WO1995006724A1 - Vip2 (vasoactive intestinal polypeptide) receptor - Google Patents

Abstract

The present invention provides a VIP2 receptor gene (seq ID No.1), and polynucleotide probes specifically binding to this gene or to naturally occurring variants thereof, as well as polypeptide probes specifically binding to the receptor polypeptide or to naturally occurring variants thereof. Expression of the gene in a host is also useful for providing means for use in the evaluation of VIP2 agonists and antagonists.

Description

VIP2 (VASOACTIVE INTESTINAL POLYPEPTIDE) RECEPTOR Background of invention Field of invention

This invention relates to the discovery of cDNA sequences encoding for a newly found member of the recently discovered secretin family of G-protein linked neuropeptide-receptors, which is a receptor for vasoactive intestinal polypeptide (VIP) , and the application of this discovery to inter alia 1) the development and use of nucleotide sequences derived from the receptor for the measurement (qualitative and quantitative) and regulation of gene expression and 2) the development and use of VIP agonist and antagonist analogues for clinical and therapeutic use in the management of human pituitary dysfunction, various cancers of the brain, pituitary, lung, adrenal, and reproductive system and VIP related disorders of brain function, and other conditions involving VIP.

Description of the Prior art

Vasoactive intestinal polypeptide (VIP) has been identified and purified from small intestine on the basis of its potent vasodilator activity[1, 2]. VIP has a number of actions in the periphery including vasodilatation, stimulation of electrolyte secretion and smooth muscle relaxation[3] . VIP is a member of a family of structurally related polypeptide hormones which include glucagon, glucagon-like peptide I (GLP) , peptide histidine isoleucine (PHI) , secretin, pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone releasing hormone (GHRH) . All of these peptides are thought to exert their actions through G- protein linked membrane receptors coupled to adenylate cyclase. Recently, a receptor for VIP[4], has been cloned from rat lung[5] and from a human colon carcinoma cell line[4]. Receptors for glucagon[6-8] , GLP[9], GHRH[10-12], secretin[13] and PACAP[14-16] have also been cloned. Together with receptors for calcitonin[17, 18] and parathyroid hormone (PTH) [19, 20] they form a protein family distinct from other G protein-linked receptors.

VIP has been shown to bind with high affinity to VIP receptors in a variety of tissues. These binding sites are present in lung, liver and intestine, as well as several regions of the brain (e.g. cerebral cortex, hypothalamus and hippocampus[21, 22]). This receptor is probably identical to a receptor known as the PACAP Type II receptor[23, 24] which recognises VIP, PACAP-27 and PACAP-38 with very similar affinities and may correspond to the previously cloned VIP receptor[4, 5] which has a similar tissue distribution[5] . This receptor is present in a variety of peripheral tissues including rat liver, rat lung, mouse splenocytes and human small intestinal epithelium[23, 25-28]. There is some evidence for the existence of other types of VIP receptor in the brain and periphery[29] . Cross-linking studies have been used to identify VIP binding proteins in tissues with molecular weights ranging from 46,000 to 73,000 depending on the tissue and species[30]. Studies with peptide analogs also suggest the existence of more than one pharmacologically distinct class of VIP receptor[31-35] . A class of VIP receptor, for which helodermin is the most potent ligand, has been identified in certain human cell lines, including the lymphoblastic cell line SUP-T1[36, 37] the THP-1 monocyte/macrophage cell line[38] and NCI-H345 lung carcinoma cells[39]. Hill et al. [40] have distinguished two subtypes (or different functional states of a single subtype) of VIP receptor which differ in their sensitivity to GTP analogs. In some brain regions, guanylyl- imidodiphosphate (GMPPNP) substantially inhibited VIP binding. In other regions, VIP binding was insensitive to GMPPNP. Both types of receptor are present in the mouse embryo[41], where they are differentially regulated by treatment with a VIP antagonist. To date, however, there have been no reports of the sequencing of any gene encoding a second VIP receptor with a distinct and different molecular structure.

The initial step in VIP action centres on the binding of VIP to a specific membrane bound receptor. Multiple steps are then involved in triggering the synthesis of intracellular cAMP (cyclic Adenosine Monophosphate) in tissues such as brain, pituitary, lung, pancreas, gastrointestinal tract, kidney, reproductive tract, blood vessels and various others and in certain tumours. In the central nervous system, VIP may play a role in the pathogenesis of psychiatric, neurological and neuroendocrine disorders. VIP may also regulate cerebral energy metabolism[42] and neuronal survival[43] . VIP stimulates prolactin secretion from the pituitary[44] , catecholamine release from the adrenal medulla[45] and in the immune system it inhibits mitogen activated proliferation of T cells by inhibiting interleukin-2 production[46] .

Knowledge of the structure and characteristics of VIP receptors in the brain and pituitary and other tissues would also be helpful both in attempting to use VIP or its analogs in pharmacotherapy and in attempting to understand the possible roles of VIP in the body.

Thus there is a particular need for providing probes and other assay materials and methods for use in the detection and/or identification, directly or indirectly, of abnormalities in VIP receptors, especially human VIP receptors and/or their expression, and/or the genes encoding them, as well as means for use in the evaluation of potential VIP agonists or antagonists for use in the treatment of such abnormalities.

It is an object of the present invention to avoid or minimise one or more of the above problems or disadvantages. Summary of Invention

We have now succeeded in the cloning and expression of a specific, adenylate cyclase-linked VIP receptor from rat brain. We have named this receptor the VIP₂ receptor to distinguish it from the receptors cloned from rat by Ishihara et. al. [5] and from human by Sreedharan et. al. [4]. The latter two receptors probably represent species variants of one gene, which we will refer to as the VIP_χ receptor. The VIP₂ receptor was identified by PCR of rat pituitary cDNA using degenerate oligonucleotide primers corresponding to the third and seventh transmembrane domains of the secretin family of G-protein linked receptors. Full length cDNAs were isolated from an olfactory bulb cDNA library. The cDNA sequence for the novel VIP receptor is presented as the basis of this invention. The sequence is presented in interleaved format (see Fig. 1) , and has been submitted to the EMBL/Gen Bank database under accession No. Z25885.

Thus we have shown for the first time that VIP receptor occurs naturally in at least two different forms with distinct tissue distributions and different molecular structures. This indicates the possibility of different physiological functions for the different receptors and the need for diagnostic means and methods for distinguishing these different forms, as well as the possibility of providing VIP2 agonists and antagonists adapted for selective interaction with the newly discovered VIP₂ receptor, for example for the purposes of controlling blood pressure and/or renal function, as well as certain tumours. Given the co-existence of both VIP₁ and VIP₂ provision by means of the present invention of cloned VIP receptor gene and systems for expression thereof, is particularly valuable in providing means for assaying VIP₂ - specific agonists and antagonists. In more detail, the present invention provides: a gene encoding VIP₂ receptor; and preferably a gene encoding VIP₂ receptor having substantially the amino acid SEQ ID No:l disclosed herewith and includes genes encoding VIP receptor which genes have substantial nucleotide sequence homology with the nucleotide sequence SEQ ID No:l disclosed herein; and in particular the genes of the present invention in a form substantially free from other genes. It will be understood that the genes of the present invention may include nucleic acid sequences (upstream and/or downstream of the receptor coding sequence) which are utilized in the expression of the gene such as promoter, operator, and terminator sequences as well as other sequences which do not inhibit its expression. Thus the expression "gene" includes DNA (including cDNA) and/or RNA sequences as well as plasmid or viral "genes" containing the receptor gene and expression vectors for the gene.

With respect to the distribution of VIP2 receptor in the body we have found that significant, and in some cases particularly high, concentrations are to be found in inter alia: the supra-chiasmatic nucleus of the brain which is involved in the control of circadian (or diurnal) and other biorhythms including those involved with sleep and arousal, feeding, drinking, various endocrine functions, and ovulation; the dorsal horn of the spinal cord and especially the substantia gelatinosa and Clark's column which are involved in the transmission and processing of pain; and tumours, especially gastro-intestinal and lung tumours.

In one aspect therefore the present invention provides new methods and means based upon the newly discovered VIP₂ receptor nucleotide sequence for use in the clinical diagnosis and therapeutic management of those processes and abnormalities thereof that involve the action of VIP and the VIP₂ receptor, including but not exclusive of: Neurological and psychiatric disorders including schizophrenia, depression, Alzheimer's disease and Parkinson's disease; Brain tumours; Pituitary tumours and neuroendocrine disorders; Cardiovascular and inflammatory disorders; Disorders of fertility, control of fertility, testicular tumours; impotence directly related to failure to achieve erection; Disorders of the kidney; Phaeochromocytoma and other tumours of neuroendocrine origin; Lung tumours; Leukaemia, immune and inflammatory disorders; Gastro-intestinal Tumours; Pancreatic tumours, pancreatitis, cystic fibrosis; Diabetes; Disorders of foetal development; especially foetal brain development; and Degenerative disorders of the nervous system.

In addition the invention provides methods and means for use in the control and/or regulation of circadian (or diurnal) and other biorhythms including those involved with sleep and arousal, feeding, drinking, various endocine functions, and ovulation; as well as of pain thereby providing valuable new alternative means for providing analgesia.

In the case of tumours there may be used anti-sense polynucleotides in order to restrict or inhibit the growth of tumours. Preferably there are used anti-sense polynucleotides capable of binding to part of the gene which includes the ATG (initiation) codon corresponding to the start of the VIP2 protien coding region.

In general the VIP2 receptor gene sequences are also useful for the design of oligonucleotide probes capable of specifically hybridising with the genes of the present invention, and for the synthesis of polypeptides which may be used in immunoassays. In addition parts of the cDNA sequence may be used to design and/or provide oligonucleotide probes for use in identifying human and other mammalian VIP receptor genes. Thus the present invention also specifically extends to a human gene DNA sequence which has been obtained directly or indirectly through recovery of a human gene by hydridisation thereof with a polynucleotide probe comprising a labelled DNA or RNA sequence capable of specifically hybridizing to a gene encoding VIP2 receptor having the amino acid sequence of SEQ ID No. 1 disclosed herein. In general any part of the VIP2 receptor coding region of the gene may be used for this probe. Both oligonucleotide probes and the polypeptides may be useful for the diagnosis of VIP2 receptor abnormalities. (It will be understood that references to oligonucleotide probes and the use thereof also include such probes as part of longer sequences) . Polypeptides encoded within the cDNA sequences may also be used to raise antibodies against selected regions of normal or abnormal VIP2 receptor polypeptide, which are particularly implicated in ligand binding i.e. the first, second, third and fourth extracellular domains of the VIP2 polypeptide (see fig 2) ; or in signal transduction i.e. the first, second, third and fourth intra-cellular domains, especially the main cytoplasmic loop or third intracellular domain, most preferably in the region of the interface between the third intracellular domain and the sixth transmembrane region, e.g. one of the four extra-cellular domains identified hereinbelow, and for the purification of antibodies directed against such regions. These antibodies may be useful in immunoassays for detecting normal or abnormal VIP₂ receptor in individuals.

Furthermore the invention provides screening means for use in the evaluation of new VIP₂ receptor agonists and antagonists, comprising a cell transformed with a recombinant expression system comprising an open reading frame (ORF) of DNA derived from a VIP₂ receptor gene or VIP₂ receptor cDNA, said ORF being operably linked to a control sequence compatible with said cell, as well as such expression systems per se. Thus the present invention further includes a method of producing receptor which method includes the step of expressing the genes of the present invention in a host, as well as VIP₂ receptor produced by such a method. Various suitable hosts are known in the art though eukaryotic hosts are generally preferred, e.g. Xenopus oocytes and COS-7 cells. Prokaryotic hosts that may be used include E. coli. and B. Subtilis. Fungi e.g. yeast may also be used. For the purposes of developing VIP₂ agonist and antagonist analogues, it is particularly preferred to use cloned cell lines expressing human VIP₂ receptor. Genes for human VIP₂ receptor can be isolated and sequenced by screening a human cDNA library preferably a human tumour (e.g. adrenal, pancreas, lung or brain) or normal adult or foetal human brain, with the aid of oligonucleotide probes from the entire rat VIP2 receptor sequence or parts thereof.

Thus the present invention also includes products and processes utilizing, directly or indirectly, human VIP₂ receptor sequences obtained in this way. In particular the invention provides means for the evaluation of new VIP₂ receptor agonists and antogonists. These may employ expression systems of the invention directly or in some cases simply the membranes (with VIP₂ receptor) thereof. Conveniently the transfected or transformed cells, or the membranes thereof, are exposed to a suitable ligand e.g. labelled VIP, and the potential agonist or antagonist and the effect of the latter on the binding of the ligand is monitored, e.g. by examining the variation of binding level with agonist/antagonist doseage. Alternatively or additionally there may be monitored a natural or artificial intra-cellular messenger system such as cAMP production, inositol phosphate turnover, cell calcium concentration, or expression of an artificial gene containing an enzyme marker producing a colour or light reaction etc. A suitable method of restriction enzyme analysis in this invention depends on Restriction Fragment Length Polymorphisms (RFLPs) . A sample is taken from any suitable tissue such as blood. DNA is extracted from the cells in any conventional way. It is then digested with an appropriate restriction enzyme e.g. one which cuts in CG-rich sequence. The fragments of different length are separated by gel electrophoresis in any conventional way. A restriction fragment pattern is generated. Probing of the fragments will generally be necessary for clearer detection of the pattern and of the fragment(s) of interest, e.g. a fragment which extends from restriction sites "n" to "n + 2" (where "n" denotes any arbitrary number) , seemingly not being restricted at the normal site "n + 1" lying between "n" and "n + 2" due to an abnormality at the "normal" restriction site. Alternatively, a polymorphism might generate restriction enzyme sites and thereby given rise to a plurality of shorter fragments where the normal DNA provides longer ones. Whether it is appropriate to probe for long or short fragments will therefore depend on the circumstances of the polymorphism. In some instances, the probe will extend outside the region designated.

Although the RFLP method is one method of assay, it cannot be ruled out that direct hybridisation of probes to the genomic region will be of interest. Thus suitable biopsy or other samples can be subjected to cloning techniques, to isolate a library of genomic DNA, or PCR of genomic DNA or cDNA. Clones containing the gene can be amplified by Polymerase Chain Reaction (PCR) and probes complementary to the said region used directly on PCR products, which need not be first restricted by enzymes.

It will be appreciated, therefore, that the cDNA of the invention also has uses in assays which are not of the RFLP type. Accordingly, the polynucleotides per se are part of this invention, as 'intermediates' suitable (when labelled) for use as probes. Both double-stranded and single-stranded polynucleotides are included as well as sense and anti-sense forms. Suitable polynucleotide probes may be oligonucleotides of from 10 to 50, preferably from 16 to 30 nucleotides in length. Shorter probes are unlikely to be sufficiently specific for the sequence of interest. Longer polynucleotide probes of from 100 to 500 nucleotides or more may also be used with longer ones (up to 2000 or more nucleotides) being useful e.g. for chro osmal in-situ hybridisation as further described hereinbelow. Preferably the probes relate to parts of the polynucleotide sequence corresponding to one or more domains, or portions thereof, of the VIP2 polypeptide, which are particularly implicated in ligand binding i.e. the first, second, third and fourth extraceullular domains of the VIP2 polypeptide (see Figs.l and 2) ; or in signal transduction i.e. the first, second, third and fourth intra-cellular domains, especially the main cytoplasmic loop or third intra-cellular domain, most preferably in the region of the interface between the third intracellular domain and the sixth transmembrane region. The probe will usually be of DNA or RNA and labelled in any suitable manner e.g. by labelling with an enzyme, radioisotope, fluorescent, luminescent, or chemiluminescent labels or biotinylation.

The fragments are probed under any appropriate conventional hybridisation conditions, the fragments being conveniently first transferred to a filter. The complexes thus formed are detected by autoradiography or other detection means appropriate to the particular kind of label used.

Abnormalities in the polynucleotide sequence of restriction fragments of the genomic DNA or cDNA coding for VIP₂ receptor which are as small as single-point mutations can also be detected by means of Temperature Gradient Gel Electrophoresis in which a temperature gradient is superimposed, parallel to or transversely of, the electrical field in gel electrophoresis. The method is based on the fact that the temperature of denaturation of double stranded (ds) DNA is altered by changes in polynucleotide sequence. Furthermore, partial denaturation of a DNA duplex causes a change in electrophoretic mobility. Further details of this technique are described in the literature by Reisner et al, 1989 (56) and, Birmse et al, 1990 (57).

We have now further found that the chromosmal location for the VIP2 receptor gene is at 7q36.3 and thus the present invention now also provides further diagnostic means for use in the detection of conditions associated with VIP₂ abnormalities, which comprises one or more of hybridisation of a polynucleotide probe of the invention with the 7q36.3 chromosome region; and examination of the 7q36.3 chromosome region for gross abnormalities. Major defects in the 7q36.3 chromosomal region have been found to be associated with the birth defect holoprosencephaly, especially the type 3 form, and thus the present invention also provides a method of diagnosis for holoprosencephaly comprising the steps of collecting a foetal sample and examining the chromosomal content thereof by one or more of the techniques described hereinbefore so as to detect the presence of defects or irregularities in the 7q36.3 chromosomal region.

The VIP₂ receptor cDNA cloned and sequenced is shown in Fig. 1A. Fig. 2 illustrates schematically the predicted transmembrane, intracellular and extracellular domains of the receptor molecule. The transmembrane domains consist of seven stretches of hydrophobic in nature amino acids which span the membrane. The extracellular domain consists of four hydrophilic in nature amino acid stretches which exist exterior to the cell membrane. This region is believed to be important for the recognition of specific ligands. The intracellular domain consists of four hydrophilic stretches of amino acids which are thought to be involved in signal transduction. It will be appreciated that abnormality in human VIP₂ receptor and/or its expression, may be "assayed" in a number of ways. Thus the DNA encoding the VIP₂ receptor may itself be assayed for the presence or absence of abnormalities or the VIP₂ receptor polypeptide may be assayed for such purposes, where this is actually expressed.

The former case generally involves the use of labelled polynucleotide probes to hybridise with DNA within the region coding for VIP₂ receptor polypeptide for the purposes of indicating the presence of absence of particular polynucleotide sequences. In the latter case antibody probes are used to form antigen-antibody complexes with regions of the expressed polypeptide for the purposes of indicating the presence or absence of particular polypeptide sequences. It will be understood that once more or less common or typical abnormalities have been specifically identified e.g. by initially probing with 'normal' polynucleotide and then sequencing, polynucleotide probes can be synthesized or otherwise produced with sequences corresponding to or complementary to the "abnormal" sequences, to allow screening of tissue samples for specific VIP₂ receptor gene abnormalities. In the case of gene abnormalities such as the complete or substantial absence of the VIP₂ receptor gene, then this may be detected by failure to yield any binding with suitable probes or absence of any product from PCR amplification using polynucleotide probes complementary to substantially spaced apart portions of the gene.

In the case of assays of the polypeptide itself, suitable stretches of amino acids based on the cDNA sequence information provided by the present information, may be synthesised on a peptide synthesiser. These peptides would generally have a length of from 10 to 50, preferably 15 to 30, amino acids but could be even shorter or longer. Alternatively the complete VIP₂ receptor polypeptide or fragments thereof may be expressed in a suitable eukaryotic or prokaryotic host such as E. Coli using an appropriate vector. Polyclonal antibodies to these peptides may be produced by conventional approaches such as the immunisation of host animals (rabbit, goat etc.) with said peptides, optionally conjugated to a protein carrier such as thyroglobulin, and recovery of the desired antibody material therefrom. Monoclonal antibodies could also be raised using conventional monoclonal antibody-production procedures.

It will also be understood that "assay" may be either qualitative or quantitative (e.g. where detection of under or over-expression of the receptor is required) . Further preferred aspects of the invention are indicated in the following patent claims.

Detailed Description Isolation and Sequencinq of VIP₂ receptor Gene Preparation of Rat Pituitary cDNA

Anterior pituitary glands from male rats (Cob Wistar, 250g) were removed and total RNA was isolated by the method of Chomczynski and Sacchi[47]. Single stranded cDNA synthesis and PCR were carried out using a commercial kit (Perkin Elmer Cetus) . lμg RNA was annealed with 2.5μM random hexanucleotide primers by heating to 95°C for five minutes, then cooling to 4°C over fifteen minutes. Single stranded cDNA was synthesised by incubating the oligonucleotide-RNA solution at 42°C for 15 minutes in 20μl lOmM Tris-HCl, pH 8.3, containing 50mM KC1, 5mM MgCl2, ImM each dNTP, 20 units RNase inhibitor and 50 units reverse transcriptase. The reaction was terminated by heating to 99°C for 5 minutes.

Selective Amplification of Rat Pituitary cDNA PCR was performed using a pair of degenerate 32-mer oligonucleotide primers (Figure 3) , corresponding to conserved regions in the third and seventh transmembrane domains of the rat secretin[13] , pig calcitonin[18] and opossum parathyroid hormone PTH[19] receptors. In more detail, each "primer" comprises a mixture of a large number of oligonucleotides with different permutations of nucleotide at particular positions within the oligonucleotide sequence corresponding to the codons for particular amino acids within the receptor peptide sequence coded for thereby. This is necessary in order to include different codons which code for the same amino acid due to the degeneracy of the genetic code. Alternative nucleotides are indicated above each other. Where it is desired to include the possibility of any one of the A, G, C, and T nucleotides (containing the bases Adenine, Guanine, Cytidine, and Thymidine) then the I nucleotide (with the base Inosine) is used. Sequences containing restriction sites (EcoRI and BamHI, respectively) were added to the ends of the two oligonucleotide primers. Sequences of the two oligonucleotides are shown, aligned with the corresponding amino acid sequences in the rat secretin, pig calcitonin and opossum PTH receptors in Fig.3.

Reactions (lOO l) contained 30pmol of each primer, 20μl reverse transcriptase reaction and 2.5 units A plitaq DNA polymerase in 50mM KCl, lOmM Tris-HCl, pH 8.3 and 2mM MgC125 cycles of PCR (60s denaturation at 94°C, 60s annealing at 45°C, 60s extension at 60°C) were followed by a further 40 cycles (60s denaturation at 94°C, 60s + 6s per cycle annealing and extension at 60°C) followed by 7 minutes at 60°C. PCR products were precipitated with ammonium acetate and ethanol after which one half of each reaction was run on a 1% agarose gel. Five bands containing cDNA sequences having a size corresponding generally to that expected of the cDNA within the receptor gene between the two primers used, and ranging in size from 500bp to 900bp were excised from the gel and purified using the Sephaglas BandPrep kit (Pharmacia) and one quarter was used for a further round of PCR ( 5 cycles of 60s denaturation at 94°C, 60s annealing at 47°C, 60s extension at 60°C followed by a further 40 cycles of 60s denaturation at 94°C, 60s + 6s per cycle annealing and extension at 60°C followed by 7 minutes at 60°C.

Preparation of cDNA clones PCR products were ethanol precipitated, digested with BamHI and EcoRI, separated on a 1% agarose/TAE gel, purified using the Sephaglas BandPrep kit (Pharmacia) , ligated into pBluescript SK-Vector (Stratagene) and used to transform competent E. coli DS941. The sequences of several of the clones were obtained on both strands (Sequenase 2.0 kit,

USB) . One of the clones (RPR4) was found to have substantial similarity to rat VIP^_^ receptor[4, 5] but was distinct from all the known members of the family.

Tissue Distribution of mRNA (Northern Blottinq) Total RNA was isolated from tissues using the guanidinium thiocyanate/caesium chloride method[48]. Approximately 20μg of each RNA was separated by electrophoresis on denaturing 1% agarose/formaldehyde gels, transferred to a nitrocellulose membrane (Hybond-C, Amersham) and baked for 2h at 80°C. The membrane was then hybridised with the insert from RPR4 that had been labelled with [32P]dCTP-using random hexanucleotide primers (Pharmacia) and the Klenow fragment of Εz. coli DNA polymerase[49] . Hybridisation was performed overnight in 50% formamide, 25mM KP04, pH7.4, 5xSSC (lxSSC=0.15M NaCl, 0.015 M Na citrate pH7.0), 5x Denhardt's solution, 50μg/ml salmon sperm DNA. They were then washed twice for 20 min in 2xSSC/0.1%5DS at 50°C, then twice more in 0.5xSSC/0.1%5DS at 50°C and exposed to Fuji RX film. This Northern blot analysis using RPR4 as a hybridisation probe revealed a mRNA transcript of approximately 3.5 kilobases expressed in the pituitary and regions of the brain, with the highest levels being observed in the olfactory bulb.

Screening Rat Olfactory Bulb cDNA Library One of the clones isolated, RPR4, was used accordingly to screen a commercial cDNA library in the Lambda Zap II vector[50] prepared from rat olfactory bulb (Stratagene Catalogue No. 936520) .

4xl0⁵ plaque forming units of the library (corresponding to one-fifth of the total number of 2xl0⁶ primary plaques) were plated onto 2Ox 140mm plates using E. coli XLl-blue as a host strain. Filters containing plaque lifts from these plates were denatured by submerging in 1.5M NaCl, 0.5M NaOH for 2 mins, followed by neutralization in 1.5M NaCl, 0.5M TrisHCl pH8.0 for 5 mins, then rinsed in 3xSSC The filters were then blotted dry on Whatman 3MM filter paper and baked at 80°C for 2 hours. They were then hybridised with the insert from RPR4 labelled using the same method found in the Northern Blot protocol as described hereinabove.

Hybridisation was performed overnight in 50% formamide, 6x SSPE, 5x Denhardt's, 0.5% SDS, lOOμg/ml salmon sperm DNA at 45°C. Positive plaques were identified, picked, and the phage purified by further rounds of plating and screening. This procedure yielded six positive clones, of which three were analysed further. Positive clones were excised by co- infecting E. coli strain XL-1 blue with ExAssist (TM) helper phage (Stratagene) Bluescript SK phagemids (packed as filamentousphage particles) were then used to generate double-stranded plasmids by infecting E.σoli strain SOLRTM, and plating on L-broth/ampicillin plates to produce colonies. The .pBluescript SK-double-stranded plasmid was then recovered using standard techniques[51] .

Three different clones were isolated and characterised by restriction mapping and sequencing. All three had sequences corresponding to RPR4 but only one cDNA, RPR4/6.3 containing an insert of 3.3 kb, encoded a complete open reading frame, encoding a protein of 437 amino acids with a predicted molecular weight of 49519 (Figure 2) . A 22 amino acid hydrophobic signal sequence is found at the amino-terminal end, with a predicted signal cleavage site between Pro²² and Glu²³[52]. A hydropathy plot shows seven hydrophobic, putative membrane-spanning domains (Figure 2) . Comparison with other members of the secretin/calcitonin/PTH receptor family (Figure 3) revealed that the predicted protein encoded by RPR4/6.3 has greatest similarity with the rat VIP_X and PACAP type I receptors (50% identity, with each) . The highest amino acid sequence identity is found in the putative transmembrane regions, whereas the sequences of the amino-terminal extracellular domains and the carboxyl- terminal cytoplasmic ends are highly divergent.

Expression of VIP₂ receptor Gene

In order to determine the pharmacological characteristics of the novel receptor, the insert from the full length clone (RPR4/6.3) was excised as an EcoRI fragment and ligated into the EcoRI site of the mammalian cell expression vector pcDNA-1 (Invitrogen) [53, 54], and transiently transfected into COS-7 cells.

COS 7 cells were grown in DMEM supplemented with 10% new- born calf serum and lOOU/ml each of penicillin and streptomycin, in a humidified atmosphere of 95% air/ 5% C02 at a constant temperature of 37°C, and were passaged every 3-4 days. Cells for transfection were trypsinised the day before the experiment and plated at a density of approximately 40-50% confluency in 75cm2 flasks.

For transfection, cells were washed twice with OptiMEM (Gibco) supplemented with lOOU/ml each of streptomycin and penicillin at 37°C before exposure to transfecting medium for 4h. The transfecting medium consisted of OptiMEM/ penicillin/ streptomycin, 400μg/ml DEAE dextran (Promega) , lOOμM chloroquine phosphate (Sigma) and 10-20μg plasmid per flask. This was replaced ith 10% DMSO in PBS for 2 min, then DMEM/ 2% UltraSer G/ penicillin/ streptomycin. Cells were grown for 24h, then trypsinised and re-plated. Cells were harvested 48h later.

Functional Assay of VIP₂ receptor Gene

Since all members of the secretin receptor family so far identified are associated with activation of adenylate cyclase (also referred to in the art as adenylyl cyclase} the transfected cells were stimulated with several potential ligands, and intracellular cAMP levels measured by radioi munoassay.

For screening purposes, cells transfected with RPR4/6.3 were seeded onto 12-well tissue culture dishes, and for dose/response experiments, onto 24-well dishes. P rior to incubation with various peptide ligands, the cultures were washed with DMEM containing 0.25% BSA, and pre-incubated at 37 C for 30 min in the presence of 0.5mM isobutyl methylxanthine (IBMX) . VIP ligand and other peptides were directly added at concentrations indicated in Figs. 4a and 4b and incubated at 37 C for 15-30 min. The reaction was stopped by adding ice cold 0.1M HC1, and the cells homogenised by trituration. The levels of cAMP in the acidic extracts were measured by radioimmunoassay using antiserum cAB4 (courtesy of K.J. Catt, NICHD, NIH, Bethesda, MD) [55] .

Results Fig. 4a shows the effect of stimulating cells transfected with RPR4/6.3 by various peptide ligands. Values of cAMP release are expressed (mean + SEM, n=3) as a percentage of the stimulation evoked by lOOnM VIP. While treatment with corticotropin releasing factor (CRF) , calcitonin gene related peptide (CGRP) , secretin and glucagon, exhibited negligible effect, treatment with VIP, PACAP27, and PACAP-38 resulted in a marked elevation of cAMP levels. PHI and rat GHRH (rGHRH) also stimulated cAMP levels but were significantly less potent. PHI, VIP and PACAP-38 failed to stimulate cAMP levels in a control experiment where COS-7 cells had been transfected with the 5HT_1A (Serotonin) receptor (data not shown) .

As shown in Fig.4b the stimulation of cAMP levels by VIP, PACAP, helodermin, and PHI was dose-dependent. The EC50 (peptide concentration for 50% of maximal effect) for cAMP accumulation was approximately O.lδnM for PACAP-38, 0.43nM for PACAP-27, 0.25nM for helodermin, 0.17nM for VIP and 2.14nM for PHI. (The values shown in Fig.4b are the mean of 3. Basal cAMP was 0.8+0.15 pmoles/well) . The maximal stimulation of cAMP accumulation by rGHRH (400nM) was only 60% of that found for VIP. We therefore conclude that the order of potency of these ligands is VIP- PACAP38- PACAP27- helodermin >PHI »rGHRH, and that accordingly, RPR4/6.3 encodes a high affinity receptor for VIP.

Description of the Figures

Fig. 1 shows the gene encoding the rat VIP₂ receptor. The polynucleotide sequences specifically elucidated thus far are indicated along with the deduced amino-acid sequence;

Fig. 2 shows schematically the 7 transmembrane domains of the rat VIP₂ receptor and the 4 extracellular and 4 intracellular domains of the amino acid sequence derived from RPR4/6.3 using standard single letter codes to represent the amino acids;

Fig.3 shows the sequences for the two degenerate oligonucleotide primers; and

Fig.4 shows cAMP accumulation monitored during functional assay of a cloned gene of the invention. [I] Mutt, V. and Said, S.I. (1974) Eur. J. Biochem. 42, 581-589.

[2] Said, S.I. and Mutt, V. (1970) Science 169, 1217- 1218.

[3] Gozes, I. and Brenneman, D.E. (1989) Mol. Neurobiol. 3, 202-235.

[4] Sreedharan, S.P., Patel, D.R. , Huang, J.-X. and

Goetzl, E.J. (1993) Biochem. Biophys. Res. Commun. 193, 546-553.

[5] Ishihara, T. , Shigemoto, R. , Mori, K. , Takahashi, K. and Nagata, S. (1992) Neuron 8, 811-819.

[6] Svoboda, M. , Ciccarelli, E. , Tastenoy, M. , Robberecht, P. and Christophe, J. (1993) Biochem. Biophys. Res. Commun. 192, 135-142.

[7] Svoboda, . ,Ciccarelli,E. ,Tastenoy,M. ,Cauvin,A. ,

Stievenart, M. and Christophe, J. (1993) Biochem. Biophys. Res. Commun. 191, 479-486.

[8] Jelinek, L.J. , Lok, S., Rosenberg, G.B. , Smith, R.A. , Grant, F.J., Biggs, S., Bensch, P.A. , Kuijper, J.L. , Sheppard, P.O., Sprecher, CA. , Ohara, P.J. , Foster, D., Walker, K.M. , Chen, L.H.J., Mckernan, P.A. and Kindsvogel, W. (1993) Science 259, 1614-1616.

[9] Thorens, B.M. (1992) Proc. Natl. Acad. Sci. USA 89, 8641-8646.

[10] Mayo, K.E. (1992) Mol. Endocrinol. 6, 1734-1744.

[II] Lin, C.R., Lin, S.C, Chang, C.P. and Rosenfeld, M.G. (1992) Nature 360, 765-768. [12] Gaylinn, B.D., Harrison, J.K. , Zysk, J.R. , Lyons, C.E., Lynch, K.R. and Thorner, M.O. (1993) Mol. Endocrinol. 7, 77-84.

[13] Ishihara, T. , Nakamura, S., Kaziro, Y., Takahashi, T. , Takahashi, K. and Nagata, S. (1991) EMBO J. 10, 1635-1641.

[14] Hosoya, M. , Onda, H. , Ogi, K. , Masuda, Y. , Miyamoto,

Y., Ohtaki, T., Okazaki, H. , Arimura, A. and Fujino,

M. (1993) Biochem. Biophys. Res. Commun. 194, 133- 142.

[15] Morrow, J.A. , Lutz, E.M. , West, K.M. , Fink, G. and Har ar, A.J. (1993) FEBS Lett. 329, 99-105.

[16] Pisegna, J. and Wank, S. (1993) Proc. Natl. Acad. Sci. USA 90, 6345-6349.

[17] Albrandt, K.G., Mull, E., Brady, E.M. , Herich, J. ,

Moore, C.X. and Beaumont, K. (1993) FEBS Lett. 325, 225-232.

[18] Lin, H.Y., Harris, T.L., Flannery, M.S., Aruffo, A.A. , Kaji, E.H., Gorn, A., Kolakowski, L.F.J., Lodish, H.F. and Goldring, S.R. (1991) Science 254, 1022-1024.

[19] Juppner, H. , Abou-Samra, A.-B.B., Freeman, M.W. , Kong, X.F., Schipani, E., Richards, J. , Kolakowski, L.F.J., Hock, J. , Potts, J.T., Kronenberg, H.M. and Segre, G. (1991) Science 254, 1024-1026.

[20] Abou-Samra, A.-B.B., Jueppner, H. , Force, T. , Freeman, M.W. , Kong, X.-F., Schipani, E. , Urena, P., Richards, J. , Bonventre, J.V. , Potts, J.T., Kronenberg, H.M. and Segre, G.V. (1992) Proc. Natl. Acad. Sci. USA 89, 2732-2736.

[21] Besson, J. , Sarrieau, A., Vial, M. , Marie, J.-C,

Rosselin, G. and Rostene, W. (1986) Brain Res. 398, 329-336.

[22] Martin, J.L. , Dietl, M.M. , Hof, P.R., Palacios, J.M. and Magistretti, P.J. (1987) Neuroscience 23, 539- 565.

[23] Shivers, B.D., Gores, T.J., Gottschall, P.E. and Arimura, A. (1991) Endocrinology 128, 3055-3065.

[24] Masuo, Y. , Ohtaki, T. , Masuda, Y., Tsuda, M. and Fujino, M. (1992) Brain Res. 575, 113-123.

[25] Gottschall, P., Tatsuno, I., Miyata, A. and Arimura, A. (1990) Endocrinology 127, 272-277. [26] Salomon, R. , Couvineau, A., Rouyer-Fessard, C. ,

Voisin, T., Lavallee, D., Blais, A., Darmoul, D. and Laburthe, M. (1993) Am. J. Physiol. 264, E294-E300.

[27] Robberecht, P., Gourlet, P., Cauvin, A., Buscail, L. , De Neef, P., Arimura, A. and Christophe, J. (1991) Am. J. Physiol. 260, G97-G102.

[28] Tatsuno, I., Gottschall, P.E. and Arimura, A. (1991) Endocrinology 128, 728-734.

[29] Robberecht, P., Cauvin, A., Gourlet, P. and Christophe, J. (1990) Arch. Int. Pharmacodyn. Ther. 303, 51-66.

[30] Laburthe, M. and Couvineau, A. (1988) Ann. NY Aσad. Sci. 527, 296-313.

[31] Blum, A.M., Mathew, R. , Cook, G.A. , Metwali, A., Felman, R. and Weinstock, J.V. (1992) J. Neuroimmunol. 39, 101-108.

[32] Gozes, I., McCune, S.K., Jacobson, L. , Warren, D., Moody, T.W., Fridkin, M. and Brenneman, D.E. (1991) Journal of Pharmcology and Experimental Therapeutics 257, 959-966.

[33] Gozes, Y., Brenneman, D.E., Fridkin, M. , Asofsky, R. and Gozes, I. (1991) Brain Res. 540, 319-321.

[34] Robberecht, P., Coy, D.H. , De Neef, P., Camus, J.C,

Cauvin, A. and Waelbroek, M. (1986) Eur. J. Biochem. 159, 45-49.

[35] Rouyer-Fessard, C, Couvineau, A., Voisin, T. and Laburthe, M. (1989) Life Sci. 45, 829-833.

[36] Robberecht, P., De Neef, P., Gourlet, P., Cauvin, A., Coy, D.H. and Christophe, J. (1989) Regul. Pept. 26, 117-126.

[37] Gourlet, P., De Neef, P., Woussen-Colle, M.-C,

Vandermeers, A., Vandermeers-Piret, M.-C, Robberecht, P. and Christophe, J. (1991) Biochim. Biophys. Acta 1066, 245-251.

[38] Gespach, C, Chedeville, A., Hurbain-Kosmath, I.,

Housset, B., Derenne, J.-P. and Abita, J.-P. (1989) Regul. Pept. 26, 158-?

[39] Luis, J. and Said, S.I. (1990) Peptides 11, 1239- 1244.

[40] Hill, J.M., Harris, A. and Hilton-Clarke, D.I. (1992) Neuroscience 48, 925-932.

[41] Gressens, P., Hill, J.M. , Gozes, I., Fridkin, M. and Brenneman, D.E. (1993) Nature 362, 155-158. [42] Magistretti, P.J., Morrison, J.H., Shoemaker, W.J. , Sapin, V. and Bloom, F.E. (1981) Proc. Natl. Acad. Sci. USA 78, 6535-6539.

[43] Brenneman, D.E. and Eiden, L.E. (1986) Proc. Natl. Acad. Sci. USA 73, 1159-1162.

[44] Reichlin, S. (1988) Ann. NY Acad. Sci. 527, 431-449.

[45] Malhotra, R.K. , Wakade, T.D. and Wakade, A.R. (1988) J. Biol. Chem. 263, 2123-2126.

[46] Ottaway, CA. (1987) Immunology 62, 291-297.

[47] Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159.

[48] Chirgwin, J. , Przbyla, A., MacDonald, R. and Rutter, W. (1979) Biochemistry 18, 5294-5299.

[49] Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13.

[50] Short, J.M. , Fernandez, J.M. , Sorge, J.A. and Huse, W.D. (1988) Nucl. Acids Res. 16, 7583-7600.

[51] Sambrook, J. , Fritsch, E. and Maniatis, T. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

[52] Von Heijne, G. Nucl. Acids Res. 14, 4683-4690.

[53] Aruffo, A. and Seed, B. (1987) Proc. Natl. Acad. Sci. USA 84, 8573-8577.

[54] Seed, B. (1987) Nature 329, 840-842. [55] Dufau, M., Watanabe, K. and Catt, K. (1973) Endocrinology 92, 6-11. [56]

[56] Reisner, D., Steger, G. , Simmat R. et al, (1989). Electrophoresis 10 (5-6) :377-389.

[57] Birmse, A., Sattler, A., Maurer, K.H. & Reisner D. (1990) . Electrophoresis 11, 795-801.

Claims

1. A gene encoding VIP₂ receptor.

2. A gene encoding VIP₂ receptor having the amino acid sequence of SEQ ID No.l.

3. An isolated DNA sequence encoding VIP₂ receptor.

4. A recombinant DNA sequence encoding VIP₂ receptor.

5. An isolated or recombinant DNA sequence for use in expression in a prokaryotic or eukaryotic host cell of a polypeptide product having an amino acid sequence corresponding to a sufficient extent to that of a naturally occurring VIP₂ receptor to provide at least one biological function of said naturally occuring VIP₂ receptor, which DNA sequence is selected from: a) the DNA sequence of SEQ ID No. 1 or a sequence complementary thereto; and b) a DNA sequence which can hybridize to a DNA sequence defined in (a) or to a fragment thereof.

6. A gene or sequence according to any one of claims 1,3,4 and 5b wherein said VIP₂ receptor is human VIP₂ receptor.

7. A gene or sequence according to claim 6 which has been obtained directly or indirectly through recovery of a human gene by hydridisation thereof with a polynucleotide probe comprising a labelled DNA or RNA sequence capable of specifically hybridizing to a gene according to claim 2.

8. A prokaryotic or eukaryotic host cell transformed or transfected with a gene or DNA sequence according to any one of the preceding claims.

9. A recombinant vector containing a DNA sequence or gene according to any one of claims 1 to 7.

10. A prokaryotic or eukaryotic host cell transformed or transfected with a recombinant vector according to claim 9.

11. A polynucleotide probe comprising a labelled DNA or RNA sequence capable of specifically hybridizing to a gene according to claim 1, claim 2 or claim 6 when dependent on claim 1 or claim 2, or a naturally occurring variant thereof.

12. A method of detecting a gene according to claim 1 or claim 2 or claim 6 when dependent on claim 1 or claim 2, which method comprises hybridizing a probe according to claim 11 with said gene, and detecting bound labelled probe.

13. An antibody probe comprising a labelled antibody raised against an amino acid sequence capable of specifically binding to VIP₂ receptor expressed by a gene according to claim 1 or claim 2 or claim 6 when dependent on claim 1 or claim 2, or a naturally occuring variant thereof.

14. An antibody probe according to claim 13 which is a monoclonal antibody.

15. A method of detecting VIP₂ receptor expressed by a gene according to claim 1 or claim 2 or claim 6 when dependent on claim 1 or claim 2, or a naturally occurring variant thereof, which method comprises allowing a probe according to claim 13 or claim 14 to bind with said receptor, and detecting bound labelled probe.

16. A method of diagnosis for a condition associated with VIP₂ receptor abnormality which comprises hybridizing a polynucleotide probe according to claim 11 with the 7q36.3 chromosomal region, and monitoring for absence of bound labelled probe.

17. A method according to claim 16 wherein is used the technique of fluorescence in situ hybridisation.

18. A method of diagnosis for a condition associated with VIP₂ receptor abnormality which comprises amplification by polymerase chain reaction using polynucleotide primers from substantially spaced apart portions of a nucleic acid sequence encoding VIP₂ receptor, and monitoring for absence of amplified polynucleotide sequence product.

19. A method of diagnosis for a condition associated with VIP₂ receptor abnormality which comprises microscopic examination of the chromosomal region for gross abnormality.

20. A method of diagnosis for holoprosencephaly comprising the steps of collecting a foetal sample and examining the chromosmal content thereof by a method according to any one of claims 16 to 19.

21. A method of evaluating a potential VIP₂ agonist or antagonist comprising the steps of: providing cell membrane of a VIP receptor expression system comprising a transformed or transfected host cell according to claim 8 or claim 10, which membrane has VIP₂ receptor; exposing said cell membrane to labelled ligand and to said agonist or antagonist; and monitoring at least one of ligand binding and an intra-cellular messenger system.

22. A method according to claim 21 which includes the preliminary step of providing a VIP₂ receptor expression system comprising a transformed or transfected host cell according to claim 8 or claim 10.