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European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale, U-413, Unité Affiliée Centre National de la Recherche Scientifique, University of Rouen (D.A., H.V., L.G., V.T., S.J., Y.A.), 76821 Mont Saint Aignan, France; Institut National de la Recherche Scientifique/Institut Armand Frappier, University of Quebec (A.F.), H9R 1GG Montréal, Canada
Address all correspondence and requests for reprints to: Dr. Youssef Anouar, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale, U-413, Unité Affiliée Centre National de la Recherche, University of Rouen, 76821 Mont Saint Aignan, France. E-mail: . youssef.anouar{at}univ-rouen.fr
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Introduction |
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Shortly after the discovery of the peptide, specific PACAP receptors were identified (4, 5) and subsequently cloned. Two types of PACAP receptors have been characterized that belong to the superfamily of G protein-coupled, seven-transmembrane domain (TMD)-containing receptors. Type I receptors, or PAC1-R, exhibit a high affinity for PACAP and a much lower affinity for VIP (6). Type II receptors, in contrast, recognize both PACAP and VIP with high affinity and include two receptor subtypes, named VPAC1-R and VPAC2-R (7, 8). All three receptors are positively coupled to adenylate cyclase (AC), whereas only PAC1-R is coupled to phospholipase C (PLC). Alternative splicing of the gene encoding PAC1-R generates different variants with distinct pharmacological properties. Thus, four different splicing events in the region encoding the third intracellular loop give rise to the molecular variants hip, hop1, hop2, and hip-hop1 of PAC1-R (6). These splice variants exhibit differences in the efficacy of AC or PLC activation by PACAP (6, 9). Alternative splicing also generates PAC1-R variants in the extracellular N-terminal domain that lack 21 or 57 amino acids or, on the contrary, that exhibit an insertion of 24 amino acids and show increased potency of PACAP27 to stimulate PLC activity (10), increased affinity for VIP (11), and altered PACAP binding and signaling transduction pathways, respectively (12). Finally, a variant with discrete amino acid modifications in TMD II and IV is no longer coupled to AC or PLC, but mediates stimulation of calcium influx through L-type channels (13).
The neuropeptide PACAP and its receptors have been also extensively studied in an amphibian species model, the European green frog Rana ridibunda (see Ref. 14 for review). Soon after the discovery of PACAP in mammals, the sequence of frog PACAP38 has been characterized, and the peptide has been shown to stimulate AC in the pituitary (15). Further studies revealed that in frog, PACAP increases the cytosolic calcium concentration in different pituitary cells (16) and stimulates the release of several pituitary hormones (17). The hypophysiotropic effect of PACAP is supported by the abundance of PACAP-encoding mRNA and PACAP immunoreactivity in the frog hypothalamus as well as the existence of a dense network of PACAP-containing fibers in the median eminence (18, 19). We recently cloned a frog PACAP/VIP receptor that displays a broad distribution in the central nervous system and peripheral tissues (20). Concurrently, a PAC1-R homolog has been recently characterized in Xenopus laevis, and this receptor has been found to be highly expressed in the brain (21). However, although several PAC1-R variants have been found in mammals, no such variants have been described in submammalian species. In the present study we characterized PAC1-R in the frog R. ridibunda and identified novel splice variants for this receptor that exhibit differential coupling to adenylate cyclase. The distribution of the mRNAs encoding PAC1-R and its variants in the brain and peripheral tissues of the frog R. ridibunda has also been determined.
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Materials and Methods |
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Cloning and sequencing
PCR was performed in 50-µl volume containing 500 ng reverse transcribed RNA, 200 µM dNTPs, 1.5 mM MgCl2, 1 U Taq DNA polymerase (Promega Corp., Charbonnières, France), and 50 pmol sense and antisense primers in the buffer (pH 9.0) supplied with the enzyme for 30 cycles (1 min at 94 C, 1 min at 44 C, and 1 min at 72 C) in a Robocycler Gradient 40 (Stratagene, La Jolla, CA). To amplify a frog PAC1-R cDNA fragment, two successive PCR were performed on reverse transcribed RNA isolated from pituitary neurointermediate lobe tissue, using oligonucleotides deduced from conserved regions of PACAP receptors in mammals. In the first PCR amplification, the sense primer 3P2 (5'-TGGYTITTYATYGARGGIYTITAYCT-3'; sequence in the third TMD) and the antisense primer HOPR (5'-GCICGYTGIGGYTTRCARTARCA-3'; sequence in the hop cassette of PAC1-R) were employed. The products obtained were used in a second PCR performed with the 3P2 primer described above and the antisense primer 45R (5'-TCDATIGTRTCCCARCANCC-3'; sequence in the second extracellular loop) to amplify a 164-bp cDNA fragment encoding a portion of the frog PAC1-R sequence. Based on the sequence of this cDNA fragment, a homologous 44-mer oligonucleotide SFPVR1 (5'-GTGAAGTCGTAGCACAGCCCATATAGTGACACATATAAGTGGGG-3') was synthesized and used to screen a frog brain cDNA library (22) to isolate a full-length cDNA encoding the frog PAC1-R.
Approximately 750,000 recombinant phages of the library were screened with the 32P-labeled SFPVR1 oligonucleotide in 50% formamide, 4x SSC (1x SSC = 0.15 M NaCl and 15 mM sodium citrate), 1x Denhardt’s (0.1% BSA, 0.1% Ficoll, and 0.1% polyvinylpyrrolidone), 50 mM phosphate buffer (pH 6.5), 0.1% SDS, 200 µg/ml salmon sperm DNA, and 100 µg/ml tRNA at 42 C. Filters were washed twice at 42 C with 0.2x SSC/0.1% SDS for 15 min. Positive clones were isolated by plaque purification, zapped in pBluescript SK (Stratagene), and sequenced on both strands on a Li-Cor 4000L DNA sequencer (ScienceTec, Les Ulis, France) using fluorescent T7 and T3 primers (MWG-Biotech, Courtaboeuf, France) and the Thermosequenase kit (Amersham Pharmacia Biotech, Les Ulis, France). DNA sequences were analyzed using DNASIS V2.1 software (Hitachi, Olivet, France).
The frog PAC1-R variants were amplified by a primary PCR on reverse transcribed RNA isolated from the brain, testis, spleen, or distal lobe of the pituitary, using the primers S3F (5'-GAAATGAACAACAATGTTGCC-3'; sequence in the second extracellular loop) and S2R (5'-AGTGACATTTGACTGTTCTC-3'; sequence in the 3'-noncoding region), followed by a secondary PCR with the S2R primer combined to the S2F primer (5'-TGCAGTCTCCAGACATAGG-3'; sequence in the third intracellular loop). The PCR products were subcloned into the pGEMT vector and sequenced on both strands.
Construction of expression plasmids
The full-length frog PAC1-R cDNA in pBluescript SK was cut by SacI, polished with T4 DNA polymerase, and digested by XhoI. The insert was subcloned into the EcoRV/XhoI sites of the pcDNA1 expression vector (Invitrogen, Leek, The Netherlands). The resulting PAC1-R-pcDNA1 plasmid was used to make expression vectors for the PAC1-R splice variants. The PAC1-R25 and PAC1-R41 cDNAs subcloned in the pGEMT vector were digested with PstI/AccIII enzymes, and the digested inserts were introduced in the PstI/AccIII-digested PAC1-R-pcDNA1 vector described above. The splice variant PAC1-Rmc was digested with AccIII/HincII enzymes, and the digested insert was ligated to the AccIII/HincII-cut PAC1-R-pcDNA1 vector.
To study the expression of PAC1-R and PAC1-Rmc in transfected cells, fusion proteins with the green fluorescent protein (GFP) were constructed. To this end, cDNA fragments encoding the C-terminal region of these two receptor variants were amplified using a common sense S3F primer and a specific antisense primer for each receptor, in which the stop codon had been mutated and replaced by an XhoI restriction site. These primers correspond to GFP1R (5'-TGCACTCGAGTGTCACCAGGTTCTCCGC-3') in the case of PAC1-R and GFP1Rmc (5'-TGCACTCGAGGGCAGTTTGGGAACTCAT-3') in the case of PAC1Rmc. The XhoI site introduced (underlined sequence) is in-frame with the GFP sequence. The PCR fragments generated were digested by XhoI at the C terminus and by an internal HindIII site at the N terminus, and subcloned into the GFP-encoding vector phrGFP-C (Stratagene). The sequence encoding the N-terminal part to the HindIII site of these receptors was cut from PAC1-R-pcDNA1 and spliced in the phrGFP-C vector constructs to produce the fusion proteins of PAC1-R or PAC1-Rmc and GFP. All constructs were confirmed by sequencing.
Cell transfection
For pharmacological studies, expression plasmids were transfected by electroporation into LLC-PK1 cells, a pig kidney proximal tubular epithelial cell line (a gift from Dr. L. Journot, Centre National de la Recherche Scientifique-UPR 9023, Montpellier, France). Cells were grown in DMEM (Sigma-Aldrich Corp., 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 plasmid DNA at 1050 µF and 210 V using the EasyJect One electroporation system (EquiBio, Angleur, Belgium).
For the analysis of receptor-GFP fusions, plasmids were transfected into LLC-PK1, CHO-K1, and PC12 cells (ECACC, Salisbury, UK) using the Lipofectamine reagent (Life Technologies, Inc., Cergy-Pontoise, France). Approximately 5 x 105 cells/well were plated on glass coverslips (diameter, 25 mm) in six-well plates and allowed to grow for 24 h before transfection. Cells were washed twice with 1 ml DMEM and transfected with 2 µg GFP fusion plasmids using 8 µl Lipofectamine in 1 ml DMEM during 3.5 h. The medium was then replaced by 2 ml DMEM supplemented with 10% FCS.
Peptides
Frog PACAP38 was synthesized by the solid phase methodology as previously described (18). Frog/chicken VIP (23) was purchased from Peninsula Laboratories, Inc. (Merseyside, UK). Peptides were dissolved in the incubation medium immediately before the experiment.
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 500 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). Peptides were added at the appropriate concentrations, and the cells were incubated for an additional 30 min at 37 C. The medium was removed, and intracellular cAMP was extracted by ice-cold ethanol. Alcohol was then evaporated in a Speed-Vac concentrator (AES 2000, Savant Instruments, Hicksville, NY), and the cAMP content was determined using a cAMP RIA kit (Amersham Pharmacia Biotech). Proteins were quantitated by the Bradford protein assay (Bio-Rad Laboratories, Inc., Marnes la Coquette, France).
Confocal laser scanning microscopy
Approximately 48–72 h after transient transfection of PAC1-Rs-GFP plasmids into LLC-PK1, CHO-K1, and PC12 cells, GFP was imaged in living cells using a CLSM NORAN OZ (Noran Instruments, Middleton, WI) and a 488-nm excitation line of an argon/krypton laser. The emission fluorescence was detected with a 525- to 552-nm bandpass filter. Images were acquired using a x60 water immersion objective (Nikon, Melville, NY) and Intervision software (Noran Instruments).
Northern blot analysis
Total RNA was prepared by the acid guanidinium thiocyanate-phenol-chloroform method described by Chomczynski and Sacchi (24) using Tri-Reagent (Sigma-Aldrich Corp.). Polyadenylated [poly(A+)] RNA was extracted with the Poly(A+) tract mRNA Isolation System (Promega Corp.), separated on a formaldehyde-agarose denaturing gel, and transferred onto a nylon membrane Hybond NX (Amersham Pharmacia Biotech). The blot was hybridized with the 32P-labeled fragment (position 1713–1938) of the frog PAC1-R cDNA in 50% formamide, 5x SSC, 5x Denhardt’s solution, 50 mM phosphate buffer (pH 6.5), 0.1% SDS, and 200 µg/ml salmon sperm DNA at 42 C and washed twice at 50 C with 0.1x SSC and 0.1% SDS for 15 min.
RT-PCR and Southern blot
Approximately 5 µg total RNA from various tissues were reverse transcribed using an oligo(deoxythymidine)12–18 primer and the SuperScript II reverse transcriptase ribonuclease H- (Life Technologies, Inc.) in the buffer supplied with the enzyme. One tenth of this reaction mixture was used for PCR with the oligonucleotides S3F and S2R described above for PAC1-R mRNA amplification. PCR was performed for 25 cycles at an annealing temperature of 55 C with a primer specific for each PAC1-R isoform and a primer common to all isoforms. The primer pair S2R/25F (5'-CAGTACTCTGTCAAGATGTC-3') was used to amplify PAC1-R25. The primer pair S2R/41F (5'-GAATCACAACGGACTGACTG-3') was used to amplify PAC1-R41. The primer pair S3F/TER (5'-TGCACCTTTAGGGAGAAA-3') was used to amplify PAC1-Rmc. The PCR products were transferred to nylon filters and hybridized with the PAC1-R cDNA fragment 1388–1937 to confirm their identity.
In situ hybridization histochemistry
Adult male frogs were anesthetized and perfused transcardially with an ice-cold solution of 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were postfixed overnight in the same solution, transferred into 0.1 M phosphate buffer containing 15% saccharose for 12 h, and frozen in isopentane at -30 C. Frontal sections (12-µm thick) were cut in a cryostat (2800 Frigocut, Leica Corp., Rueil-Malmaison, France) and used for in situ hybridization as described previously (19). Sense and antisense riboprobes were generated using T3 and T7 RNA polymerases in the presence of [35S]UTP (Riboprobe Combination Systems, Promega Corp.) and a 695-bp fragment of the frog PAC1-R cDNA (position 1–695) subcloned into pBluescript II KS (Stratagene) between the PstI and SmaI sites. Hybridization was performed overnight at 60 C using 1.5 x 107 cpm/ml heat-denatured RNA probes. Brain slices were washed in 2x SSC at 60 C and treated with ribonuclease A (50 µg/ml) for 60 min at 37 C. Five final washes were performed in 0.1x SSC containing 14 mM 2-mercaptoethanol and 0.05% sodium pyrophosphate. The slices were dehydrated in ethanol and exposed to Hyperfilm ß-max (Amersham Pharmacia Biotech) for 5 d. Tissue slices were subsequently dipped into Kodak NTB2 liquid emulsion at 40 C, exposed for 30 d, and developed. To identify anatomical structures, sections were stained with hematoxylin and eosin. Nomenclature of frog brain structures was based on the atlas of Neary and Northcutt (25).
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Results |
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). Comparison with the PACAP/VIP receptor sequences revealed that the frog receptor had the greatest sequence identity with PAC1-R of X. laevis (88%), human PAC1-R (78%), and goldfish PAC1-R (71%; Fig. 1). R. ridibunda PAC1-R exhibited 47% amino acid identity with the previously characterized frog VPAC receptor (20). The PAC1-R domain encompassing the transmembrane segments and the C-terminal tail is strongly conserved (>90%), whereas the N-terminal region shows less identity (50–70%) in the different species (Fig. 1). Within the N-terminal domain, a short 21-amino acid sequence that is present in mammalian PAC1-Rs and not in other receptors of the VIP/secretin/glucagon family is only partially conserved (Fig. 1). Ten extracellular cysteine residues in the PAC1-R sequence, i.e. seven in the N terminus and three in the first and second extracellular loops, are strictly conserved across the different vertebrate species. Finally, the R. ridibunda PAC1-R sequence possesses five consensus N-glycosylation sites, two of which are conserved in all species studied, and the others are found in one or the other species (Fig. 1).
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). RT-PCR analysis of different tissues confirmed the intense expression level of PAC1-R mRNA in the brain and revealed moderate expression levels in the spinal cord, the distal lobe of the pituitary, and the spleen (Fig. 2B). In other tissues, very low or no mRNA signal was detected. Note that more than one PCR product was amplified, indicating the existence of molecular variants in frog tissues. When reverse transcriptase was omitted in the RT-PCR, no amplification product was observed (Fig. 2B).
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). Low to moderate PAC1-R mRNA levels were detected in the striatum and the septum nuclei (Fig. 3B).
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). Caudally, the central thalamic nucleus and the ventral hypothalamus were moderately labeled, whereas the lateral thalamic nucleus and the lateral hypothalamic nucleus exhibited only a weak signal (Fig. 3D). More caudally, an intense mRNA signal was observed in the nucleus of the periventricular organ and in the posterior thalamic nucleus (Fig. 3E).
In the mesencephalon, a positive signal was observed in the optic tectum and in the dorsal and the ventral parts of the tegmentum (Fig. 3F). No hybridization signal could be detected in the pituitary gland (Fig. 3F).
Control sections, taken at different levels of the brain and treated with the sense PAC1-R probe, did not show any hybridization signal, as illustrated in Fig. 3G.
In all of the autoradiograms shown in Fig. 3, a robust signal was observed in the periventricular regions. Microscopic examination of emulsion-coated slices showed strong accumulation of silver grains over the ependymal cells bordering the ventricles (Fig. 4). For instance, in the telencephalon, intense labeling was seen around the ventricle, whereas neighboring cells of the distal pallium only displayed a weak signal (Fig. 4A). In the diencephalon, a high density of silver grains was observed in the ependymal cell layer and in the ventral hypothalamus (Fig. 4B), whereas the posterior thalamic nucleus was only moderately labeled (Fig. 4C). Incubation of consecutive sections with the sense probe only produced weak background staining (Fig. 4, D–F).
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) have been found in the brain, the distal lobe of the pituitary, the testis, or the spleen. The frog PAC1-R25 presents a 25-amino acid insertion after the Leu336 residue. The inserted sequence is highly homologous (68% amino acid identity) to the human hop cassette (6) (Fig. 5B). The frog PAC1-R41 contains an insertion of 41 residues at the same location as in the PAC1-R25 variant; however, the sequence of this cassette is completely different from those of the hip and hop cassettes of mammalian PAC1-Rs (Fig. 5C). The third variant, PAC1-Rmc, contains an insertion of 13 nucleotides located at the C-terminal end of the seventh TMD sequence, at the level of residue Glu393. This insertion alters the reading frame leading to a completely different C-terminal sequence (Fig. 5D).
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). PACAP induced a dose-dependent stimulation of cAMP accumulation in cells transfected with frog PAC1-R with an EC50 of approximately 10-8 M, whereas VIP was far less potent (Fig. 6). In cells transfected with PAC1-R, PAC1-R25, and PAC1-R41, PACAP also stimulated cAMP formation with an EC50 of approximately 10-8 M and similar efficacy (Fig. 7), although slightly greater cAMP production was systematically observed with PAC1-R25 (Fig. 7). Conversely, in PAC1-Rmc-transfected cells, PACAP and VIP were totally unable to stimulate cAMP formation (Fig. 7 and data not shown), suggesting that the sequence of the C-terminal tail of PAC1-R is crucial for its coupling to Gs proteins.
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, A and B). In contrast, transfection of the plasmid encoding GFP alone resulted in evenly distributed fluorescence labeling (Fig. 8C). The PAC1-Rs-GFP fusion proteins were also targeted to the cell surface in transfected CHO-K1 and PC12 cells (Fig. 8, D–F). These results indicate that the frog PAC1-R variants are expressed properly and targeted to the plasma membrane, and that the loss of Gs coupling observed with the PAC1-Rmc variant is not due to a deficiency in the expression or the compartmentalization of this receptor.
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). PAC1-R and PAC1-R25 mRNAs exhibited similar distribution patterns, and both messengers were detected in peripheral tissues such as the testis, spleen, and heart (Fig. 9, A and B). In contrast, PAC1-R41 mRNA was not detected in these peripheral tissues (Fig. 9C). Finally, PAC1-Rmc mRNA was detected in the testis, but not in the spleen (Fig. 9D). When reverse transcriptase was omitted in the RT-PCR, no amplification product was observed (Fig. 9).
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Discussion |
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In mammals, a 21-amino acid domain is present in the N terminus of PAC1-R, but not at the equivalent position of other members of this receptor family, including VPAC-Rs (9). A similar 21-amino acid insertion is present in the Xenopus PAC1-R (21). In the frog R. ridibunda, sequence alignment showed the occurrence of an insertion at the same position (Fig. 1). However, the sequence of frog PAC1-R encompasses only 12 residues (amino acids 88–99) at this position. In fact, it has been shown that this region in the human PAC1-R gene could be alternatively spliced to give rise to 2 receptor variants lacking either the 21-amino acid cassette (10) or a 57-amino acid cassette that includes the previous one (11). The insertion of these cassettes in the N-terminal domain of PAC1-R leads to a decrease in the ability of PACAP27 to stimulate PLC activity (10) and in the affinity for VIP (11) in transfected cells. R. ridibunda PAC1-R, which has a 12-amino acid cassette, exhibited a low affinity for VIP, as revealed by cAMP accumulation data (Fig. 6). Although the coupling efficacy of frog PAC1-R to PLC remains to be determined, the fact that the length of this cassette seems to impinge on the pharmacological properties of PAC1-R in mammals suggests that the frog receptor may exhibit some particular functional features.
The third intracellular loop is another site of alternative splicing in the mammalian PAC1-R. This region of the heptahelical receptors is involved in the coupling to and activation of G proteins (30). In R. ridibunda, two variants of the receptor also occur as the result of cassette insertion in the third intracellular loop, at the very position where the hip and hop cassettes occur in mammals. One of the frog isoforms, PAC1-R25, contains a cassette that is homologous to the hop cassette in mammals and exhibits a pharmacological profile similar to PAC1-R, in agreement with previous data reported in mammals (6). In contrast, the second variant of the third intracellular loop of the frog receptor, PAC1-R41, contains a cassette whose sequence is completely different from those of the mammalian hip and hop cassettes. The efficacy of PACAP to stimulate cAMP formation through PAC1-R41 in transfected cells was comparable to that observed with the two previous variants. It remains to be established whether these frog PAC1-R isoforms display any differential coupling efficacy to PLC or calcium mobilization, as has been shown for PAC1-R variants in mammals (6, 13).
The third variant of frog PAC1-R that we have identified, PAC1-Rmc, is unique in that it carries a cytoplasmic C-terminal tail with a sequence completely different from that in PAC1-R. This modification is the consequence of a 13-nucleotide insertion at the cytoplasmic extremity of the seventh TMD that shifts the open reading frame of the receptor and thus alters the sequence and the size (10 amino acids longer than in PAC1-R) of the intracellular C-terminal domain. The variations at the C terminus of G protein-coupled receptors that have been described to date correspond to extended or shortened C-terminal tails, as in the somatostatin-2 receptor (31), the neurokinin-1 receptor (32), or the GnRH receptor (33). To our knowledge this is the first report describing a receptor variant with a totally different C-terminal tail in the superfamily of G protein-coupled receptors.
The modification of the C-terminal tail of the frog PAC1-R abolished the stimulatory effect of PACAP on adenylate cyclase in transfected cells, suggesting that PAC1-Rmc was either not expressed at the plasma membrane or had lost its coupling efficacy to a Gs protein. Given that PAC1-Rmc retains all its hydrophobic domains and its potential posttranslational modifications, it seemed unlikely that this receptor was not addressed properly. Indeed, the fusion of this receptor variant to GFP showed that PAC1-Rmc is targeted to the cell surface in LLC-PK1 and CHO cells, as is the PAC1-R, and in PC12 cells, a neuroendocrine cell line that naturally expresses PAC1-R, confirming that PAC1-Rmc is actually expressed in transfected cells and correctly targeted to the plasma membrane. These findings, which show that the C-terminal domain is instrumental in the coupling of PAC1-R to adenylate cyclase, are consistent with a recent report demonstrating the critical role of the proximal C-terminal region of the human PAC1-R for signal transduction (34). In addition, it has been shown that mutation or deletion of amino acids within the C terminus or even complete truncation of this part of PAC1-R does not alter the ligand binding to the receptor (34), suggesting that the C-terminus modification in PAC1-Rmc should not substantially modify its affinity for the ligand. In agreement with these observations, there are multiple examples showing that alteration of the intracellular C-terminal domain of seven TMD receptors mainly impairs coupling efficiency to G proteins (35, 36).
It is noteworthy that within the mammalian PAC1-R gene an intron is located exactly at the position where the insertion occurs in the frog PAC1-Rmc. Indeed, the 14th intron in the rat and mouse PAC1-R genes occurs after the Glu residue located at the C-terminal end of the seventh TMD (37, 38), i.e. at the site where the modification of PAC1-Rmc is found. This structural feature of the gene supports the existence of an isoform of PAC1-R that is alternatively spliced in this region.
The transduction pathways associated with activation of PAC1-Rmc are currently unknown. Whether this receptor may stimulate PLC and/or MAPK or provoke calcium mobilization remains to be investigated. It should be mentioned that the modification of the C terminus in this receptor causes the loss of a putative protein kinase C phosphorylation site and the appearance of a potential casein kinase II phosphorylation site. This molecular switch may have important functional consequences, because the phosphorylation status of the C-terminal domain plays a crucial role in the activation, desensitization, and internalization of G protein-coupled receptors (39).
The expression of PAC1-R and its variants in the frog R. ridibunda was studied in the brain as well as in peripheral tissues by Northern blot, RT-PCR, and in situ hybridization. The PAC1-R mRNA and the different isoforms were predominantly expressed in the central nervous system. Moderate to low levels of PAC1-R mRNA were observed in the distal lobe of the pituitary, the spleen, the testis, and the lung as revealed by RT-PCR. The other tissues examined were virtually devoid of PAC1-R mRNA. This expression pattern substantially differs from that of the frog VPAC receptor that we have characterized previously and which exhibits a widespread expression in brain and peripheral tissues (20). The distribution of frog PAC1-R mRNA is reminiscent of that of the frog PACAP precursor mRNA, which is also primarily expressed in the central nervous system (19), strongly suggesting that PACAP plays important regulatory functions in the brain and spinal cord in amphibians.
In situ hybridization analysis showed that in the brain of the frog R. ridibunda, intense expression of PAC1-R mRNA occurs in the olfactory bulb, the anterior preoptic area, the suprachiasmatic nucleus, the nucleus of the periventricular organ of the hypothalamus, and the optic tectum. These observations suggest that in the frog, PACAP may be involved in the regulation of the olfactory system, the neuroendocrine control of the hypothalamic-pituitary complex, and the processing of visual information. A remarkable feature regarding the distribution of PAC1-R mRNA in the frog brain is the high expression in the ependymal cells bordering the ventricles. This is noticeable because the frog VPAC receptor is not expressed in these cells (20), nor is PACAP precursor mRNA (19). The expression of PAC1-R mRNA in the ependymal cell layers suggests that PACAP acting on these receptors may originate from the cerebrospinal fluid and thus may participate in volume transmission (40).
Besides their occurrence in the central nervous system, PAC1-R and its variants are also expressed in peripheral tissues. Both PAC1-R and PAC1-R25 mRNA occur in the frog testis, spleen, and heart. In contrast, PAC1-Rmc mRNA was observed in the testis, but not in the spleen, whereas PAC1-R41 expression could not be detected in any of the peripheral tissues examined. The predominant expression of the PAC1-R variants in the central nervous system or in certain peripheral tissues suggests that they may allow PACAP to exert specific effects in particular cells that possess these isoforms. It has previously been shown that a particular variant of PAC1-R that is expressed in ß-cells of the pancreas mediates the effect of PACAP on insulin release through calcium influx only (13). In seminiferous tubules, high expression of another variant has been observed, and this variant has been postulated to play a specific role during spermatogenesis (12). Finally, it has been shown that PAC1-R isoform expression may determine the opposing mitogenic effects of PACAP observed in different neuroblasts (41).
In conclusion, we have characterized a type I PACAP receptor and three splice variants in the frog R. ridibunda, and we have shown that one of these receptor isoforms fails to stimulate adenylate cyclase upon PACAP treatment. In addition, the PAC1-R variants displayed differential relative abundance in various frog tissues. The novel variants described herein extend the family of PAC1-R isoforms, making this receptor one of the most alternatively spliced receptors among G protein-coupled receptors. The characterization of these variants will contribute to elucidation of the structure-function relationships of PAC1-R. The fact that several variants of this receptor exist in different vertebrate taxa indicates that these isoforms are involved in the physiological effects of PACAP. We have also shown that PAC1-R and its variants are intensely expressed in the frog central nervous system. Along with previous studies showing that high concentrations of PACAP mRNA and PACAP-immunoreactive elements are found in the brain of adult frogs and tadpoles (19, 42, 43), these data strongly suggest that PACAP plays a major role in the activity and development of the central nervous system of amphibians.
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Acknowledgments |
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Footnotes |
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H. V. is an Affiliated Professor at the Institut National de la Recherche Scientifique-Institut Armand Frappier (Montréal, Canada).
L. G. is supported by a doctoral fellowship from the Conseil Régional de la Vallée d’Aoste, Italy.
Abbreviations: AC, Adenylate cyclase; GFP, green fluorescent protein; PACAP, pituitary adenylate cyclase-activating polypeptide; PLC, phospholipase C; poly(A+), polyadenlated; SSC, standard saline citrate; TMD, transmembrane domain; VIP, vasoactive intestinal polypeptide.
Received July 2, 2001.
Accepted for publication March 1, 2002.
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 L. Grumolato, A. G. Elkahloun, H. Ghzili, D. Alexandre, C. Coulouarn, L. Yon, J.-P. Salier, L. E. Eiden, A. Fournier, H. Vaudry, et al. Microarray and Suppression Subtractive Hybridization Analyses of Gene Expression in Pheochromocytoma Cells Reveal Pleiotropic Effects of Pituitary Adenylate Cyclase-Activating Polypeptide on Cell Proliferation, Survival, and Adhesion Endocrinology, June 1, 2003; 144(6): 2368 - 2379. [Abstract] [Full Text] [PDF]
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), conserved cysteine residues (
), and the short amino acid insertion present only in the N-terminal domain of the PAC1-R (+). Putative transmembrane regions are labeled TMD I–VII. The arrow shows the putative cleavage site of the signal peptide. Amino acid alignment was performed using DNASIS version 2.1 software. GenBank accession no. 
) or frog VIP (
), and cAMP contained in the cell extracts was measured by RIA. Data are from one representative experiment and are expressed as the mean ± SEM of three determinations (PACAP) or two determinations (VIP). Three independent experiments were conducted, and similar results were obtained.
), PAC1-R25 (
), and PAC1-Rmc (
