s,:ores. Conditions related to obesity may also be treated by the compounds which bind to and activate the receptor polypeptides of the present invention including hyperlidimeia, type II diabetes and certain cancers.
These compounds may also be employed to treat and/or prevent other conditions related to an underexpression of the receptor polypeptide of the present invention or ligands which bind thereto, for example, to stimulate neuronal growth.
Specific examples of compounds which inhibit activation of the receptor polypeptides of the present invention include an antibody, or in some cases an oligonucleotide, which binds to the receptor but does not elicit a second messenger response such that the activity of the receptbr is prevented.
Another example is proteins which are closely related to S. the ligands of the receptor, i.e. a fragment of the ligand, which have lost biological function and when binding to the receptor, elicit no response.
Another example 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 polynucleotie to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature -24- :0,0*6 s* polypeptides of the present invention, is used -s des:m.n a 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. Ac: is 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 a neuropeptide receptor polypeptide of the present invention. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the receptor (antisense Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of.Gene Expression, CRC Press, Boca Raton, FL (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 the receptors.
Another example is a small molecule which binds t a neuropeptide receptor polypeptide of the present invention, making it inaccessible to ligands such that normal biological activit is prevented Examples of small molecules include but are not limited to small peptides or peptide-like molecules and neuropeptide Y fragments and/or derivatives.
Soluble forms of a neuropeptide receptor polypeptide of Sthe present invention, a fragment of the receptor, which binds to the ligand and prevents the ligand from interacting with membrane bound receptors may also inhibit activation of the receptor polypeptides of the present invention.
This invention additionally provides a method of utilizing such compounds which inhibit activation for treating abnormal conditions related to an excess of activity of a neuropepuide receptor polypeptide of the present invention for treating obesity since the neuropeptide receptor polypeptides of the present invention may bind neuroopepide Y whZ. Is the mos: ocen o<rown subsance 0t cause an increase in feeding behavior and type II Diabetes Mellitus since neuropeptide Y may play a role in the genetic basis of this disease.
The compounds which inhibit activation of the receptor polypeptides of the present invention may be employed to treat and/or prevent hypertension since neuropeptide
Y
stimulates renin release and neuropeptide Y is known to have potent vasoconstrictor activity when involving the coronary and cerebral vessels.
The compounds may also be employed to treat Alzheimer's disease since neuropeptide Y receptors are prevalent in the central nervous system and are localized predominantly within interneurons where they appear to have regulatory roles in memory and Alzheimers disease.
The compounds may also be employed to suppress excitatory transmission by neuropeptide Y in the hippocampus and therefore may be employed to treat epileptic seizure, stress and anxiety.
The prevalence of neuropeptide Y receptors in the central nervous system indicates that the compounds which inhibit the neuropeptide receptor polypeptides of the present invention may be used as an antipsychotic drug by regulating neurotransmission.
The compounds which inhibit the receptor polypeptides of the present invention may also be employed to treat pathological vasospasm involving coronary and cerebral vessels.
This invention also provides a method for determining whether a ligand not known to be capable of binding to a neuropeptide receptor of the present invention can bind thereto which comprises contacting the ligand to be identified with a cell comprising the coding sequence of a neuropeptide receptor and expressing same on its surface under conditions sufficient for binding of ligands previously S* -26identified as binding co such a receptor. In other embodiments cell membrane fractions comprising the receptor or isolated receptors free or immobilized on solid supports may be used to measure binding of the ligand to be tested.
When recombinant cells are used for purposes of expression of the receptor it is preferred to use cells with little or no endogenous receptor activity so that binding, if any, is due to the presence of the expressed receptor of interest.
Preferred cells include human embryonic kidney cells, monkey kidney (HEK-293 cells), fibroblast (COS) cells, Chinese hamster ovary (CHO) cells, Drosophila or murine L-cells. It is also preferred to employ as a host cell, one in which a receptor responsive second messenger system exists. Well known second messenger systems include increases or decreases in phosphoinositide hydrolysis, adenylate cyclase, guanylate cyclase, or ion channel activity in response to ligand binding to extracellular receptor domains. In a further embodiment a specifically designed indicator of receptor binding can be constructed. For example, a fusion protein can be made by fusing the receptor of this invention with a protein domain which is sensitive to receptor ligand binding.
Such a domain referred to here as an indicator domain is capable, itself, or in association with accessory molecules, of generating an analytically detectable signal which is S indicative or receptor ligand binding.
This invention also provides a method of detecting expression of a neuropeptide 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 or a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence of mRNA *-27 -27hvbricized to the crobe, and hereby detctring m e:xression of the receptor by the cell.
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 neuropeptide 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 neuropeptide receptor polypeptides of the present invention.
Fragments of the genes may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the genes of the present invention, or which have similar biological activity. Probes of this type preferably have 50 bases or more. 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 of the present invention including regulatory and promoter regions, exons and introns. An example of a screen of this type 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 S the genes 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.
The neuropeptide receptor polypeptides and compounds identified above 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." 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 *28 S-28- ยฐo provided co a poaent to be treated wi:h the polyeopide.
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.
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.
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, gibbqn 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.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechni us, Vol. 7, No. 9, 980-990 (1989) or any other promoter cellular promoters such as eukaryotic cellular promoters including, but not limited to, the -29histone, pol III, and 3-act:n promoters). ther vrapromoters which may be employed include, but are nce 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 ApoAl 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 O-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 PE501, PA317, 4-2, -AM, PA12, T19-14X, VT-19-17-H2, CRE, CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Theraoy, 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 CaPO, 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 ifcous re:rovral vector oarticles 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.
The soluble neuropeptide receptor polypeptides and comoounds which bind to and activate or inhibit activation of a receptor of the present invention may also be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the soluble neuropeptide receptor polypeptide or compounds, 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.
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 -anufacture, use or sale fcr human administration. In addition, the soluble neuropeptide receptor polypeptides or compounds of the present invention may be employed in conjunction with other therapeutic compounds.
The pharmaceutical compositions may be administered in a convenient manner such as by the topical, intravenous, -31intraoeritoneal, intramuscular, subcutan-.ous, intranasa 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 oharmaceutical compositions will be administered in an amount of at least about 10 g/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 xg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
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 colorimetric assay, expression on MacConkey plates, complementation experiments, in a receptor deficient strain of HEK293 cells) as yet another means to verify or identify mutations. Once "mutant" genes have been identified, one can then screenv 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 S" 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., 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 -32instant 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 Sl protection or the chemical cleavage method Cotton, et al., PNAS, USA, 85:4397-4401 1985).
In addition, some diseases are a result of, or are characterized by changes in gene expression which can be detected by changes in the mRNA. Alternatively, the genes of the present invention can be used as a reference to identify individuals expressing a decrease of functiops associated with receptors of this type.
-33- The present invention also relates to a diagnostic assay for detecting altered levels of soluble forms of the neuropeptide receptor polypeptides of the present invention in various tissues. Assays used to detect levels of the soluble receptor polypeptides in a sample derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive-binding assays, Western blot analysis and preferably as ELISA assay.
An ELISA assay initially comprises preparing an antibody specific to antigens of the neuropeptide receptor polypeptides, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or in this example a horseradish peroxidase enzyme. A sample is now removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any neuropeptide receptor proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to neuropeptide receptor proteins. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of neuropeptide receptor proteins present in a given volume of patient sample when compared against a standard curve.
The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on -34- SUsTsITUFE SHEET (RULE 261 an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process.
These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases.
For a review of this technique, see Verma et al., Human Chromosomes: Manual of Basic Techniques, Pergamon Press, New York (1988).
The above techniques were utilized to ma .Zne ge.
corresponding to the neuropeptide receptor of the present invention to chromosome 1 position 31-34.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetid map data. 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.
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 kb).
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as San immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
The oresent 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 he used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be -36obtained by direct injecion of the ?olypepi a es in:o an animal or by administering the polypep=ides t: 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.
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 EBVhybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies Patent 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.
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.
In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.
"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 -37constructed from available plasm-s ir. accord w.i. s..
procedures. In addition, equivalen plasmids to :hose described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only a: 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 pg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 il of buffer solution. For the purpose of isolating
DNA
fragments for plasmid construction, typically 5 to 50 pg 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 37C 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.
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).
,Oligonucletides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide S* strands which may be chemically synthesized. Such synthetic Soligonucleotides have no 5' phosphate and thus will nit 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. for "Ligation" refers to the process of forming :phosphodiester bonds between two double stranded nucleic acid 38fragments (Maniatis, et al., Id., p. 146) Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase") per 0.5 Ag 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) Example 1 Bacterial Expression and Purification of the Neuropeptide Receptor The DNA sequence encoding for neuropeptide receptor, ATCC 97128 is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the processed neuropeptide receptor gene (minus the signal peptide sequence) and the vector sequences 3' to the gene.
Additional nucleotides corresponding to neuropeptide receptor nucleotide sequence are added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence CACTAAAGCTTAATGGAGCCCTCAGCCACC 3' (SEQ ID NO:7) contains a Hind III restriction enzyme site followed by 18 nucleotides of neuropeptide receptor coding sequence starting from the presumed terminal amino acid of the processed protein codon.
The 3' sequence 5' ACAAGTCCTTGTCCTTCTAGAGGGC 3' (SEQ ID NO:8) and contains an Xbal site. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, CA). pQE-9 encodes antibiotic resistance a bacterial origin of *o replication (ori), an IPTG-regulatable promoter operator a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 is then digested with Hind e*7. TII and Xbal. The amplified sequences are ligated into pQE-9 and are inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture is then used -39to transform c sran Mi5/repD 4 Qa c-v procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid OREP4, which exoresses the lacI repressor and also confers kanamycin resistance (Kan') Transforma.ts are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis. Clones containing the desired constructs are grown overnight 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 are crown to an optical density 600 of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression. Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HC1. After clarification, solubilized neuropeptide receptor is 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) The protein is eluted from the column in 6 molar guanidine HC1 pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine SHCl, 100mM sodium phosphace, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the protein is dialyzed to mmolar sodium phosphate.
S
Example 2 Expression of Recombinant NeuroDeDtide Receptor in COS cells 4
S
The expression of plasmid, neuropeptide receptor HA is derived from a vector pcDNA3/Amp (Invitrogen) containing: 1) 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 neuropeptide receptor precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described 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: The DNA sequence encoding for neuropeptide receptor, ATCC 97128, is constructed by PCR using two primers: the primer 5' CCTAGGATGCCCCTCTGCTGCAGCGG 3' (SEQ ID NO:9) contains a BamHI site; the 3' sequence 5' ACAAGTCCTTGT CCTTCTAGAGGGC 3' (SEQ ID NO:10) contains complementary sequences to an XbaI site, translation stop codon, and the last 17 nucleotides of the neuropeptide receptor coding sequence (not including the stop codon). Therefore, the PCR product contains a BamHI site, coding sequence, a translation termination stop codon and an Xbal site. The PCR amplified DNA fragment and the vector, pcDNA3/Amp, are digested with BamHI and XbaI restriction enzymes and ligated. The ligation mixture is transformed into E. coli strain SURE (Stratagene S" Cloning Systems, La Jolla, CA) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the -41correct fragment. For expression of the recombinant neuropeptide receptor, COS cells are transfected with the expression vector by DEAE-DEXTRAN method Sambrook,
E.
Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the neuropeptide receptor HA protein is detected by radiolabelling and immunoprecipitation method Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hours with "S-cysteine two days post transfection. Culture media are then collected and cells are lysed with detergent
(RIPA
buffer (150 mM NaC1, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, Tris, pH (Wilson, I. et al., Id. 37:767 (1984)).
Both cell lysate and culture media are precipitated with a HA specific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE gels.
Example 3 Cloning and expression of Neuropeptide Receptor using the baculovirus expression system The DNA sequence encoding the full length neuropeptide receptor protein, ATCC 97128, is amplified using
PCR
oligonucleotide primers corresponding to the 5' and 3' sequences of the gene: The 5' primer has the sequence 5' CGGGATCCGCCATCATGGAG CCCTCAGCCACC 3' (SEQ ID NO:11) 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 Mol. Biol. 1987, 196, 947-950, Kozak, The initiation codon for translation "ATG" is underlined) The prime: has the sequence 5' ACAAGTCCTTGTCCTTCT AGAGGGC 3' (SEQ ID NO:12) and contains the cleavage site for the restriction endonuclease Xbal and 5 nucleotides complementary to the 3' non-translated sequence of the -42neuropeptide receptor gene. The amplifed se.ences are isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.) The fragment is then digested with the endonucleases BamlqI and Xbal and then purified as described in Example 1. This fragment is designated F2.
The vector pA2 (modification of pVL941 vector, discussed below) is used for the expression of the neuropeptide receptor 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:1, 3 and 5555). This .expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhidrosis virus (AcMNPV) followed by the recognition sites for the restriction endonucleases BamHI and XbaI. 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 co-transfected 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, Virology, 170:31-39).
The plasmid is digested with the restriction enzymes BamHI and XbaI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The S: DNA is then isolated from a 1% agarose gel as described in o*o" Example 1. This vector DNA is deFignated V2.
Fragment F2 and the dephoFphorylated plasmid V2 are ligated with T4 DNA ligase. DH5a are then transformed and bacteria identified that contained the plasmid (pBac -43ooo neurooeotJide recepzcr) W,7: -h neuroO_30--Ce rcozr gene using the enzymes DamH;- and Xba1-. Te seciee of:hecoe fragment is confirmed by DNA sequencing.
Aig of the plasmid cBac neuropeptide receptor are cotransfected with 1.0 Aig of a commercia 2 -l-y available linearized baculovir~s ("BaculoGOld- baculovirus DN -A, Pharmingel, San Diego, CA using the lipofeccion method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
ipg of BaculoGold- virus DNA and 5 ยฑg of the plasrnid pBac neuropeptide receptor are mixed in a sterile well of a microtiter dlate contafiing 50 Lj of serum free Grace's medium (Life Technologies Inc., Gaithersburg,
MD).
Afterwards 10 41 Licofectin plus g0 Al Grace's medium are added, mixed and incubated f or 15 minutes at room temperature. Then the transfect--ion mixture is added drop wise to the Sf9 insect cells (ATOC CRL 1711) seeded in a mmn tissue culture plate with 1 m-l Grace' medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27 0 C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation continlued'at 27 0 C for four days.
After four days the supernata.-r is collected and a plaque assay performed similar as described by Summurers and Smith (supra). As a modification an agarose gel with "Blue Gal" (Life Technlologies Inc., Gaithersburg) is used which allows an easy isolat:ion of blue stained plaques.
(A
detailed description of a "plaque assay" can also be found in the user's guile for insect cell culture and baculovirology distributed by Life Techn--ologies Inc., n~itersburg, page 9- -44- S.
S
S
S
S
S
S.
S
S
Four days after the serial dilu:in the v-rus is added to the cells and blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 ul of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculoviruses is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then stored at Sf9 cells are grown in Grace's medium supplemented with heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-neuropeptide receptor at a multiplicity of infection (MOI) of 2. Six hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 1 Ci of "S-methionine and pCi "S cysteine (Amersham) are added. The cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS- PAGE and autoradiography.
Example 4 Expression via Gene Therapy 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 Sto the bottom of the flask and fresh media Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added.
1.0" This is then incubated at 37 0 C for approximately one week.
At this time, fresh media is added and subsequently changed ooโ€ขโ€ขo every several days. After an additional zc weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
pMV-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 containing an EcoRI site and the 3' primer having contains a HindIII site.
Equal quantities of the Moloney murine sarcoma virus linear backbone and the EcoRI and HindIII fragment are added together, in the presence of T4 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 agarcontaining kanamycin for the purpose of confirming that the vector had the gene of interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles' Medium (DMEM) with 10% calf serum
(CS)
S 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 harveted from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore 0 filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from -46- -46a 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 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.
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.
Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the Sinclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
e o**ee *ee -47- SEQUENTCE L'sTIN- GENERA.L
INFORMATION:
Ci) APPLICANT: LI, ET AL.
(ii) TITLE OF INVENTION: B:urin~ Neuropez~c.de Deceo _or (iii) NUM3ER. OF SEQUENCES: 12 (iv) CORRESPONDENCE
ADDRESS:
ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECG{I, STEWART
OLSTEIN
STREET: 6 BECKER FARM ROAD CITY:
ROSELAND
STATE: NEW JERSEY CE) COUNTRY:
USA
ZIP: 07068 COMPUTER READABLE
FORM:
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MS-DOS
SOFTWARE: WORD PERFECT 5.2.
(vi) CURRENT APPLICATION
DATA:
CA) APPLICATION
NUMBER:
FILING DATE: concurrently CC) CLASSIFICATION: (vii) AITORNEY/AGENT INFORMATION1.1: N AME: FERRARO, GREGORY
D.
REGISTRATION NUJMBER: 36,134 CC) REFERENCE/DOCKET NUMIBER: 325800-268 L vi TELECOMMUNICATION
INFORMATION:
CA) TELEPHONE: 201-994-1700 B B) TELEFAX 201-994-1744 INFORMATION FOR SEQ ID NO:1: i) SEQUENCE CHARACTERIST7CS A) LENGTT-{: 1209 BASE PAIRS TY.PE: NUCLEIC
ACID
CC) STRANDEDNESS:
SINGLE
TOPOLOGY:
LINEAR
MOLECULE TYPE: cDNA xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: *ATGGAGCCCT CAG CCACCCC AG4GGGCCCAG AT-!GC-G4GGTCC CCCCGGCAG CAGACZAG ccG TCCCTTC C-1CCAGACrA TGAAGATGIAG T-.CTCCGC-i ATCTG7GGCC TGA77ATCTG 120 -48- TACCCAAAA,;C AGTATGAG?3S CrGGTGGCA
ACACGCTGG?-
ACCAACTACr TCA~rGTCAA CCGGCCAGCC
TGCTGGTGGA
GTCATCCCCT
ATCTACAGGC
GCCC1'GGAC'C
GCTGGTATGC
GCCCGTGG CT- CC-ATCCTGGG GCAGTCATGG AATG CAGCAG GTCTGTCATG A.ACGC7,GGGC ATTGTCACCT
ACCTGGCCCC
A~ccTCTGGG GcCGCCAGAT cccTCAGACC
AGCTGCGGGA
CGCGCCI=CC
TGGCTGAAGT
ATGGTGGTGC
TGCTGGT=F
?AGAGGTGTGT
TCGGGATGTT
ACCTI'CTCCC ACrGGCrGGT CTCAGTGGC-A AA~rCCGGGA GGTCCCTGCG GCTCrTGAA
TCCTGTAG
CTGCC7TG-GCC CCTGTCCCTiG
CATCACTGAG
TGTGTCCG7G CAT CTG CCAC CAT GG C?-
TGTGCW-GCC?
AGAT.GACCTC
ACTGGGCCTC
CCCCGGCACC
CCrGGAGCAG
GAAGCAGATG
CGCCCTCTGC
CCGCCAAGCC
GTACGCCAAC
GCAGTTI'AAG
GGCCCCTAGT
CCAGCCC?7A CT3ThGCG-A ACCA CCACA GC-.aACG=IC TG'7TGACTGC TC-r GrGCT GT TCGG-CCATGC ?-AGTGGC-AG
TGC-,ACTCT
c,C- ATAG T TcAAGAGCAC GCGTCG CTGG CO-- ATOATO CAGC-AGCCA
ACCGCACACG
TATCCCAAGA
-CTACCACAG
ATG~C CATGG CC-TA=TC CA ACCTCAGCAC
TGGTGCGC-A
GGCC-TGAGTG
GAGAGCCCCA
CGTGCACGGA
GGAAGACAGC
TACCTGCCCA
TCAGCGTCCT
AGTGACCG CG AAGCTGTCrA AGCGCTGCCA
ACCCCATCAT
GCTGCCFCT CCTGCTGCCr CC.-CGCrCCT- CrGCcZAGCcA GAGGIACAG7 C
TATCOGCCTG
C CT -CTCCAAZ
CAGCTCT
AGCCCGGCGG
GCCCCAC-OC?
GCCTCTCA
TAC C CC C
CTIGGAAGCGC
GccccGGc-GC
CAAGATGCTG
CAATGTCCTI
CGCCT.GCC
CTACAA=C
GCCTGGCCTG
CGTCCTT=G
300 360 420 480 540 600 660 720 840 900 960 1020 1080 1140 1200 1209 INFORMATION FOR SEQ ID NO:2: SEQUENCE CH.ARACTrERISTICS LENGTH: 402 AMINO ACIDS TYPE: AMINO ACID
STRANDEDNESS:
TOPOLOGY:
LINEA-R
(ii) MOLECULE TYPE:
PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: t- (7Ai, Pro Sr Ala Thr Pro Gly Ala Gin Met Gly Val Pro Gly Ser Arg Giu Phe Leu A-r; Tyr Glu Tr-p Val Leu Leu Val Gly Asn His Met- Ar; Thr Ala Asp Val Leu Val Asp Ile Thr Val Ile Pro TIyr Thr Leu Ser Phe Pro Leu Leu Phe Leu Gly Ile Trp Pro 20 Leu 35 Ile so Thr 6S Val 80 Val 95 Glu 110 Leu 125 Ile 140 Lys 155 Ala 170 Ser Pro Val Pro Trp Ar; Asp Tyr Ala Ala Tyr Val Leu Val Cys Leu Ttir Asn Tyr Phe Thr Ala le Cys Ser Tr= Leu Phe Gin Ala Val 5cr Ala Leu Asp Ar; 5cr Thr Ala Ar; Val 5cr Leu Ala Pro- 25 Leu 40 Al a 55 Ala 70 lie 85 Leu 100 Gly 115 Vai 130 Trp 145 Ar; 160 Ile 175 AS-~ Tyr Glu Asp Tyr Pro Lys Val Phe Val Gin Val Val Trp Ar; Asn Val Asn Leu 5cr Pro Ala Ser Leu His Ala Leu Cys 5cr Val Ala Val Tyr Aia Ile Cys Ala Ar; Gly Ser Mer- Val Pro Gin Pro Giu Tyr Ala His Leu Leu 105 Lys 120 Leu 135 His 150 Ile 165 Al a 180 -49- -49- Ala Val Me: GL Thr Ar; Leu Phe Tyr Pro Lys le Ala Pro Leu Gly Lys Leu Tro Gly Ar; Asn Trp Lys Gly Leu Ser Gly Giu Val Lys Gin Met Val Val Leu Val Leu ASfl Val Ser ASp Ar; Glu.
Leu Val Tyr Ala Leu Ser Gly Lys Cys Leu Pro Gly Pro Arg Ser Ser 185 Ser 200 Tyr 215 Leu 230 A-r g 245 A-rg 260 Glu 275 Met 290 Leu 305 Leu 320 Al a 335 Asn 350 Phe 365 Leu 380 Ala 395 5cr Ser Val Cys His Ser Met_ Ala Gin Ile Pro Ser Pro Gin Ar; Ala Val Phe Lys A-r; Val Tyr Ser Ala Arg Giu Gly Pro Ser His Val Leu Pro G Iu e-; 190 Aso Gl.. Ar; Tro-t Ala 205 Cvs Phe Phe Ile Val 220 Met Ala Tyr phe Girn 235 Pro Gly Thr Th~r Ser- 250 Asp Gin Leu Gly AS p 265 Pro Arg Gly Ar; Ala 280 Arg Ar; Lys Thr Ala 295 Ala Leu Cys TIyr Leu 310 Val Phe Gly Met Phe 325 Ala Cys Phe Thr Phe 340 Ala Asn Pro Ile Ile 355 Gin Phe Lys Ala Ala 370 Cys Gly Ser Leu Lys 385 Lys Ser Leu Ser Leu 400 a A S n ASO ASO Thr Tvr Ile Phe Ala Leu Leu Giu Phe Leu Lys Met Pro Ile Ar; Gin Scr His TPyr Asn Phe Ser Ala Pro Arg 195 Leu 210 Leu 225 Ar; 240 Val1 255 Gin 270 Ala 285 Leu 300 Ser 315 Ala 330 Trp 345 Phe 360 Cys 375 Ser 390 INFORMATION FOR SEQ ID NO;3-: SEQUENCE
CHARACT'ERISTICS
LENGTH: 1110 BASE PAIRS TYPE: NUCLEIC
ACID
STRANDEDNESS:
SINGLE
TOPOLOGY:
LINEAR
C CT
TCCCCTGT.GC
TACCCAAAAC
cTC-GTGGG CA
ACCAA.CTACT
CCGGCCAGCC
GT CATC C CCT CCCC'rGGAcc
GCCCG-TGOCT
GCAGTCATGC
(rGTCATG A G TCAC AAC CTCTCG
CAGCCACCCC
CTCCAGACTA
AGTATC-AOTG
ACACGCTGGT
TCATTGTCAA
TGCTGGTG(SA
ATCTACAGGC
G=G-CTGC'~O
CCATCCTGG
AATCCAGCAG
AACGTGGIGC
ACCTGGCCCC
G CCC CCAGAT
AGGGGCCCAG
TGAAGATG. G GGTC CT CAT C cTGCCTGGCC
CCGTCCCTG
CATCACTGG
TGTGTCCGTG
CAT CrGCCAC CAT CTGC-IGCT
TGTGCTGCCT
AGATGACCTC
ACTOTGOC CT C ATGGCOfG-1CC CCCCT-GGCAC-- =TCTCCGC-, ATcCCTGGCG
GTOGCCGAT
O CrcACGTC
TC:CTGGCTGT
TCAGTGGCAG
CcACTA
T
G
GTGT CGCTGG
G.ACCTAGCCA
TATCCCA-A
ATCOCCATGG
ACCACCACAT
TGGTG.ACTC
TC-GCCATGC:
TGCTrAAcT'-- TCAACX.-7 CAC CCA TC.-GCT
ACCOGCACACG
TCTACCACAG
CCTA=CCA
C;AC1;AOCCC
CGTGTGOCC
GAGACAOTC
TATCTGCCT.G
CCTCTGCAAG
CAG=rCATC ArccccGCC-3 CCCCAGC CT G .CTrCTCA T7GCcr
GATACTC.CCC
CTGGAAGCGC
120
ISO
240 300 360 420 480 540 600 660 720 780 MOLECULE TYPE: CDNA SEQUEN~CE DESCRIPTION: SEQ ID NO:3: CCC~cGGCACC AC--CAGCAC TGGT(GCGU'.
CC=TCAGACC AGZrGG C.-GGAGCAG c30VTGAG~G GAGCAGCZ A 1 CGCGC rTCC TGGC-1 C X TCA T GAAGG0AAGAC-; C OA~A7G0C 300 ATGGTGGTGC TGCTGGT= CGCCCTC1GC 'ACCTCCCC TCAGCGCCT OA:C, 960 AAGAGGGTGT TCGGGATGT-, CCGCCAAGCC AGTGACCGCG AACCTGTCTA CGCCGL. iC 1020 ACCTOTCCC ACTGGCGGT GTACGCCAAC AGCGJTGCCA ACCCCATCAT CAC C 1080 CTCAGTGGCC ,CCCTGGAG TCrCTCTAA 1110 INFORMATION FOR SEQ ID NO:4: SEQUENCE
CHARACTERISTICS
LENGTH: 369 BASE PAIRS TYPE: AMINO ACID STRANDEDNESS:
SINGLE
TOPOLOGY:
LINEAR
(ii) (xi) MOLECULJE TYPE: cDNA SEQUENCE DESCRIPTION: SEQ ID NO:4: Met C Glv 5 Phe Glu Leu His I Ala Val Val Thr Pro Leu Ala Tyr Ala ,lu 3er eu rrp Jal Met Asp Asp Ile Leu Leu Gly Val Ar g Pro Pro Pro Ser Ala Arg Glu Pro Arg Tyr Leu Val Leu Ile Gly Asn Thr Arg Thr Val Val Leu Val Ile Thr Glu 110 Pro Tyr Leu 125 Ser Phe Ile 140 Leu Phe Lys 155 Ile Trp Ala 170 Met Glu Cys 185 Leu Phe Ser 200 Lys Ile Tyr 215 Leu Gly Leu 230 Thr Ser Trp Ala Leu Thr Thr Ser Gin Ala Ser Val Ser Val His Met Cir Pro Gly Ala Pro Val Pro Arg Asp Tyr Ala Tyr Val Val Cys Leu Asn Tyr Phe Ala Ile Cys Trp Leu Phe Ala Val Ser Leu Asp Arg Thr Ala Arg Ser Leu Ala Ser Val Leu Cys Asp Glu Ser Cys Phe Ala Met- Ala Ile Pro Gly G1n Met Gly Val Pro Pro 10 Pro 25 Leu 40 Ala 55 Ala 70 Ile 85 Leu 100 Gly 115 Val 130 Trp 145 Arg 160 Ile 175 Pro 190 Asp Tyr Tyr Pro Val Phe Val Trp Val As Pro Ala His Ala Ser Val Tyr Ala Ala Arg Met Val Glu Leu Glu Asp Lys Gin Val Val Arg Asn Leu Ser Ser Leu Leu Cys Ala Val Ile Cys Gly Ser Pro Gin Ala Asn is Glu Tyr Ala His Leu Leu 105 Lys 120 Leu 135 His 150 Ile 165 Ala 180 Arg 195 A-rg Trp Ala Asp Asp Leu 205 210 Phe Ile Val Thr Tyr Leu 220 225 Tyr Phe Gin Ile Phe Arg 235 240 Thx Thr Ser Ala Leu Val 250 255 Leu Gly Asp Leu Glu Gin 265 270 Lys Leu Trp Gly Arg Asn Trp Lys ar 245 Arg Pro Ser 260 Asp Gin -51- Gly Leu Ser Gly Glu 275 Glu Val Lys Gin Met 290 Met Val Val Leu Leu 305 Val Leu Asn Val Leu 320 Ser Asp Arg Glu Ala 335 Leu Val Tyr Ala Asn 350 Pro GIn Pro APrg Ar; Ala Ar; Ar; Val Phe Ala Leu Lys A-r; Val Pne Val Tyr Ala Cys Ser Ala Ala Asn Gly 280 Lys 295 Cys 310 Gly 32'5 Phe 340 Pro 355 A Aa eu_ Ttr Ala Lys Met Tyr Leu Pro Ile me-, phje Ar;g Gin Thr Phe Ser His Ile Ile Tyr Asn Ala 235 Leu 300 Ser 315 Ala 330 Tr= 345 Phe 360 Leu Ser Gly Leu Pro Trp Ser Let' Leu 365 INFORMATION FOR SEQ ID SEQUENCE
CHARACTERISTICS
LENGTH: 1133 BASE PAIRS TYPE: NUCLEIC
ACID
STRANDEDNESS:
-SINGLE
TOPOLOGY:
LINEAR
(i i) MOLECUE TYPE: cDNA (xi) ATGGAG C CCT
TCCCCTGTGC
TACCCAAAAC
CTrGGTGGGCA
ACCAACTACT
CCGGCCAGCC
GTC-ATCCCCr
GCCCTGGACC
GCC CGTGG CT GCAC :CATGG
GTCT-GTGATG
AITGTCACCT
AAG CTCTGGG CCCT CAGACC
CGCGC=TCC
ATGGTGGTGC
AAGAGCTGTGT
AC~c--rCCC CTCAGTGGA7T S EQUENC t-AG CCACCCC
CTCCAGACTA
AGTATGAGTG
ACACGCTGGT
TCATTGTCAA
TGCTGGTGGA
ATCTACAGGC
GCTGGTATGC
C CAT CCTGGG AATG CAG GAG AACGCrGGGC
ACCTGGCCCC
GCCG CCAGAT
AGCTGGGGG
TGG CTGAAZT TcGcGGATGT
ACT.GGCTGGT
E DESCRIPTION:
SE(
AGGGGCCOAG ATGGGGGTCC TGAAGATGAG =rCTCCGCT GGTCCTCATC GCAGCCTrATG CTGCCTGGCC
GTGTGGCGGA
CCTrGTCCCTG
GCTGACGTITC
CATCACTGAG
TCCTGGCTGT
TGTGTCCGTG
TCAGTGGCAG
CATCTGCCAC CCACTATTrGT CATCTGG-GCT
GTGTCGCTGG
TGTGCTGCCr
GAGCTAGCCA
AGATGACcTrC TATCCCAAGA ACTGGGCCTC
ATGGCCATG
ccCCGGCACC
ACCTCAGCAC
CCTGG-AGCAG
GGCCTGAGTG
GAAGCAGATG
CGTGCACGGA
CGCCCTCTGC TACCTGC~CCA CCGCCAAC-CC
AG"TGACCGCG
GTACGCCAAC
AGCGCTGCCA
GAGTCTrAG'
CTTCCTGAC
ID NO:
CCCCTGGCAG
ATCTGTGGCG
I
TGCTGTG=T
ACCACCACATC
TGGTGACTGC
TCG-GCCATGC
TGCTAACTCT
TCAAGAGC-A-
CCATCATGGT
ACCGCACACG
TCTACC-ACAG
CCATCCA.
TGGTGCGGAA
GAGAGCCCCZA
GGAAGACAGC
TCAGZCGTCCT
AAG=GTTCTA
ACCCCATCAT
CATCGTGCCC
:AGAGACCCC
~GACTATCTrG
:GTCGTGGCC
AGGACAGTC
CATCrGCCTG CTCrGCAAG CAG=rCA6TC
,GCCC-GGCG
3CCCCAGGCT 3cTrrcCA rrdcr
GATAT'CCGC
CrGGAAGCGC
GCCCCGGGGC
CAGCATGCT-G
CAATGTC=r CGCCT-G=Cr CTACAA~cT
CG
120 IS0 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1133 t 0* 4.
S
C
INFORMATION FOR SEQ ID NO:6: Ci) SEQUENCE
CHARACTERISTICS
CA) LENGTH: 377 BASE PAIRS TYPE: AMINO ACID STRANDEDNESS:
SINGLE
TOPOL(")GY:
LINEAR
(ii) (Xi MOLECULE TYPE: cDNA SEQUENCE DESCRIPTION: SEQ ID NO:6: -52- Me: Glu Pro Gly Ser Arg Phe Leu Arg Glu Trn Val Leu Val Gly His Me: Arg Ala Asp Val Val AsD Ile Val Ile Pro Thr Leu Ser Pro Leu Leu Leu Gly Ile Ala Val Me: Thr Arg Leu Tyr Pro Lys Ala Pro Leu Lys Leu Trp Arg Asn Trp Gly Leu Ser Glu Val Lys Me: Val Vai Val Leu Asn Ser Asp Axg Leu Val Tyr Leu Ser Gly Pro Gly Ser Glu Tyr Leu Asn Thr Leu Thr Tyr Phe Phe Trp Glu Phe Ile G1y Gly Lys Gly Gin Leu Val Glu Ala Cys Ala Pro Leu Ile Thr Va1 Va1 Glu 110 Leu 125 Ile 140 Lys 155 Ala 170 ys 185 Ser 200 Tyr 215 ieuL 230 Arg 245 Arg 260 Glu 275 Met 290 Leu 305 Leu 320 Ala 335 Asr Tlys 365 Thr Pro Ser Pro Tro Arg Ala Ala Leu Val Thr Asn Thr Ala Ser Trp Gin Ala Ala Leu Ser Thr Val Ser Ser Ser Val Cys His Ser Met Ala Gin Ile Pro Ser Pro Gin Arg Ala Val Phe Lys Arg Val Tyr G1lv Val Asp Tvr Cys Tyr Ile Leu Val Asp Ala Leu Val Asp ys Me: Pro Asp Pro Arg Ala Val Ala kia Gin Met 10 Pro Pro Asp 25 Pyr Leu Tyr 40 Vai Ala Val 55 Leu Ala Val 70 Phe Ile Val 85 -ys Leu Pro 100 Phe Giy His 115 Ser Val Ser 130 krg Trp Tyr 145 krg Arg Ala 160 kia Ile Met 175 Leu Pro Glu 190 Glu Arg Trp 205 Phe Phe Ile 220 Ala Tvr Phe 235 Gly Thi Thr 250 Gin Leu Gly 265 Arg Gly Arg 280 Arg Lys Thr 295 Leu Cys TYr 310 Phe Gly Met 325 Cys Phe Thr 340 Asn Pro Ile 355 Leu Val Leu 370 C- 1 Tyr Pro Phe Trp Asn Ala Ala Val Ala Arg Val Leu Ala Val Gin Ser Asp Ala Ala Leu Phe Phe Ile Ser Val Pro Pro Glu Asp Glu Lys Gin Pyr Val Val Ala Arg Asn His Leu Ser Leu Ser Leu Leu 105 Leu Cys Lys 120 Ala Vai Leu 135 Ile Cys His 150 Gly Ser Ile 165 Pro Gin Ala 180 Ala Asn Arg 195 Asp Asp Leu 210 Thr Tyr Leu 225 Ile Phe Arg 240 Ala Leu Val 255 Leu Giu Gin 270 Phe Leu Ala 285 Lys Me: Leu 300 Pro Ile Ser 315 APrg Gin Ala 330 Ser His Trp 345 Tyr Asn Phe 350 Pro Ser Cys 375 *s Ser Ala Ala Glu Lys Ser a. -53- 1INFORMATION FOR SEQ ID INO: 7: SEQUENCE
CHAPLACTERISTICS
LENGTH: 30 BASE PAIRS TYPE: NUCLEIC ACID STRANLEDNESS: S INGLE TOPOLOGY: LI NEAR (ii) MOLECUL-E TYPE: Oligonucleocide (xi) SEQUTENCE DESCRIPTION: SEQ ID NO:7: CACTAAAGCI' TAATGGAGCC CTCAGCCACC INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACT'ERISTICS ()LENGTH: 25 BASE PAIRS TYPE: NUCLEIC
ACID
STRAND'LEDNESS:
-SINGLE
TOPOLOGY: LINEA!kR (ii) MOLECULE TYPE: Oligonucleoc.ide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ACAAGTCCI= GTCC=TCTAG, AGGOC INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS LENGTH: 26 BASE PAIRS TYPE: NUCLEIC ACID STRANDEDtTESS:
SINGLE
TOPOLOGY: LINEAR (ii) MOLECULE TYPE: oligcflucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CJPAGGATGC CCCTCTGCTG CAGCGG 26 INFORMATION FOR SEQ ID SEQUENCE QiARACrERISTICS LENGTH: 25 BASE PAIRS TYPE: NUCLEIC
ACID
STRANDEDNESS:
SINGLE
TOPOLOGY:
LINEAR
(ii) MOLECULE TYPE: oligonucleOtide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:210: -54- A CAAG T CJ= G T CJ7AG A GG~GC INFORMATION FOR SEQ ID NO:11: SEQUENCE CiTARACERISTICS LENGTH: 32 BASE PAIRS TYPE NUJCLEIC ACID STR.ANDEDN-ESS
SINGLE
CD) TOPOLOGY: L INEA- lR (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CGGGATCCGC CATCATOGAG CCCrCAGCCA CC 32 INFORMATION FOR SEQ ID NO:12: SEQUENCE
GIARLACTERISTICS
LENGTH: 25 BASE PAIRS TYPE: NUCLEIC
ACID
CC) STRANDEDNESS:
SINGLE
TOPOLOGY: L INEAR (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ACAAGTC=I GTCCCAG AGGGC

Claims (30)

1. An isolated antibody or portion thereof that specifically binds to a protein selected from the group consisting of: a. a protein whose sequence consists of amino acid residues 1-46 of SEQ ID NO:2; b. a protein whose sequence consists of amino acid residues 190-213 of SEQ ID NO:2; c. a protein whose sequence comprises at least 30 contiguous amino acid residues of SEQ ID NO:2; and d. a protein whose sequence comprises at least 50 contiguous amino acid residues of SEQ ID NO:2, wherein said antibody or portion thereof specifically binds to the amino acid sequence of SEQ ID NO:2.
2. An isolated antibody or portion thereof that specifically binds to a protein selected from the group consisting of: a. a protein whose sequence consists of the amino acid sequence of the full S* length polypeptide encoded by the cDNA in ATCC Deposit No. 97128; b. a protein whose sequence consists of amino acid residues 1 to 402 of SEQ ID NO:2; c. a protein whose sequence consists of amino acid residues 1 to 369 of SEQ ID NO:4; and d. a protein whose sequence consists of amino acid residues 1 to 372 of SEQ ID NO:6.
3. An isolated antibody or portion thereof that specifically binds to a protein expressed on the surface of a cell, wherein said protein is selected fom the group consisting of: a. a protein whose sequence consists of the amino acid sequence of the full length polypeptide encoded by the cDNA in ATCC Deposit No. 97128; b. a protein whose sequence consists of amino acid residues 1 to 402 of SEQ ID NO:2; c. a protein whose sequence consists of amino acid residues 1 to 369 of SEQ ID NO:4; and d. a protein whose sequence consists of amino acid residues 1 to 372 of SEQ ID NO:6.
4. The antibody or portion thereof of any one of claims 1, 2 or 3, wherein said protein specifically bound by said antibody or portion thereof is glycosylated. The antibody or portion thereof of any one of claims 1,2 or 3 which is a monoclonal antibody.
6. The antibody or portion thereof of any one of claims 1,2 or 3, which is a polyclonal antibody.
7. The antibody or portion thereof of any one of claims 1, 2 or 3 which is a chimeric antibody.
8. The antibody or portion thereof of any one of claims 1,2 or 3 which is a humanized antibody.
9. The antibody or portion thereof of any one of claims 1, 2 or 3 which is a human antibody.
10. The antibody or portion thereof of any one of claims 1, 2 or 3 which is a single chain antibody.
11. The antibody or portion thereof of any one of claims 1, 2 or 3 which is a Fab fragment.
12. The antibody or portion thereof of any one of claims 1, 2 or 3 which is labeled.
13. The antibodyof claim 12, wherein the label is selected from the group consisting of: a. an enzyme label; b. a radioisotope; and c. a fluorescent label.
14. A composition comprising the antibody or portion thereof of any one of claims 1, 2 or 3 and a carrier. The composition of claim 14, wherein the antibody or portion thereof is a monoclonal antibody.
16. The composition of claim 14, wherein the antibody or portion thereof is a chimeric antibody.
17. The composition of claim 14, wherein the antibody or portion thereof is a humanized antibody.
18. The composition of claim 14, wherein the antibody or portion thereof is a human antibody. .i 19. The composition of claim 14, wherein the antibody or portion thereof is a single chain antibody. The composition of claim 14, wherein the antibody or portion thereof is a Fab fragment.
21. The composition of claim 14, wherein the antibody or portion thereof is labeled.
22. The composition of claim 21, wherein the label is selected fiom the group consisting of: a. an enzyme label; b. a radioisotope; and c. a fluorescent label.
23. An isolated cell that produces the antibody of any one of claims 1, 2 or 3.
24. A hybridoma that produces the antibody of any one of claims 1, 2 or 3. A hybridoma that produces the antibody of claim
26. A method of detecting Human Neuropeptide Receptor protein in a biological sample comprising: a. contacting the biological sample with the antibody or portion thereof of any one of claims 1, 2 or 3; and b. detecting the Human Neuropeptide Receptor protein in the biological sample.
27. The method of claim 26, wherein the antibody is a monoclonal antibody.
28. The method of claim 26, wherein the antibody is a polyclonal antibody.
29. The method of claim 26, wherein the antibody is a chimeric antibody.
30. The method of claim 26, wherein the antibody is a humanized antibody.
31. The method of claim 26, wherein the antibody is a human antibody.
32. The method of claim 26, wherein the antibody is a single chain antibody.
33. The method of claim 26, wherein the antibody is a labeled antibody.
34. The method of claim 33, wherein the label is selected from the group consisting of: a. an enzyme label; oo a radioisotope; and c. a fluorescent'label. An isolated antibody or portion thereof produced by immunizing an animal with a protein selected from the group consisting of: a. a protein whose sequence consists of amino acid residues 1-46 of SEQ ID NO:2; b. a protein whose sequence consists of amino acid residues 190-213 of SEQ ID NO:2; c. a protein whose sequence comprises at least 30 contiguous amino acid residues of SEQ ID NO:2; and d. a protein whose sequence comprises at least 50 contiguous amino acid residues of SEQ ID NO:2, wherein said antibody or portion thereof specifically binds to the anino acid sequence of SEQ ID NO:2.
36. An isolated antibody or portion thereof produced by immunizing an animal with a protein selected from the group consisting of: a. a protein whose sequence consists of the amino acid sequence of the full length polypeptide encoded by the cDNA in ATCC Deposit No. 97128; i b. a protein whose sequence consists of amino acid residues 1 to 402of SEQ S ID NO:2; c. a protein whose sequence consists of amino acid residues 1 to 369 of SEQ ID NO:4; and d. a protein whose sequence consists of amino acid residues 1 to 372 of SEQ ID NO:6.
AU27737/00A 1995-05-05 2000-04-13 Human neuropeptide receptor Ceased AU763711B2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU27737/00A AU763711B2 (en) 1995-05-05 2000-04-13 Human neuropeptide receptor
AU2003259580A AU2003259580A1 (en) 1995-05-05 2003-10-29 Human Neuropeptide Receptor

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AU24707/95A AU715286B2 (en) 1995-05-05 1995-05-05 Human neuropeptide receptor
AU715286 1995-05-05
AU27737/00A AU763711B2 (en) 1995-05-05 2000-04-13 Human neuropeptide receptor

Related Parent Applications (1)

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AU24707/95A Division AU715286B2 (en) 1995-05-05 1995-05-05 Human neuropeptide receptor

Related Child Applications (1)

Application Number Title Priority Date Filing Date
AU2003259580A Division AU2003259580A1 (en) 1995-05-05 2003-10-29 Human Neuropeptide Receptor

Publications (2)

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AU2773700A AU2773700A (en) 2001-02-01
AU763711B2 true AU763711B2 (en) 2003-07-31

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Family Applications (1)

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