Molecular Evolution of Vertebrate VIP Receptors and Functional Characterization of a VIP Receptor from GoldfishCarassius auratus

General and Comparative Endocrinology 105, 176–185 (1997) Article No. GC966818

Molecular Evolution of Vertebrate VIP Receptors and Functional Characterization of a VIP Receptor from Goldfish Carassius auratus1,2 Billy Kwok-Chong Chow,3 Tony Tat-Hung Yuen, and Koon-Wing Chan Department of Zoology, The University of Hong Kong, Hong Kong Accepted September 3, 1996

Vasoactive intestinal polypeptide (VIP) is a neuropeptide that has numerous physiological actions and is widely distributed in the body. However, as yet, there is no sequence information about VIP receptors in lower vertebrates. Partial cDNA fragments spanning transmembrane domains 2 to 6 of VIP receptors were isolated from six nonmammalian vertebrate species, including chicken, pigeon, frog, lizard, salmon, and goldfish. Sequence comparison of these receptors revealed essential structural motifs responsible for receptor function. In addition, the first nonmammalian full-length VIP receptor cDNA was obtained by screening a goldfish brain and pituitary cDNA library. Functional expression of this receptor in mammalian COS-7 cells showed that it is coupled to cAMP production in a VIP and PACAP concentration-dependent manner; the EC50 of VIP was determined to be 1 nM. At 100 nM peptide, the relative potency of various peptides in stimulating cAMP in the transfected cells was VIP G PACAP G GHRH 5 secretin G PHM G PTH G glucagon G GLP-1 G GIP. Characterization of the VIP receptors in lower vertebrates should

1 This work was presented in part at the 3rd International Symposium of Fish Endocrinology. 2 The nucleotide sequence data reported in this report have been deposited with the GenBank Data Library under Accession No. U56391. 3 To whom correspondence should be addressed at Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong. Fax: 852-2857-4672. E-mail: [email protected].

176

enhance our understanding of the molecular evolution and physiology of VIP in vertebrates. r 1997 Academic Press Vasoactive intestinal polypeptide (VIP) is a 28-aminoacid peptide which shares sequence identity to a family of peptide hormones including pituitary adenylate cyclase activating polypeptide (PACAP), secretin, growth hormone releasing hormone (GHRH), peptide histidine isoleucine (PHI), peptide histidine methionine (PHM), glucagon, glucagon like peptide (GLP), gastric inhibitory polypeptide (GIP), and parathyroid hormone (PTH). VIP was originally found in the gastrointestinal system (Said and Mutt, 1970) and VIP exerts a number of physiological actions in the gut such as the stimulation of water and electrolyte secretion from intestinal mucosa and elevation of exocrine secretion from pancreas (Said, 1982). VIP was subsequently located in both the peripheral and the central nervous systems; it is now considered a widely expressed neuropeptide that is present throughout the body. It is important in numerous biological processes, for example, vasodilatation in cerebral, pulmonary, and peripheral blood vessels, relaxation of gastrointestinal, bronchial, and uterine smooth muscle, and embryonic development (Said, 1982; Gressens et al., 1993). In the central nervous system, VIP regulates cerebral energy metabolism, neuronal survival, and prolactin secretion from pituitary (for reviews, see Rawlings and Hezareh, 1996; Gozes and Brenneman, 1989). In the

0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

177

Goldfish VIP Receptor

immune system, VIP inhibits interleukin-2 production but stimulates the growth of B cells and the production of immunoglobulin (Ottaway, 1987; Ishioka et al., 1992). The primary sequence of most mammalian (except guinea pig) VIPs isolated from human, rat, porcine, dog, goat, and bovine were found to be identical, while nonmammalian VIPs from chicken, alligator, frog, trout, bowfin, dogfish, cod, and goldfish differ from the human VIP at four or five positions (Chartrel et al., 1995; Wang and Conlon, 1995; Uesaka et al., 1995). Not just that the amino acid sequences of all VIPs are highly conserved, the species variations usually involve conservative substitutions that do not greatly affect the biological activities of the peptide (Chartrel et al., 1995). The comparative studies of VIP indicated that the evolutionary pressure to conserve the VIP molecule in vertebrates has been high, and this finding is consistent with its importance in biological function. The action of VIP is mediated via its specific interaction with cell surface receptors. In fact, the whole VIP–secretin–glucagon peptide superfamily interacts with a distinct family of G-protein-coupled receptors that are coupled to adenylyl cyclase and the production of intracellular cAMP. Like other family members, the VIP receptor is a glycoprotein with a large hydrophilic extracellular domain followed by seven highly conserved hydrophobic transmembrane helices. VIP receptors were purified to homogeneity from porcine liver and were found to be 53 kDa in size (Couvineau et al., 1990). Rat and human VIP receptor cDNAs were recently cloned and functionally characterized from lung (Ishihara et al., 1992), pituitary (Lutz et al., 1993), HT-29 cells (Sreedharan et al., 1991, 1993), small intestine (Couvineau et al., 1994), and liver (Gagnon et al., 1994). Pharmacological evidence indicating the presence of multiple receptors was confirmed by the cloning of at least two VIP receptor subtypes (VIP1 and VIP2) with different but related primary amino acid sequences, and each receptor was found to be expressed in a tissue-specific manner (Usdin et al., 1994; Sheward et al., 1995). Despite the rapid progress in the area of VIP receptor cDNA cloning, there is still little information with respect to the molecular evolution and structure–function relationships of these receptors, especially in nonmammalian vertebrates. The present study aims to fill a significant gap in our knowledge in the understanding of VIP and its recep-

tor in lower vertebrates. In this report, partial cDNA clones spanning the transmembrane domain (TMD) 2 to 6 were cloned and sequenced from representative species lower vertebrate, including chicken (Gallus gallus), pigeon (Columba livia), frog (Rana tigrina rugulosa), lizard (Gekko gecko), salmon (Oncorhynchus tschawytscha), and goldfish (Carassius auratus). In addition, the first nonmammalian full-length VIP receptor cDNA was characterized by screening a goldfish brain and pituitary cDNA library. Sequence comparison of the clones revealed essential structural motifs responsible for VIP-specific receptor function. Goldfish was chosen in this study since it is one of the most well-studied teleost species. The understanding of VIP and its receptor in goldfish will pave the path for solving problems related to the physiology and pathophysiology in fish reproduction and metabolism in the future.

METHODS Cloning of the Partial Receptor cDNAs from Various Vertebrate Species Partial cDNA clones corresponding to the TMD 2 to 6 of the receptors were obtained by a novel 2-step polymerase chain reaction (PCR) approach. Total (Maniatis et al., 1982) and poly(A)1 mRNA from pancreas and brain tissues were prepared from various animals, and first-strand cDNA was prepared and were used as template for PCR amplification. The primers used for PCR were designed according to the consensus regions among receptors of the VIP–secretin receptor family. The sequence of the primers are: G2 (TGCAYTGYACNMGNAAYTAYATYCA), G6 (AGSGGGATSAGSRKNAGNGTGGAYTT), and G7 (TGSACCTCNCCRTTNASRAARCARTA). After the first PCR using G2 and G7 as primers, 1 µl of the first PCR products were reamplified in the second PCR using nested primers G2 and G6. PCR products between 500 and 600 bp were agarose gel purified and blunt-end cloned into pUC-18 for DNA sequence analysis using a T7 sequencing kit (Pharmacia). The PCR typically contained 50 pmol of primers, 200 µM dNTPs and 2.5 units of Taq polymerase (GIBCO/BRL) in the buffer provided by the manufacturer. The reaction times were 1 min at 94, 58, and 72°, respectively, for 30 cycles.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

178

The deduced amino acid sequences from the VIP1 receptor partial cDNA clones were analyzed by using the GeneWorks (IntelliGenetics) protein alignment program. The program uses a progressive alignment method which produces approximate phylogenetic order by a scoring algorithm similar to FASTA. The program begins by placing each sequence to be aligned in a list and scoring each sequence in the list against every other. The two most similar sequences with the best pairwise scores are removed from the list and aligned again with all the remaining sequences in the list. Once again, the best alignment is removed and aligned again until a single alignment of all the sequences in the list is included. In addition, the program uses a PAM-250 scoring matrix and it scores mismatches rather than matches. This means that the lower the score, the better the alignment. The score is then represented by the horizontal distance in the phylogenetic tree, i.e., the shorter the distance, the more related the two sequences should be.

Cloning of the Goldfish VIP Receptor cDNA The partial cDNA clones corresponding to the TMD 2 to 6 of goldfish VIPA and VIPB receptor were used as probes to screen the goldfish brain and pituitary cDNA library (0.5 million primary clones) according to Maniatis et al. (1982). The library was constructed using the Stratagene ZAP-Express cDNA library system. Five micrograms of poly(A)1 mRNA was used and 2.5 million primary clones were obtained. A full-length cDNA clone encoding the goldfish VIPA receptor was isolated from the library and then excised to the phagemid form pBK-CMV-gfVIP according to Stratagene’s instructions. The clone was sequenced from both strands using a T7 sequencing kit (Pharmacia) by synthetic primers and by subcloning of restriction fragments. The DNA sequences were analyzed by DNasis (Hitachi).

Functional Expression of the Cloned Receptor To make use of the unique flanking NotI and ApaI restriction sites in pBS-SK1, a 2.9-kb EcoRI–XhoI DNA fragment corresponding to the full-length goldfish VIP receptor was initially subcloned into pBS-SK1 (Stratagene). From this plasmid, a NotI–ApaI 2.9-kb fragment was then released and directionally subcloned into the

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

Chow, Yuen, and Chan

eukaryotic expression vector pRC-CMV (Invitrogen) to form pRC-CMV-gfVIP. A permanent cell line, COSgfVIPR, with the putative goldfish VIP receptor expressed was obtained by transfecting pRC-CMV-gfVIP (10 µg) into 1 million COS-7 cells using Lipofectin reagent (GIBCO/BRL) and followed by G418 selection at 500 µg/ml (BRL/GIBCO) for 2 weeks. Functional expression and cAMP assays were performed as described earlier (Chow, 1995). In summary, 0.2 million COS-gfVIPR cells were seeded into 6-well plates (Costar) 2 days before cAMP assays. The cells were washed once with MEM with 1 mg/ml of bovine serum albumin and then incubated with the same medium containing 0.2 mM 1-methyl-3-isobutylxanthine (RBI) and the desired concentrations of peptide for 45 min at 37°. After incubation, the medium was removed and the cells were lyzed by the addition of 1 ml of iced ethanol. The cell debris was pelleted by centrifugation (10,000g for 10 min) and the supernatant was dried by a vacuum concentrator. The cAMP levels were then quantified by a radioimmunoassay kit (Amersham). All peptides used in this study were purchased from Bachem Fine Chemicals, Inc. (CA).

RESULTS Cloning of Partial VIP cDNA Clones from Various Nonmammalian Vertebrates The approach used to obtain partial VIP receptor clones was based on the amplification of G-proteincoupled receptor cDNAs using degenerate oligonucleotide primers (Chow, 1995). These primers corresponding to TMDs 2, 6, and 7 were designed according to the consensus sequence of the family of mammalian VIP– secretin receptors. Partial cDNA clones corresponding to the TMD 2 to 6 of VIP receptors were obtained from six vertebrate species by reamplification of the first PCR products using nested primers G2 and G6. The sequence identities of these receptors within the same class are very high (mammal, 83%; bird, 92%; fish VIPA, 87%) (Table 1); while the human VIP1 receptor shares from 62% (goldfish B) to 70% (frog) identities when compared to nonmammalian species (Table 1). Of the 161 (average of 159 to 163) amino acids spanned in all the species compared, there is identity at 70

179

Goldfish VIP Receptor

TABLE 1 Amino Acid Similarity (%) of Partial VIP1 Receptor in Vertebrates

Human Rat Chicken Pigeon Lizard Frog Goldfish A Goldfish B Salmon A

Rat

Chicken

Pigeon

Lizard

Frog

Goldfish A

Goldfish B

Salmon A

Salmon B

83.5

67.7 76.4

69.5 75.1 92.5

65.0 67.7 77.6 76.4

70.6 75.8 82.0 79.5 76.3

68.1 70.0 66.9 67.5 67.5 68.7

62.0 61.9 65.2 64.6 61.9 63.8 74.2

65.5 68.5 68.5 66.7 66.0 68.5 87.1 73.5

62.0 66.5 68.5 65.8 62.5 66.3 74.2 74.4 74.1

(43.5%) positions (Fig. 1). When conservative changes are included, the amino acid similarity increases to 119 (73.9%) positions. The most conserved regions of the receptors are the putative TMDs and the intracellular loops between TMD 3/4 and TMD 5/6. The least conserved areas are the extracellular loops between TMD2/3 and TMD4/5. A phylogenetic tree was generated by comparison of the deduced amino acid sequences of the partial receptor clones (Fig. 2). Phylogenetic analysis of these partial receptors indicated that they are closely related to each other and also to human and rat VIP1 receptors. While these receptors are less related to other mammalian receptors within

the same family, these VIP1 receptors form a subbranch within the VIP–glucagon receptor superfamily.

Isolation of the Goldfish VIP Receptor cDNA The partial cDNA clones corresponding to TMD 2 to 6 of goldfish VIPA and VIPB were then used as probes to screen the goldfish brain and pituitary cDNA library. A clone 2610 bp in length was obtained which showed a consistent hybridization signal under highstringency wash conditions using the VIPA probe. Nucleotide sequencing of the putative VIP receptor cDNA clone revealed a single open reading frame of

FIG. 1. Comparison of partial amino acid sequences of VIP1 receptors from different vertebrates. The putative TMD are labeled with arrows. The amino acid residues that are identical and conserved among all receptors are boxed. The conserved cysteines and the putative protein kinase C site are labeled ‘‘*’’ and ‘‘1,’’ respectively. The Gsa protein coupling motif (Basic-L/A-L/A/V/S-Basic) is shown in boldface. The putative VIP1 receptor signatures, cysteine and ‘‘P-D-I/V’’ motifs, are underlined.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

FIG. 2. Phylogenetic tree of partial VIP1 receptors and other members within the same gene family. The alignment was done using partial protein sequences from the ‘‘FMSFI’’ motif within the TMD2 to the Gsa protein coupling motif ‘‘RLAK’’ that immediately precedes TMD6. These two motifs are present in all members of the secretin–VIP–glucagon receptor family. The tree was generated using the default setting of the GeneWorks (IntelliGenetics) protein alignment program: cost to open a gap 5, cost to lengthen a gap 25, minimum diagonal length 4, and maximum diagonal offset 10. The alignment program scores mismatches rather than matches; in other words, the lower the score, the better the alignment. The score between two protein sequences, which is a measure of the relative phylogenetic relationship between the two receptors, is represented by the horizontal distance in this phylogenetic tree (see Methods). References: receptor sequences for rat VIP (Ishihara et al., 1992), human VIP (Gagnon et al., 1994), human PACAP (Ogi et al., 1993), human secretin (Chow, 1995), rat VIP2 (Lutz et al., 1993), human GHRH (Mayo, 1992), human glucagon (Lok et al., 1993), rat GIP (Usdin et al., 1993), human GLP-1 (Thoren et al., 1993), and human PTH (Schipani et al., 1993).

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

180

181

Goldfish VIP Receptor

1341 bp (from nucleotides 917 to 2258) encoding a protein of 447 amino acids with a predicted molecular weight of 51 kDa. Computer analysis of the deduced amino acid sequence of this clone indicated that it belongs to the VIP–secretin receptor family. The goldfish VIP1 receptor was found to have 65 and 62% sequence homology at the cDNA level with human and rat VIP1 receptor and 60% sequence homology at the amino acid level for both species (see Fig. 3). A Kyte–Doolittle hydrophobicity analysis of the receptor indicated that the protein belongs to the G-proteincoupled receptor family with seven segments of hydrophobic amino acids presumably forming the transmembrane spanning regions (Fig. 4).

were found to inhibit cAMP production in the COSgfVIPR cells consistently; the significance of this observation remains to be investigated. It was also found that VIP and PACAP were able to stimulate the production of cAMP in a dose-dependent manner. In order to generate the same response, VIP is about 100-fold more potent than PACAP. The EC50 of VIP in stimulating cAMP production was determined to be between 1 and 3 nM (Fig. 5B).

DISCUSSION Sequence Comparison of VIP Receptors

Functional Expression of Goldfish VIP Receptor To demonstrate that the isolated goldfish VIP receptor could transduce a physiological signal, the cAMP response in the presence of various related peptides was measured (Fig. 5). At a 100 nM peptide concentration, the relative potencies of various peptides in stimulating cAMP in the transfected cells were VIP . PACAP-38 . GHRH 5 secretion . PHM . PTH . glucagon . GLP-1 . GIP (Fig. 5A). GIP and GLP-1

Partial cDNA clones corresponding to TMD 2 to 6 of VIP receptors were obtained from six vertebrate species. It is interesting to note that two highly related VIP clones (VIPA and VIPB) were obtained from salmon and goldfish. This is likely due to the fact that both goldfish and salmon are tetraploids, and it is not uncommon that these fishes express two different mRNAs from duplicate gene loci. The most conserved regions of the receptors are the putative TMDs and the

FIG. 3. Amino acid sequence alignment of goldfish (top), human, and rat VIP1 receptors by GeneWorks (IntelliGenetics). The arrows represent the seven putative TMDs. Identical and conservative sequences are boxed.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

182

Chow, Yuen, and Chan

FIG. 4. Hydropathy plot of the predicted amino acid sequence of goldfish VIP1 receptor. The plot was obtained by the method of Kyte and Doolittle using DNASIS (Hitachi). The X-axis indicates the position of the amino acid residue of the precursor protein. The putative TMDs are labeled I to VII as indicated in the graph.

intracellular loops between TMD 3/4 and TMD 5/6. These regions are likely to be responsible for contributing to the proper conformation, structure, and function of the receptor. A consensus phosphorylation sequence for protein kinase C is located in the second intracellular loop between TMDs 3 and 4, and this serine residue may be important for receptor activation/inactivation through secondary messenger systems. There is recent

evidence indicating that VIP receptors can be downregulated by protein kinase C in human fetal ciliary epithelial cells. Treatment of cells with protein kinase C agonists resulted in a 25–40% reduction in the number of VIP binding sites but there was no change in the dissociation constant (Crook and Yabu, 1994). Interestingly, this conserved ‘‘S-E0–1-R/K’’ motif is also found in several other members in the same receptor family,

FIG. 5. Stimulation of cAMP production in COS-gfVIPR cells. (A) The cells were incubated with 100 nM various human peptides as indicated. cAMP level was measured and expressed as fold stimulation compared to the control (no peptide). In the positive control column, a cell line expressing the human secretin receptor was used and the cells were stimulated by 10 nM secretin (Chow, 1995). (B) cAMP production in COS-gfVIPR cells stimulated with various concentrations of VIP and PACAP. Data presented here were from a single experiment. The assays were done in triplicate, and the values represent the mean 6 SD.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

183

Goldfish VIP Receptor

including secretin, GHRH, glucagon, GLP-1, GIP, and PTH receptors, suggesting a more universal role for this phosphorylation site in this family of receptor regulation. The ‘‘RLAK’’ motif immediately in front of TMD 6 and within the third intracellular loop between TMDs 5 and 6 matches the consensus found in other members of the family of G-protein-coupled receptors, such as the b2-adrenergic receptor, at the same position. This motif (Basic-L/A-L/A/V/S-Basic) containing two basic amino acids separated by two hydrophobic residues has been indicated to be essential to Gsa protein coupling (Okamoto et al., 1991). The least conserved areas are the first and second extracellular loops between TMD2/3 and TMD4/5. However, even within these two less conserved regions, there are three cysteine residues that are identical in all VIP1 receptors; these cysteine residues may form disulfide linkages for a proper receptor function. On the other hand, a conserved cysteine residue within TMD5, just in front of the ‘‘IIRIL’’ motif, was found to be unique to VIP1 receptors: none of the other receptors in the same gene family has this cysteine residue at the same position. This cysteine residue, together with a second motif ‘‘P-D-I/V’’ found only in VIP binding receptors, including VIP1, VIP2, and PACAP receptors, at the third intracellular loop between TMDs 5 and 6, may be used as signature sequences for all VIP1 receptors.

Isolation of the Goldfish VIP Receptor cDNA A goldfish VIP receptor which shares a high degree of sequence similarity with human and rat VIP1 receptor was obtained by standard library screening techniques. This receptor contains the characteristic seven hydrophobic transmembrane spanning regions found in all G-protein-coupled receptors (Fig. 3). It is interesting to note that the goldfish VIP receptor lacks a putative N-terminal hydrophobic leader peptide region, as indicated by both hydrophobicity plot and amino acid sequence comparison with human and rat VIP1 receptors (Figs. 3 and 4). Even in the absence of a putative signal peptide, the goldfish VIP receptor was able to mediate cAMP response (Figs. 5A and 5B), suggesting that the receptor is functional and is expressed onto the cell surface. The consensus ‘‘signature’’ for the mammalian VIP–glucagon receptor superfamily located at the seventh TMD (Lok et al., 1994) is also found in this goldfish VIP receptor. The consensus

is FQGBBVAXBYCFXNXEVQ, where X and B represent any amino acid residue and any hydrophobic amino acid residue, respectively. Within the Nterminal extracellular domain, there are two conserved and two nonconserved putative N-glycosylation sites at residues 22 and 64 and at residues 18 and 91, respectively. The VIP receptor in the human melanoma cell line IRG 39 was shown to be a 60-kDa glycoprotein containing 20 kDa of N-linked carbohydrates (Chochola et al., 1993). The absence of N-glycosylation decreased VIP binding in these cells due to the reduction in the expression of functional surface receptors. The conserved N-linked glycosylation sites at residues 22 and 64 are therefore likely to be important for receptor–ligand interactions. There are 7 conserved cysteine residues (positions 2, 15, 27, 36, 50, 69, and 86) within the N-terminal extracellular domain, 2 within the first extracellular loop between TMDs 2 and 3 (positions 170 and 177), and 1 within the second extracellular loop between TMDs 4 and 5 (position 247) (Fig. 4). Mutational analysis of cysteine residues of the human VIP1 receptor identified seven mutants that were defective in VIP binding (positions 15, 27, 36, 50, 69, 86, and 247) (Gaudin et al., 1995). Five (position 27, 36, 50, 170, 247) of these 10 cysteine residues are conserved among all receptors within the superfamily so that these residues are probably important for receptor function by the formation of disulfide bonds. Pretreatment of the cell line expressing rat secretin receptor with a reducing agent, dithiothreitol, led to a reduction in binding capacity, suggesting that integrity of the disulfide bridges is required for this type of receptor–ligand interaction (Vilardaga et al., 1994). The aspartic acid residue at position 32 is also conserved among all receptors. A missense mutation of this residue in the GHRH receptor in mouse leads to little mouse phenotype (Godfrey et al., 1993). In addition to this aspartic acid residue, mutation of two other highly conserved residues, tryptophan 37 and glycine 73, was also detrimental to the binding to VIP to its receptor (Couvineau et al., 1995), again indicating the importance of these residues in receptor function.

Functional Expression of Goldfish VIP Receptor Defining receptor identity in the VIP/PACAP system is difficult because of the existence of multiple receptors and peptides. The effect of VIP on the

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

184

activation of adenylyl cyclase in mammalian systems is mediated by at least two receptors, VIP1 and VIP2 (or PACAP type II). These two receptors can be activated with comparable potency by VIP and PACAP, while pharmacologically, the two receptors can be distinguished by the modest effect of secretin on only the VIP1 receptor. In the present study, the goldfish VIP receptor was expressed in mammalian COS-7 cells and was coupled to the cAMP pathway for signal transduction. The fact that secretin is an agonist for this receptor (Fig. 5A), and together with the sequence comparison data (Figs. 1–3), leads to the conclusion that the receptor characterized in this study is a species variant of the mammalian VIP1 receptors. The EC50 of VIP in stimulating cAMP production was determined to be 1 nM, which is comparable to human and rat homologs; the EC50 values of human VIP receptors reported previously were 0.5 (Couvineau et al., 1994) and 1 nM (Sreedharan et al., 1993). These experimental data reflect the conservation of both VIP and the VIP receptor in evolution: (1) human VIP is able to stimulate the fish VIP receptor at nanomolar concentrations; and (2) the goldfish VIP receptor is able to be functionally coupled to a mammalian G-protein in COS-7 cells. These conservations are suggested by the structure of the molecules: some of the substitutions between human and goldfish VIP are conservative (10V toY, 11T toS, 13L to Y, 19VtoA, 26I toV, and 28NtoA) (Uesaka et al., 1995). Four (positions 11, 19, 26, and 28) of these six changes are also found in other nonmammalian species including chicken, alligator, frog and trout, suggesting that the evolutionary pressure to conserve the primary structure of the VIP molecule has been strong. In both mammalian and piscine test systems, these structural differences seem to have little effect on their biological activities. Porcine and dogfish VIPs were equipotent in stimulating amylase secretion and displacement of radioactively labeled ligand from dispersed guinea pig pancreatic acini preparation (Dimaline et al., 1987). In the fish, porcine VIP was also able to inhibit cholecystokinin-induced gall bladder contraction in trout (Aldman and Holmgren, 1992), and to relax smooth muscle in rectum and swim bladder in cod (Lundin and Holmgren, 1992). The receptor conservation is indicated by the high degree of sequence similarities among partial VIP1 receptors characterized in this study; up to 74% identity is observed when

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

Chow, Yuen, and Chan

conservative changes are included (Fig. 1). Also, the functional coupling of the goldfish VIP receptor with mammalian COS-7 Gsa protein may be reflected by the conservation of the ‘‘RLAK’’ motif. In fact, this motif is also present in other goldfish receptors within the same family, including glucagon, PACAP, and GHRH receptors (unpublished data).

ACKNOWLEDGMENTS The authors thank E. Lau for her excellent technical help in this project. We thank J. Brown and T. Mommsen for providing the salmon tissues for RNA extraction. The work was supported by HK government RGC HKU 309/93M.

REFERENCES Aldman, G., and Holmgren, S. (1992). VIP inhibits CCK-induced gall bladder contraction involving a beta-adrenoreceptor mediated pathway in the rainbow trout, Oncorhynchus mykiss, in vivo. Gen. Comp. Endocrinol. 88, 287–291. Chartrel, N., Wang, Y., Fournier, A., Vaudry, H., and Conlon, J. M. (1995). Frog vasoactive intestinal polypeptide and galanin: Primary structures and effects on pituitary adenylate cyclase. Endocrinology 136, 3079–3086. Chochola, J., Fabre, C., Bellan, C., Luis, J., Bourgerie, S., Abadie, B., Champion, S., Marvaldi, J., and Battari, A. (1993). Structural and functional analysis of the human VIP receptor glycosylation. J. Biol. Chem. 268, 2312–2318. Chow, B. K. C. (1995). Molecular cloning and functional expression of a human secretin receptor. Biochem. Biophys. Res. Commun. 212, 204–211. Couvineau, A., Vosin, T., Guijarro, L., and Laburthe, M. (1990). Purification of VIP receptor from porcine liver by a newly designed one-step affinity chromatography. J. Biol. Chem. 265, 13386–13390. Couvineau, A., Fessard, C., Darmoul, D., Maoret, J. J., Carrero, I., Ogier, Denis, E. and Laburthe, M. (1994). Cloning of a common VIP-PACAP receptor from human intestine. Biochem. Biophys. Res. Commun. 200, 769–776. Couvineau, A., Gaudin, P., Maoret, J. J., Rouyer-Fessard, C., Nicole, P., and Laburthe, M. (1995). Highly conserved Aspartate 68, tryptophan 73 and glycine 109 in the N-terminal extracellular domain of the human VIP receptor are essential for its ability to bind VIP. Biochem. Biophys. Res. Commun. 206, 246–252. Crook, R. B., and Yabu, J. M. (1994). Down-regulation of VIP receptors by protein kinase C in fetal human non-pigmented ciliary epithelial cells. Exp. Eye Res. 59, 31–39. Dimaline, R., Young, J., Thwaites, D. T., Lee, C. M., Shuttleworth, T. J.

Goldfish VIP Receptor

and Thorndyke, M. C. (1987). A novel vasoactive intestinal peptide (VIP) from elasmobranch intestine has full affinity for mammalian pancreatic VIP receptors. Biochim. Biophys. Acta 930, 97–100. Gagnon, A. W., Aiyar, N., and Elshourbagy, N. A. (1994). Molecular cloning and functional expression of a human liver VIP receptor. Cell Signal. 6, 321–333. Gaudin, P., Covuinea, A., Maoret, J. J., Rouyer-Fessard, C., and Laburthe, M. (1995). Mutational analysis of cysteine residues within the extracellular domains of the human VIP1 receptor identifies seven mutants that are defective in VIP binding. Biochem. Biophys. Res. Commun. 211, 901–908. Gressens, P., Hill, J. M., Gozes, I., Fridkin, M., and Brenneman, D. E. (1993). Growth factor function of vasoactive intestinal polypeptide in whole cultured mouse embryo. Nature 362, 155–157. Ishihara, T., Shigemoto, R., Mori, K., Takahashi, K., and Nagata, S. (1992). Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide. Neuron 8, 811–819. Ishioka, C., Yoshida, A., Kimata, H., and Mikawa, H. (1992). VIP stimulates immunoglobulin production and growth of human B cells. Clin. Exp. Immunol. 87, 504–508. Jensen, J., and Holmgren, S. (1985). Neurotransmitters in the intestine of the Atlantic cod, Gadus morhua. Comp. Biochem. Physiol. C 82, 81–89. Langer, S., Van Noorden, S., Polak, J. M., and Pearse, A. G. E. (1979). Peptide hormone-like immunoreactivity in the gastrointestinal tract and endocrine pancreas of eleven teleost species. Cell Tissue Res. 199, 493–508. Lok, S., Kuijper, J. L., Jelinek, L. J., Kramer, J. M., Whitmore, T. E., Sprecher, C. A., Mathewes, S., Grant, F. J., Biggs, S. H., Sheppard, P. O., Rosenberg, G. B., O’Hara, P. J., and Foster, D. C. (1993). Human glucagon receptor: cDNA sequence, gene structure and chrosomal location. Gene 140, 203–209. Lundin, K., and Holmgren, S. (1984). Vasoactive intestinal polypeptide-like immunoreactivity and effects of VIP in the swimbladder of the cod, Gadus morhua. J. Comp. Physiol. B 154, 627–633. Lutz, E. M., Sheward, W. J., West, K. M., Morrow, J. A., Fink, G., and Harmar, A. J. (1993). The VIP2 receptor: Molecular characterization of a cDNA encoding a novel receptor for VIP. FEBS Lett. 334, 3–8. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). ‘‘Molecular Cloning: A Laboratory Manual,’’ 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Mayo, K. E. (1992). Molecular cloning and expression of a pituitaryspecific receptor for growth hormone-releasing hormone. Mol. Endocrinol. 6, 1734–1744. Okamoto, T., Murayama, Y., Hayashi, Y., Inagaki, M., Ogata, E., and Nishimoto, I. (1991). Identification of a Gs activator region of the beta2-adreneric receptor that is autoregulated via protein kinase A-dependent phosphorylation. Cell 67, 723–730. Ottaway, C. A. (1987). Role of sulfhydryl groups in the binding of VIP to its receptor on murine lymphocytes. Immunology 62, 291–297. Rawlings, S. R., and Hezareh, M. (1996). Pituitary adenylate cyclaseactivating polypeptide (PACAP) and PACAP/vasoactive intesti-

185 nal polypeptide receptors: Actions on the anterior pituitary gland. Endocr. Rev. 17(1) 4–29. Godfrey, P., Rahal, J. O., Beamer, W. G., Copeland, N. G., Jenkins, N. A., and Mayo, K. E. (1993). GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function. Nature Genet. 4, 227–232. Gozes, I., and Brenneman, D. E. (1989). VIP: Molecular biology and neurobiological function. Mol. Neurobiol. 3, 201–236. Ogi, K., Miyamoto, Y., Masuda, Y., Habata, M., and Christophe, J. (1993). Molecular cloning and functional expression of a cDNA encoding the human pituitary adenylate cyclase-activating polypeptide receptor. Biochem. Biophys. Res. Commun. 196, 1511–1521. Said, S. I., and Mutt, V. (1970). Polypeptide with broad biological activity: Isolation from small intestine. Nature 225, 863–864. Said, S. I. (1982). In ‘‘Advances in Peptide Hormone Research’’ (S. I. Said, Ed.), Vol. 1, Raven Press, New York. Schipani, E., Karga, H., Karaplis, A. C., Potts, J. T., Kronenberg, H. M. Abou-Samra, A-B. B., Segre, G. V., and Jueppner, H. (1993). Identical complementary deoxribonucleic acids encode a human renal and bone parathyroid hormone (PTH)/PTH-related peptide receptor. Endocrinology 132, 2157–2165. Sheward, W. J., Lutz, E. M., and Harmar, A. J. (1995). The distribution of VIP2 receptor messenger RNA in the rat brain and pituitary gland as assessed by in situ hybridization. Neurscience 67, 409–418. Sreedharan, S. P., Robichon, A., Peterson, K. E., and Goetzl, E. J. (1991). Cloning and expression of the human vasoactive intestinal peptide receptor. Proc. Natl. Acad. Sci. USA 88, 4986–4990. Sreedharan, S. P., Patel, D. R., Huang, J. X., and Goetzl, E. J. (1993). Cloning and functional expression of a human neuroendocrine vasoactive intestinal peptide receptor. Biochem. Biophys. Res. Commun. 193, 546–553. Thoren, B., Porret, A., Bhler, L., Deng, S., Morel, P., and Widmann, C. (1993). Cloning and expression of the human GLP-1 receptor: Demonstration that exendin-4 is an agonist and exendin-(9-39) an antagonist of the receptor. Diabetes 42, 1678–1682. Uesaka, T., Yano, K., Yamasaki, M., and Ando, M. (1995). Somatostatin-, vasoactive intestinal peptide- and granulin-like peptides isolated from intestinal extracts of goldfish, Carassius auratus. Gen. Comp. Endocrinol. 99, 298–306. Usdin, T. B., Mezey, E., Button, D. C., Brownstein, M. J., and Bonner, T. I. (1993). Gastric inhibitory polypeptide receptor, a member of the secretin-vasoactive intestinal peptide receptor family, is widely distributed in peripheral organs and the brain. Endocrinology 133, 2861–2870. Usdin, T. B., Bonner, T. I., and Mezey, E. (1994). Two receptors for VIP with similar specificity and complementary distributions. Endocrinology 135, 2662–2680. Vilardaga, J. P., Ciccarelli, E., Dubeaux, C., De, Neef, P., Bollen, A., and Robberecht, P. (1994). Properties of chimeric secretin and VIP receptor proteins indicate the importance of the N-terminal domain for ligand discrimination. Mol. Pharmacol. 45, 1022–1028. Wang, Y., and Conlon, J. M. (1995). Purification and structural characterization of vasoactive intestinal polypeptide from trout and bowfin. Gen. Comp. Endocrinol. 98, 94–101.

Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.