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Endocrinology Vol. 140, No. 3 1285-1293
Copyright © 1999 by The Endocrine Society

ARTICLES

A Cloned Frog Vasoactive Intestinal Polypeptide/ Pituitary Adenylate Cyclase-Activating Polypeptide Receptor Exhibits Pharmacological and Tissue Distribution Characteristics of Both VPAC1 and VPAC2 Receptors in Mammals1

David Alexandre, Youssef Anouar, Sylvie Jegou, Alain Fournier and Hubert Vaudry

European Institute for Peptide Research 23, Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, Centre National de la Recherche Scientifique, University of Rouen (D.A., Y.A., S.J., H.V.), 76821 Mont-Saint-Aignan, France; and Institut National de la Recherche Scientifique-Santé, Université du Québec (A.F.), Pointe Claire, Canada H9R 1G6

Address all correspondence and requests for reprints to: Dr. H. Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Three receptor subtypes for the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been identified in mammals: the PAC1 receptor (PAC1-R) which is selectively activated by PACAP, and two VPAC receptors (VPAC1-R and VPAC2-R), which are equally stimulated by PACAP and VIP. The structures of PACAP and VIP have been well conserved during evolution, but little is known about VIP/PACAP receptors in nonmammalian species. An amphibian VIP/PACAP receptor complementary DNA (cDNA) has been cloned and characterized from a frog (Rana ridibunda) pituitary cDNA library. The predicted protein contains seven putative transmembrane domains and exhibits the highest sequence identity (65%) with the human VPAC1-R. The cloned cDNA was transiently expressed in LLC-PK1 cells, and its pharmacological profile was determined in comparison with the human VPAC1-R. Both PACAP and VIP stimulated cAMP accumulation through the cloned receptor with an EC50 of about 30 nM. In contrast, secretin, at concentrations that stimulate the human VPAC1-R, had no effect on cAMP production. RT-PCR analysis revealed the widespread distribution of this frog VIP/PACAP receptor in peripheral tissues. In situ hybridization histochemistry using a complementary RNA probe showed that the receptor gene is highly expressed in several hypothalamic and thalamic nuclei and to a lesser extent in the pallium and striatum. In the pituitary, the highest messenger RNA levels were detected in the distal lobe. Taken together, these data show that the cloned frog receptor shares several common features with both the VPAC1-R and VPAC2-R of mammals; the frog receptor exhibits the highest sequence identity with mammalian VPAC1-R, but the lack of effect of secretin and the brain distribution of the receptor are reminiscent of the characteristics of the mammalian VPAC2-R. The sequence of the frog receptor should thus prove useful to decipher the structure-activity relationships of the VIP/PACAP receptor family.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VASOACTIVE intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two closely related neuropeptides that belong to the secretin/glucagon/GH-releasing hormone family of peptide hormones (1, 2). VIP is a 28-amino acid polypeptide, whereas PACAP occurs as two molecular forms of 38 and 27 amino acids (3, 4). VIP and PACAP27 exhibit 68% sequence identity, and their structures have been remarkably conserved during evolution. In particular, the sequences of almost all mammalian PACAP38 or VIP are identical (5, 6). In nonmammalian species, only minor amino acid changes have been found, such as the substitution of only one residue in the structure of PACAP38 in frog (7), three residues in catfish (8), or five residues in chicken PACAP (9). In the tunicates, two PACAP27 forms have been described that differ by one and four amino acids, respectively, from the human homolog (10). The structure of VIP shows four or five conservative substitutions in frog, chicken, or trout (11) compared with the mammalian peptides. The high conservation of the sequences of PACAP and VIP is consistent with the important physiological functions exerted by these neuropeptides (1, 12, 13, 14).

PACAP and VIP act through three types of G protein-coupled receptors, which also share a high degree of sequence similarity (15). Two of these receptors, VPAC1-R and VPAC2-R (16), bind PACAP and VIP with equal affinity (17, 18, 19, 20), whereas the third receptor, PAC1-R, selectively binds PACAP with high affinity (21, 22). In the central nervous system and in peripheral tissues, these receptors show both complementary and overlapping distributions (17, 19, 23, 24, 25).

Recent studies have attempted to delineate the structural domains of the VIP/PACAP receptors that specify their ligand recognition and activation. The data revealed that the amino-terminal extracellular domain, transmembrane domains I and II, and the extracellular loops are important for peptide binding and receptor responsiveness (26, 27, 28). However, only a few studies have identified specific amino acids that could be important for the function of these receptors (27, 29, 30). Cloning of VIP/PACAP receptors from distant species should prove useful to establish the structure-activity relationships of these receptors, because molecular evolution would have acted to preserve amino acid residues that are essential for ligand binding and activation. Moreover, characterization of the structure of VIP/PACAP receptors in nonmammalian vertebrate species and determination of their functional properties and expression pattern should provide crucial information for elucidating the evolutionary process of this family of receptors.

In this report we describe the molecular cloning of a frog (Rana ridibunda) PACAP/VIP receptor (fPVR) from a pituitary complementary DNA (cDNA) library. We have established the pharmacological profile of fPVR compared to that of the human VPAC1-R, and we have determined its tissue distribution in the frog brain and peripheral organs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning and sequencing
PCR amplifications were performed on both a frog pituitary cDNA library (31) and reverse transcribed RNA isolated from frog brain, adrenal, or heart. PCR was performed in a 50-µl volume containing 150 ng of the library DNA or 500 ng reverse transcribed RNA, 200 µM deoxy-NTPs, 1.5 mM MgCl2, 1 U Taq DNA polymerase, and 50 pmol sense and antisense primers in the buffer supplied with the enzyme (Eurobio, Les Ulis, France). A cDNA fragment of fPVR was amplified from the pituitary cDNA library using the sense primer 3P1 [5'-TGG(C/T)TI(C/T)TIGTIGA(A/G)GGI(C/T)TITA(C/T)CT-3'] and the antisense primer 7P1 [5'-TGIAC(C/T)TCICC(A/G)TTIA(A/G)(A/G)AA(A/G)CA(A/G)TA-3'], deduced from conserved regions in the third and seventh transmembrane domains of rat PACAP, VIP/PACAP, and secretin receptors. The same primers also amplified a cDNA fragment encoding part of a putative frog secretin receptor from reverse transcribed heart RNA. Finally, another pair of primers, namely 12F [5'-CA(C/T)TG(C/T) AC(A/C/G/T)(A/C)G(A/C/G/T)AA(C/T)T(A/T)(C/T)AT(A/C/T)CA-3'] deduced from the first intracellular loop and 3DR [5'-A(A/G) (A/G)TA(A/C/G/T)A(A/G)(A/C/G/T)CC(C/T)TC(A/C/G/T) A(C/T)(A/C/G/T)A(A/G)(A/C/G/T)A(A/G)CC-3'] deduced from the third transmembrane domain of the rat VIP/PACAP and PACAP receptors, amplified a 225-bp DNA fragment from the frog pituitary cDNA library. This DNA fragment was extended by rapid amplification of 3'-cDNA end reaction using a homologous oligonucleotide primer and a T7 universal primer. The latter PCR was nested using the 3P1 and 7P1 primers described above, and a DNA fragment encoding part of a putative frog glucagon receptor was amplified. All PCR amplifications were carried out for 30 cycles (1 min at 94 C, 1 min at 48 C, and 2 min at 72 C) in a Robocycler Gradient 40 (Stratagene, La Jolla, CA). The PCR products were purified and subcloned in the pGEMT vector (Promega Corp., Madison, WI). DNA was sequenced on both strands on a Li-Cor 4000L DNA sequencer (ScienceTec, Les Ulis, France) using fluorescent T7 and SP6 primers and the Thermosequenase kit (Amersham, Les Ulis, France).

Isolation of a full-length frog PACAP/VIP receptor cDNA
The frog pituitary cDNA library was screened with the 32P-labeled fPVR fragment in 50% formamide, 5 x SSC (1 x SSC = 0.15 M NaCl and 15 mM sodium citrate), 5 x Denhardt’s solution (0.1% BSA, 0.1% Ficoll, and 0.1% polyvinylpyrrolidone), 50 mM phosphate buffer (pH 6.5), 0.1% SDS, and 200 µg/ml salmon sperm DNA at 42 C, followed by two washes at 50 C with 0.1 x SSC-0.1% SDS for 15 min. Positive clones were isolated, purified, and sequenced as described above. DNA sequences were analyzed using DNASIS V2.1 software (Hitachi, Olivet, France).

Cell transfection
A full-length fPVR clone was inserted between the NotI and XbaI sites of the pcDNA1 expression vector (Invitrogen, Leek, The Netherlands) and transfected into kidney proximal tubule LLC-PKI cells (a gift from Dr. L. Journot, CNRS-UPR 9023, Montpellier, France) by electroporation. Cells were grown in DMEM (Sigma Chemical Co., Saint Quentin-Fallavier, France) containing 10% FCS (Bioproducts, Gagny, France) at 37 C in 5% CO2. About 2.5 x 106 cells in 500 µl culture medium were transfected with 20 µg of plasmid DNA at 1050 µF and 210 V using the EasyJect One electroporation system (EquiBio, Angleur, Belgium).

cAMP measurement
Transfected LLC-PK1 cells were plated in 24-well culture dishes at a density of 105 cells/well and cultured for 72 h. Cells were preincubated for 15 min at 37 C in 600 µl Krebs buffer (11.8 mM NaCl, 0.25 mM CaCl2, 0.12 mM KH2PO4, 0.12 mM MgSO4, 2.4 mM NaHCO3, 0.47 mM KCl, 1 mM HEPES, and 0.2 g/liter glucose, pH 7.3) containing 1 mg/ml BSA and 0.2 mM 3-isobutyl-1-methylxanthine (Sigma Chemical Co.). Peptides were dissolved in the incubation medium immediately before the experiment. Peptides were added at the appropriate concentration, and the cells were incubated for an additional 30 min at 37 C. The medium was removed, and cAMP contained in the cells was extracted by ice-cold ethanol. Alcohol was then evaporated in a Speed-Vac concentrator (AES 2000, Savant, Hicksville, NY), and the cAMP content was determined using a cAMP RIA kit (Amersham). Frog and human PACAP38 and porcine VIP were synthesized as previously described (13). PACAP27 was obtained from American Peptide Co. (Sunnyvale, CA). Chicken VIP and peptide histidine methionine (PHM) were purchased from Peninsula (Merseyside, UK), and porcine secretin was obtained from Sigma Chemical Co.

RT-PCR analysis
Total RNA was isolated from various frog tissues by the method of Chomczynski and Sacchi (32) using Tri-Reagent (Sigma Chemical Co.). Approximately 5 µg RNA were reverse transcribed using oligo(deoxythymidine)12–18 primer and SuperScript II reverse transcriptase ribonuclease H- (Life Technologies, Cergy Pontoise, France) in the buffer supplied with the enzyme. For fPVR messenger RNA (mRNA) detection, PCR was performed with the forward and reverse primers (5'- CACAATCTGCTCGTCATCTCC-3' and 5'-GTTGTCAGGGAAGAAGGCG-3', respectively), which amplified a 369-bp fragment of the frog receptor cDNA. Amplification was carried out for 30 cycles of 1 min at 94 C, 1.5 min at 65 C, and 1 min at 72 C. PCR products were separated on a 1.5% agarose gel, transferred to Hybond-N membrane (Amersham), and hybridized with a 32P-labeled fPVR probe under the conditions described above for screening of the library.

In situ hybridization histochemistry
In situ hybridization was performed as described previously (31, 33). Briefly, frogs were anesthetized and perfused transcardially with an ice-cold solution of 4% paraformaldehyde. Brain sections (15 µm thick) were cut on a cryostat and mounted onto poly-L-lysine- and gelatin-coated slides. A 763-bp fragment of the frog receptor cDNA (position 1329–2092) was subcloned into pBluescript II KS (Stratagene) between EcoRI and BamHI sites and used to generate sense and antisense riboprobes with T7 or T3 RNA polymerase, respectively, in the presence of [35S]UTP (Riboprobe Combination Systems, Promega Corp.). Hybridization was performed overnight at 55 C with 107 cpm/ml sense or antisense probe. The slides were exposed on Hyperfilm-ßmax films (Amersham) for 14 days. Tissue sections were then stained with hematoxylin and eosin for identification of anatomical structures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Screening for PACAP/VIP receptors in frog
To isolate PACAP/VIP receptor clones, a frog pituitary cDNA library and reverse transcribed RNA extracted from several frog tissues were screened by PCR using degenerate oligonucleotide primers deduced from conserved regions within the third and seventh transmembrane domains of mammalian PACAP-VIP-secretin receptors. Of more than 50 positive clones analyzed, 3 groups could be identified after sequencing and determination of the highest homology to members of the PACAP-VIP-secretin receptor family (Fig. 1Go). The first group of clones exhibited 65% amino acid identity with the human VPAC1-R (Fig. 1AGo) and was named fPVR, the second group showed 58% amino acid identity with the human secretin receptor (Fig. 1BGo), and the third group showed 70% identity with the human glucagon receptor (Fig. 1CGo). The putative frog VIP/PACAP and glucagon receptor clones were both amplified from the pituitary cDNA library, whereas the putative frog secretin receptor clone was amplified from heart cDNA. The clones obtained from other tissues, such as brain or adrenal, corresponded to fPVR.



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  		Figure 1. Amino acid sequences deduced from three partial frog cDNA clones obtained by PCR amplification. Each sequence was aligned with that of the corresponding human receptor homolog. A, fPVR exhibits the highest degree of homology with the human VPAC1-R (65% identity). B, Frog secretin receptor (fSecR) exhibits the highest degree of homology with the human secretin receptor (58% identity). C, Frog glucagon receptor (fGR) exhibits the highest degree of homology with the human glucagon receptor (70% identity). Conserved amino acids are shaded. Numbers on the right refer to the amino acid positions in the human sequences (18 42 43 ).

 
Cloning and sequence analysis of the frog PACAP/VIP receptor
The partial fPVR clone obtained by PCR was used as a probe to isolate full-length cDNA clones. Sequencing of the longest clone, which contained a 2.1-kb insert, revealed the presence of an open reading frame of 1332 bp encoding a 444-amino acid protein with a predicted molecular mass of 50,953 daltons. A hydropathy plot of this deduced protein identified eight hydrophobic regions, including a signal peptide encompassing the 19 N-terminal amino acid residues with a predicted signal cleavage site between residues Cys19 and Val20 (34) and seven putative transmembrane domains. Alignment of the deduced amino acid sequence with those of the different members of the mammalian PACAP/VIP receptors revealed that this fPVR has the greatest homology with the human VPAC1-R devoid of hip/hop cassettes (63% identity), followed by the human VPAC2-R (53% identity) and the rat PAC1-R (48% identity). Comparison of fPVR with human PACAP/VIP receptors revealed 64–77% amino acid identity within the putative seven membrane-spanning domains and 47–67% identity at the C-terminus, whereas the N-terminal domain showed only 32–42% identity (Fig. 2Go). Ten conserved cysteine residues are present in the extracellular domains of fPVR, seven of which are located in the N-terminal segment, two in the first extracellular loop, and one in the second extracellular loop. Five putative N-glycosylation consensus sites (-Asn-X-Ser/Thr-) were identified in the N-terminal segment of fPVR at Asn45,56,88,92,115. Four of these sites also occur in at least one of the human PACAP/VIP receptors, whereas the putative frog Asn115 glycosylation site is not present in any of the human receptors.



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  		Figure 2. Alignment of the amino acid sequences of the full-length fPVR with the human VPAC1 receptor (hVIP1-R), the human VPAC2 receptor (hVIP2-R), and the human PAC1 receptor (hPACAP-R). Putative transmembrane regions are overlined and labeled I–VII. Conserved amino acids are shaded. Arrowheads and dots indicate potential N-glycosylation sites and conserved cysteine residues, respectively. The arrow shows the putative cleavage site of the signal peptide of fPVR.

 
Pharmacological characterization
To demonstrate that the protein encoded by the cDNA isolated is a functional VIP/PACAP receptor, we examined the abilities of PACAP and VIP to stimulate adenylate cyclase in LLC-PK1 cells transfected with fPVR. Incubation of cells with graded concentrations of PACAP27, frog PACAP38, or VIP increased intracellular cAMP content in a dose-dependent manner (Fig. 3Go). All three peptides exhibited nearly the same potency, with a half-maximal concentration (EC50) of approximately 30 nM, and the same efficacy (15-fold increase at a concentration of 10-6 M). Human PACAP38 and porcine VIP produced similar dose-response curves (data not shown).



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  		Figure 3. Measurement of cAMP accumulation in LLC-PK1 cells transiently expressing fPVR. Transfected cells were incubated with graded concentrations of frog PACAP38 (•), PACAP27 ({blacksquare}), and VIP ({diamondsuit}) as described in Materials and Methods, and cAMP contained in the cell extracts was measured by RIA. Data are expressed as the mean ± SEM of three independent experiments performed in triplicate.

 
Although fPVR exhibited the highest sequence homology with the mammalian VPAC1-R, some of its amino acids are found in the human VPAC2-R or in the PAC1-R but not in the VPAC1-R (Fig. 2Go). This observation prompted us to compare the pharmacological profile of fPVR with that of the human VPAC1-R transfected in LLC-PK1 cells. PACAP and VIP stimulated cAMP formation in human VPAC1-R- and fPVR-transfected cells with the same efficacy, but the EC50 values for the human VPAC1-R (~2–4 nM) were lower than those for the fPVR (Fig. 4Go). In LLC-PK1 cells transfected by the human receptor, PHM and secretin were also able to stimulate cAMP production, albeit with much less efficiency than PACAP or VIP (Fig. 4AGo). In cells transfected with fPVR, PHM stimulated cAMP production, whereas secretin was totally devoid of effect, even at concentrations up to 5 x 10-6 M (Fig. 4B).



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  		Figure 4. Measurement of cAMP accumulation in LLC-PK1 cells transiently expressing the human VPAC1-R (A) or fPVR (B). Transfected cells were incubated with graded concentrations of mammalian PACAP38 (•), porcine VIP ({diamondsuit}), PHM ({blacktriangleup}), and porcine secretin (X). Data are expressed as the mean ± SEM of three (PACAP, VIP, and PHM) or two (secretin) independent experiments performed in triplicate.

 
Tissue distribution of fPVR
fPVR expression was investigated by RT-PCR on RNA isolated from various frog tissues. Strong amplification was observed for all tissues examined with the exception of the neurointermediate lobe of the pituitary, the pancreas, and the jejenum, which showed only a weak signal. No amplification was observed when reverse transcriptase was omitted (Fig. 5Go).



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  		Figure 5. Analysis of fPVR expression in various frog tissues by RT-PCR. RNA from the tissues indicated was incubated with a RT mixture in the presence (RT+) or absence (RT-) of reverse transcriptase. The cDNAs obtained were then amplified by PCR using specific fPVR primers to generate DNA products of 369 bp. The lane labeled fPVR corresponds to a PCR performed on the fPVR cDNA clone used as a positive control. Autoradiographs of Southern blots performed on the PCR products and hybridized with the fPVR cDNA are shown.

 
The distribution of fPVR mRNA was studied in the frog brain by in situ hybridization histochemistry. Antisense or sense 35S-labeled riboprobes were generated and used for hybridization to frog brain sections. A strong hybridization signal with the antisense probe was observed in discrete areas of the frog brain, such as the bed nucleus of the pallial commissure, several hypothalamic nuclei, and the lateral septum, whereas other brain regions exhibited only moderate labeling (Fig. 6Go). In the telencephalon, a moderate fPVR mRNA expression was found in the internal granular layer of the olfactory bulb (Fig. 6AGo), the lateral and dorsal pallium, and the dorsal and ventral striatum. A high concentration of fPVR mRNA was observed in the lateral and medial septum (Fig. 6BGo), the bed nucleus of the pallial commissure, and the anterior preoptic area (Fig. 6CGo). In the diencephalon, an intense hybridization signal was detected in the ventral hypothalamic nucleus and the nucleus of the periventricular organ (Fig. 6Go, D and E). The signal intensity was lower in several thalamic nuclei, the tectal lamina six, the pretectal gray, and the pretoral gray (Fig. 6Go, D and E). In the mesencephalon, a moderate concentration of fPVR mRNA was observed in the optic tectum (Fig. 6FGo). In the pituitary gland, the pars distalis was moderately labeled, whereas the pars intermedia and the pars nervosa were totally devoid of fPVR mRNA (Fig. 6FGo). When hybridization was performed with a sense probe, no labeling was observed (Fig. 6GGo).



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  		Figure 6. In situ hybridization analysis of fPVR mRNA in the brain and pituitary gland of Rana ridibunda using a specific complementary RNA probe. The autoradiographs show the distribution of fPVR mRNA in frog brain hemisections at the level of the telencephalon (A–C), diencephalon (D and E), and mesencephalon (E and F). Two brain hemisections at the level of the telencephalon hybridized with either an antisense or a sense probe are shown in G. No hybridization signal was observed with the control sense probe. The schematic parasagittal section of the frog brain shows the level of the frontal sections. AT, Anterior thalamic nucleus; AD, anterodorsal tegmental nucleus; AV, anteroventral tegmental nucleus; BN, bed nucleus of the pallial commissure; BON, basic optic nucleus; CNT, central thalamic nucleus; CO, subcommissural organ; DH, dorsal hypothalamic nucleus; DP, dorsal pallium; Dst, dorsal striatum; E, epiphysis; Ea, anterior entopeduncular nucleus; Ep, posterior entopeduncular nucleus; EPL, olfactory bulb, extragranular plexiform layer; GL, olfactory bulb, glomerular layer; IGL, olfactory bulb, internal granular layer; LA, lateral amygdala; La, lateral thalamic nucleus, anterior division; LH, lateral hypothalamic nucleus; LP, lateral pallium; LS, lateral septum; ML, olfactory bulb, mitral cellular layer; MP, medial pallium; MS, medial septum; NA, nucleus accumbens; NDB, nucleus of the diagonal band of Broca; NIP, nucleus interpeduncularis; NMLF, nucleus of the medial longitudinal fasciculus; NPM, nucleus profundus mesencephali; NPv, nucleus of the periventricular organ; OC, optic chiasma; ON, optic nerve; OT, optic tectum; Pdis, pars distalis; PE, postolfactory eminence; PI, pars intermedia; PN, pars nervosa; Poa, anterior preoptic area; PtG, pretectal gray; PtrG, pretoral gray; RIS, nucleus reticularis isthmi; SC, suprachiasmatic nucleus; TS, torus semicircularis; VH, ventral hypothalamic nucleus; VM, ventromedial thalamic nucleus; VN, vomeronasal nerve; Vst, ventral striatum; III, oculomotor and trochlear nuclei; 6, tectal lamina six.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have characterized, from the European green frog Rana ridibunda, a cDNA that encodes a 444-amino acid protein with seven putative membrane-spanning domains, and we have determined the pharmacological profile of the corresponding receptor. The predicted protein exhibited the highest degree of homology with the human VPAC1-R, followed by VPAC2-R and PAC1-R. The highest sequence homology was found in the putative transmembrane regions (64–77% amino acid sequence identity), whereas the N-terminal region exhibited a low degree of homology (32–42% identity). It is worth noting that this amino-terminal extracellular region has been shown to confer the binding selectivity of the mammalian VPAC1-R toward VIP (26, 28). The low conservation of the amino acid sequence of this region indicates that a few residues are presumably sufficient to impart VIP and PACAP recognition. Consistent with this idea, site-directed mutagenesis studies have recently shown that several of the residues conserved from frog PVR to mammalian VPAC1-R are essential for VIP binding (27, 29, 30). As in mammalian VIP/PACAP receptors (17, 18, 19, 20), 10 cysteine residues were found in the sequence from the N-terminus to the second extracellular loop of fPVR. Mutational studies performed with the mammalian VPAC1-R revealed that the disulfide bonds between the cysteine residues contribute to the active conformation of the receptor (35, 36). The conservation of these cysteine residues from frog to mammals emphasizes their importance. Five potential N-glycosylation sites were found in the amino-terminal part of fPVR. The three most N-terminal sites (Asn45, Asn56, and Asn88) matched the N-glycosylation sites found in VPAC1-R, which have been shown to be used for N-glycosylation, a modification necessary for correct delivery of this receptor to the plasma membrane (37). The fourth potential N-glycosylation site (Asn92) of fPVR is conserved in VPAC2-R and PAC1-R, but its importance has not yet been studied. The last putative N-glycosylation site (Asn115) of fPVR is not found in any mammalian PACAP/VIP receptor. Thus, fPVR possesses common as well as unique potential glycosylation sites that may influence its functional properties compared with each of the mammalian receptors.

Although fPVR exhibited the highest degree of identity with the VPAC1-R, substantial sequence similarity with the three mammalian PACAP/VIP receptors was observed. The pharmacological profile of fPVR was established through transient expression in LLC-PK1 kidney cells compared to human VPAC1-R. PACAP and VIP stimulated cAMP accumulation with the same efficacy in LLC-PK1 cells transfected by frog or human receptor. However, although the human receptor exhibited EC50 values in the nanomolar range as was previously reported (17, 18), fPVR showed higher EC50 values, probably reflecting the level of receptor expression or the different efficacies of receptor coupling to adenylate cyclase. These data clearly indicated that fPVR is a functional frog VIP/PACAP receptor, but they were not sufficient to distinguish between the VPAC1-R-like and VPAC2-R-like phenotypes. It has previously been shown that secretin is a weak, but selective, agonist of the mammalian VPAC1-R and that it does not activate the VPAC2-R. Consistently, secretin could stimulate cAMP formation in LLC-PK1 cells transfected with the VPAC1-R. In contrast, secretin was totally devoid of effect on cells transfected with fPVR, indicating that the latter possesses a pharmacological profile similar to that of mammalian VPAC2-R (17, 18, 19, 23) even though it shares the highest degree of homology with VPAC1-R of mammals. The structural determinants for the differential effect of secretin on VPAC receptors are currently unknown. The sequence of fPVR may thus prove useful for identification of the amino acid residues that are involved in determining the selectivity of the VPAC receptors for secretin.

Structure-activity studies have shown that three nonadjacent amino acids present between the first extracellular loop and the third transmembrane domain of mammalian VPAC1-R, i.e. H208, A212, and V220 in the rat (17) and Q207, G211, and M219 in the human (18), are responsible for the higher affinity of the rat vs. the human receptor for the VIP-related peptide histidine-isoleucine (PHI) (38). Our data revealed that fPVR, like human VPAC1-R, exhibits a low affinity for PHM. The three amino acids of fPVR that align with those involved in PHI recognition in mammalian VPAC1 receptors are H194, G198, and M206. Interestingly, two of these residues (G198 and M206) are identical to those present in human VPAC1-R (18).

We have also investigated the distribution of fPVR mRNA in frog tissues to correlate it to that of mammalian VPAC1 and VPAC2 receptors. RT-PCR analysis revealed that fPVR mRNA is expressed in all frog tissues studied. In mammalian species, RT-PCR and ribonuclease protection assay analyses have shown that liver expresses exclusively VPAC1-R, whereas stomach and skeletal muscle express mostly VPAC2-R (17, 20, 23, 24). Thus, it appears that fPVR is more widely expressed in peripheral tissues, including liver, stomach, and skeletal muscle, than its mammalian homologs. At the brain level, in situ hybridization histochemistry showed that fPVR mRNA is highly expressed in several thalamic and hypothalamic nuclei and at lower levels in the pallium (equivalent to the hippocampus in mammals) (39). Mammalian VPAC1-R mRNA is highly expressed in the cerebral cortex and hippocampus, but is not expressed in the hypothalamus, whereas VPAC2-R mRNA is predominantly expressed in several thalamic and hypothalamic nuclei (17, 23, 25). Thus, the expression pattern of fPVR mRNA in the frog brain exhibits similarities with those of both VPAC1-R and VPAC2-R in mammals. In the frog pituitary, RT-PCR analysis followed by Southern blotting revealed the presence of high levels of fPVR mRNA in the distal lobe, whereas only a faint band was detected in the neurointermediate lobe. These results were confirmed by in situ hybridization histochemistry. Such a distribution is reminiscent of that previously described for the mammalian VPAC2-R. Specifically, in the rat pituitary, in situ hybridization studies have shown that the anterior lobe contains mainly VPAC2-R mRNA and little or no VPAC1-R mRNA (23). In the rat neurointermediate lobe, only a few cells express the VPAC2-R gene (23). Using RT-PCR, Rawlings et al. (40) have shown that the anterior lobe of the pituitary expresses mainly PAC1-R and VPAC2-R.

The fact that the cloned fPVR exhibited structural, pharmacological, and regional distribution features common to both VPAC1-R and VPAC2-R of mammals raised the question of the existence of a unique VIP/PACAP receptor in frog that would be more widely expressed in the brain and peripheral tissues than its mammalian homologs. We have thus attempted to clone other PACAP/VIP receptors from several other frog tissues using different sets of primers for PCR amplifications. These experiments led to the isolation of the frog homologs of the mammalian secretin and glucagon receptors as well as a frog PAC1-R (our unpublished results). Despite our efforts, we could not obtain evidence for the expression of a second form of a VIP/PACAP receptor in frog. These negative data would suggest that the gene duplication that yielded the two VPAC-R variants identified in mammals took place after the separation of amphibians from the lineage leading to reptiles, birds, and mammals. Recently, a VIP/PACAP receptor was cloned from the goldfish (41). However, although the researchers concluded that the cloned receptor is a VPAC1-R homolog, no precise pharmacology or tissue distribution was reported for this goldfish receptor that could substantiate its relationship to fPVR.

In conclusion, we have isolated a frog VIP/PACAP receptor that exhibits the highest sequence homology with the mammalian VPAC1-R but possesses pharmacological and tissue distribution characteristics of both VPAC1-R and VPAC2-R of mammals. The cloning of this frog receptor provides the molecular basis for identification of the structural determinants for ligand binding and activation of PACAP/VIP receptors.


   Acknowledgments
 
We thank Dr. M. Laburthe for his kind gift of human VPAC1 receptor cDNA, and Dr. L. Journot for LLC-PK1 cells. The expert technical assistance of P. Bizet is gratefully acknowledged.


   Footnotes
 
1 This work was supported by grants from INSERM (U-413) and the Conseil Régional de Haute-Normandie. The nomenclature of the VIP and PACAP receptors used in the present study is that recommended by IUPHAR in 1998. The sequences reported in this study have been deposited in GenBank: frog VIP/PACAP receptor, accession no. AF100644; frog glucagon receptor, accession no. AF100642; and frog secretin receptor, accession no. AF100643. Back

Received August 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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