Abstract: The human VPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) belongs to the class II family of G protein coupled receptors with seven transmembrane segments. It recognizes several VIP-related peptides and displays a very low affinity for secretin despite >70% homology between VIP and secretin. Conversely, the human secretin receptor has high affinity for secretin but low affinity for VIP. We took advantage of this reversed selectivity to identify a domain of the VPAC1 receptor responsible for selectivity toward secretin by constructing human VPAC1-secretin receptor chimeras. A first set of chimeras consisted of exchanging the entire N-terminal ectodomain or large parts of this domain. They were constructed by overlap PCR, transfected in COS-7 cells, and their ligand selectivity, expressed as the ratio of EC50 for secretin/EC50 for VIP (referred to as S/V), in stimulating cAMP production was measured. Two very informative chimeras respectively referred to as S144V and S123V were obtained by replacing the entire ectodomain or only the first 123 amino acids of the VPAC1 receptor by the corresponding sequences of the secretin receptor. Whereas S144V no longer discriminated between VIP and secretin (S/V = 1.2), S123V discriminated between the two peptides (S/V = 300) in the same manner as the wild-type VPAC1 receptor. The motif responsible for discrimination was determined by introducing small blocks or individual amino acids of secretin receptor in the 123–144 sequence of the S123V chimera. The data obtained from 14 new chimeras sustained that two nonadjacent pairs of amino acids, Gln135 Thr136 and Gly140Ser141 in the C-terminal end of the N-terminal VPAC1 receptor ectodomain constitute a selective filter that strongly restricts access of secretin to the VPAC1 receptor. The human VPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) belongs to the class II family of G protein coupled receptors with seven transmembrane segments. It recognizes several VIP-related peptides and displays a very low affinity for secretin despite >70% homology between VIP and secretin. Conversely, the human secretin receptor has high affinity for secretin but low affinity for VIP. We took advantage of this reversed selectivity to identify a domain of the VPAC1 receptor responsible for selectivity toward secretin by constructing human VPAC1-secretin receptor chimeras. A first set of chimeras consisted of exchanging the entire N-terminal ectodomain or large parts of this domain. They were constructed by overlap PCR, transfected in COS-7 cells, and their ligand selectivity, expressed as the ratio of EC50 for secretin/EC50 for VIP (referred to as S/V), in stimulating cAMP production was measured. Two very informative chimeras respectively referred to as S144V and S123V were obtained by replacing the entire ectodomain or only the first 123 amino acids of the VPAC1 receptor by the corresponding sequences of the secretin receptor. Whereas S144V no longer discriminated between VIP and secretin (S/V = 1.2), S123V discriminated between the two peptides (S/V = 300) in the same manner as the wild-type VPAC1 receptor. The motif responsible for discrimination was determined by introducing small blocks or individual amino acids of secretin receptor in the 123–144 sequence of the S123V chimera. The data obtained from 14 new chimeras sustained that two nonadjacent pairs of amino acids, Gln135 Thr136 and Gly140Ser141 in the C-terminal end of the N-terminal VPAC1 receptor ectodomain constitute a selective filter that strongly restricts access of secretin to the VPAC1 receptor. vasoactive intestinal peptide pituitary adenylate cyclase-activating peptide the ratio of EC50 for secretin/EC50 for VIP peptide histidine isoleucineamide peptide histidine methionineamide parathyroid hormone growth hormone-releasing factor transmembrane domain The human VPAC1 receptor represents together with VPAC2 one of the two subtypes of vasoactive intestinal peptide (VIP)1 receptors (1Laburthe M. Couvineau A. Gaudin P. Rouyer-Fessard C. Maoret J.J. Nicole P. Ann. N. Y. Acad. Sci. 1996; 805: 94-111Crossref PubMed Scopus (123) Google Scholar, 2Ulrich C.D. Holtmann M. Miller L.J. Gastroenterology. 1998; 114: 382-397Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 3Harmar A.J. Arimura A. Gozes I. Journot L. Laburthe M. Pisegna J.R. Rawlings S.R. Robberecht P. Said S.I. Sreedharan S.P. Wank S.A. Washeck J.A. Pharmacol. Rev. 1998; 50: 265-270PubMed Google Scholar, 4Laburthe M. Couvineau A. Marie J.C. Recept. Channel. 2002; (in press)PubMed Google Scholar). The VPAC1 receptor (for official nomenclature see Ref. 3Harmar A.J. Arimura A. Gozes I. Journot L. Laburthe M. Pisegna J.R. Rawlings S.R. Robberecht P. Said S.I. Sreedharan S.P. Wank S.A. Washeck J.A. Pharmacol. Rev. 1998; 50: 265-270PubMed Google Scholar) mediates the actions of the two neuropeptides VIP (5Said S.I. Trends Endocrinol. Metab. 1991; 2: 107-112Abstract Full Text PDF PubMed Scopus (83) Google Scholar) and pituitary adenylate cyclase-activating polypeptide or PACAP (6Arimura A. Shioda S. Front. Neuroendocrinol. 1995; 16: 53-88Crossref PubMed Scopus (361) Google Scholar) in a large variety of tissues through coupling to G proteins and subsequent activation of adenylyl cyclase (1Laburthe M. Couvineau A. Gaudin P. Rouyer-Fessard C. Maoret J.J. Nicole P. Ann. N. Y. Acad. Sci. 1996; 805: 94-111Crossref PubMed Scopus (123) Google Scholar). It belongs to a recently emerged subfamily within the superfamily of G protein-coupled heptahelical receptors, referred to as class II or family B (1Laburthe M. Couvineau A. Gaudin P. Rouyer-Fessard C. Maoret J.J. Nicole P. Ann. N. Y. Acad. Sci. 1996; 805: 94-111Crossref PubMed Scopus (123) Google Scholar, 4Laburthe M. Couvineau A. Marie J.C. Recept. Channel. 2002; (in press)PubMed Google Scholar). This subfamily of peptide receptors comprises receptors for VIP-related peptides including secretin, PACAP, GRF, gastric inhibitory polypeptide, glucagon, glucagon-like peptides 1 or 2, and also receptors for peptides with no sequence homology with VIP, such as PTH, calcitonin, and corticotropin-releasing factor (CRF) (1Laburthe M. Couvineau A. Gaudin P. Rouyer-Fessard C. Maoret J.J. Nicole P. Ann. N. Y. Acad. Sci. 1996; 805: 94-111Crossref PubMed Scopus (123) Google Scholar). Class II receptors for peptides share several specific properties, the most important of which is the presence of a large N-terminal ectodomain that contains some highly conserved residues including a set of six cysteines that are necessary for ligand binding (1Laburthe M. Couvineau A. Gaudin P. Rouyer-Fessard C. Maoret J.J. Nicole P. Ann. N. Y. Acad. Sci. 1996; 805: 94-111Crossref PubMed Scopus (123) Google Scholar, 4Laburthe M. Couvineau A. Marie J.C. Recept. Channel. 2002; (in press)PubMed Google Scholar). A three-dimensional model of a large part of the N-terminal ectodomain of human VPAC1 has recently been developed, suggesting the existence of an electronegative binding groove with an outspanning tryptophan shell at one end (7Lins L. Couvineau A. Rouyer-Fessard C. Nicole P. Maoret J.J. Benhamed M. Brasseur R. Thomas A. Laburthe M. J. Biol. Chem. 2001; 276: 10153-10160Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The current view of VIP binding to VPAC1 implies the interaction of the peptide with this N-terminal ectodomain binding groove and a still poorly characterized juxtamembrane domain comprising at least the junction between the N-terminal ectodomain and transmembrane domain (TM) I, the first extracellular loop, and the upper part of TM2 (4Laburthe M. Couvineau A. Marie J.C. Recept. Channel. 2002; (in press)PubMed Google Scholar, 8Laburthe M. Couvineau A. Nicole P. Endo. Series. 2002; (in press)Google Scholar). The human VPAC1 receptor binds VIP and PACAP with high affinities but also recognizes some VIP-related peptides with lower affinities,i.e. VIP = PACAP-27 > PACAP-38 > helodermin > GRF = PHI > secretin (4Laburthe M. Couvineau A. Marie J.C. Recept. Channel. 2002; (in press)PubMed Google Scholar). Based on the observation that PHI has a much higher affinity for rat than for human VPAC1 receptors, chimeras between rat and human VPAC1 receptors have been constructed in order to identify the critical domain responsible for such selectivity. It has been shown that three nonadjacent amino acids at the junction of extracellular loop I and TM3 ensured low affinity of PHI for the human VPAC1 receptor (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). This supports that domains involved in high affinity VIP binding and domains involved in the selectivity toward low affinity VPAC1 agonists may be functionally and topologically different (4Laburthe M. Couvineau A. Marie J.C. Recept. Channel. 2002; (in press)PubMed Google Scholar, 9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In the present work, this subject was further pursued by investigating the molecular determinant(s) responsible for ensuring the very high selectivity of human VPAC1 receptor toward secretin, though VIP and secretin have 9 identical residues and 10 homologous residues out of 27. For this purpose we constructed a series of chimeras between human VPAC1 and secretin receptors based on the observation that the human VPAC1 receptor has a high affinity for VIP and very low affinity for secretin (10Couvineau A. Rouyer-Fessard C. Darmoul D. Maoret J.J. Carrero I. Ogier-Denis E. Laburthe M. Biochem. Biophys. Res. Commun. 1994; 200: 769-776Crossref PubMed Scopus (150) Google Scholar), whereas the reverse is true for the human secretin receptor (11Jiang S. Ulrich C. Biochem. Biophys. Res. Commun. 1995; 207: 883-890Crossref PubMed Scopus (44) Google Scholar). We provide evidence that a motif composed of two nonadjacent pairs of residues at the C-terminal end of the N-terminal ectodomain is involved in the ability of the human VPAC1 receptor to restrict access to secretin. Restriction enzymes and culture medium were obtained from Invitrogen (Cergy-Pontoise, France). DNA sequenase kit and radioactive reagents were from Amersham Biosciences (Les Ulis, France). Cloned Pfu DNA polymerase and Taq DNA polymerase were purchased from Stratagene (La Jolla, Ca, United States) and Promega (Charbonnière, France), respectively. The transformer site-directed mutagenesis kit was from CLONTECH (Palo Alto, Ca, United States). Synthetic peptides were from Neosystem (Strasbourg, France). 125I-VIP, 125I-secretin, and 125I-cAMP were prepared and purified in our laboratory as previously described (12Laburthe M. Rousset M. Rouyer-Fessard C. Couvineau A. Chantret I. Chevalier G. Zweibaum A. J. Biol. Chem. 1987; 262: 10180-10184Abstract Full Text PDF PubMed Google Scholar). Synthetic oligonucleotides were from Eurogentec (Seraing, Belgium). The human VPAC1 receptor cDNA was cloned in our laboratory (10Couvineau A. Rouyer-Fessard C. Darmoul D. Maoret J.J. Carrero I. Ogier-Denis E. Laburthe M. Biochem. Biophys. Res. Commun. 1994; 200: 769-776Crossref PubMed Scopus (150) Google Scholar). The human secretin receptor cDNA (11Jiang S. Ulrich C. Biochem. Biophys. Res. Commun. 1995; 207: 883-890Crossref PubMed Scopus (44) Google Scholar) was a gift from Dr. C. Ulrich (University of Cincinnati College of Medicine, OH). Other highly purified chemicals used were from Sigma (Saint-Quentin-Fallavier, France). The human secretin-VPAC1 chimeric receptors were designed to replace portions of one wild-type receptor cDNA with the corresponding portions of the other receptor according to the amino acid sequence alignment of the two receptors (10Couvineau A. Rouyer-Fessard C. Darmoul D. Maoret J.J. Carrero I. Ogier-Denis E. Laburthe M. Biochem. Biophys. Res. Commun. 1994; 200: 769-776Crossref PubMed Scopus (150) Google Scholar, 11Jiang S. Ulrich C. Biochem. Biophys. Res. Commun. 1995; 207: 883-890Crossref PubMed Scopus (44) Google Scholar). The strategies included overlap PCR and site-directed mutagenesis. The coding sequence of all the receptor constructs were modified at the 5′ and 3′ ends by adding the EcoRI site and were subcloned into the expression vector pcDNA I/Amp (Invitrogen, Groningen, Netherlands) with a marker dodecapeptide sequence (Tag) at the C terminus as described (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A first set of chimeric receptors were constructed using the two-step overlapping polymerase chain reaction as described (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), with oligonucleotides consisting of secretin and VPAC1 receptor cDNA sequences specifying the splicing junction. The composition of wild-type and chimeric receptors is shown schematically in Fig. 1 with the following nomenclature in which amino acids are numbered according to the VPAC1 and secretin receptor sequences (10Couvineau A. Rouyer-Fessard C. Darmoul D. Maoret J.J. Carrero I. Ogier-Denis E. Laburthe M. Biochem. Biophys. Res. Commun. 1994; 200: 769-776Crossref PubMed Scopus (150) Google Scholar, 11Jiang S. Ulrich C. Biochem. Biophys. Res. Commun. 1995; 207: 883-890Crossref PubMed Scopus (44) Google Scholar): VPAC1 receptor (1–457); Secretin receptor (1–440); S123V, secretin receptor (1–123), VPAC1 receptor (123–457); S144V, secretin receptor (1–144), VPAC1 receptor (145–457); V144S, VPAC1 receptor (1–144), secretin receptor (145–440). The polymerase chain reactions were performed withTaq polymerase or Pfu polymerase using a “hot start” procedure: 95 °C for 5 min, one cycle; 55 °C for 1 min, 72 °C for 3min, 94 °C for 30 s for 15 cycles (first step) or 10 cycles (second step); followed by one cycle at 72 °C for 5 min. PCR products were separated on 1% agarose gel and purified by Nucleotrap extraction kit (Macherey-Nagel, Ilkirch, France). Successful construction of chimeras was confirmed by sequencing. Additional chimeric receptors were the following: S127V, secretin receptor (1–127), VPAC1 receptor (128–457); S131V, secretin receptor (1–131), VPAC1 receptor (132–457); S134V, secretin receptor (1–134), VPAC1 receptor (135–457); S139V, secretin receptor (1–139), VPAC1 receptor (140–457); S140V, secretin receptor (1–140), VPAC1 receptor (141–457); S141V, secretin receptor (1–141), VPAC1 receptor (141–457); and S142V, secretin receptor (1–142), VPAC1 receptor (142–457). They were constructed by several steps of oligonucleotide site-directed mutagenesis of the S123V chimera (see above) as previously reported (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Couvineau A. Rouyer-Fessard C. Darmoul D. Maoret J.J. Carrero I. Ogier-Denis E. Laburthe M. Biochem. Biophys. Res. Commun. 1994; 200: 769-776Crossref PubMed Scopus (150) Google Scholar). The double-strand chimeric receptors' cDNA cloned in expression vector pcDNA1 was used as template. Synthetic oligonucleotides 5′-GGGAGACCCACGCGTGGTACCGAG-3′ or 5′-GGGAGACCCAAGCTTGGTACCGAG-3′ was used as selection primer, which switches a unique restriction site on the vector fromHindIII to MluI or from MluI toHindIII, respectively. The sequencing confirmed mutants were directly used for subsequent transfection into COS cells. Other mutants were obtained by site-directed mutagenesis exactly as reported (10Couvineau A. Rouyer-Fessard C. Darmoul D. Maoret J.J. Carrero I. Ogier-Denis E. Laburthe M. Biochem. Biophys. Res. Commun. 1994; 200: 769-776Crossref PubMed Scopus (150) Google Scholar). Receptor constructs were transfected into COS-7 cells by electroporation as previously described (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Couvineau A. Rouyer-Fessard C. Darmoul D. Maoret J.J. Carrero I. Ogier-Denis E. Laburthe M. Biochem. Biophys. Res. Commun. 1994; 200: 769-776Crossref PubMed Scopus (150) Google Scholar). Briefly, 4 × 106 cells were preincubated on ice for 5 min with 15 μg of vector construction in 0.5 ml of phosphate-buffered saline. After electroporation (330 V, 500 μF, infinite resistance), cells were put on ice for 5 min and then transferred into culture medium containing 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin-streptomycin before seeding in 12-well trays for cAMP assay, 10 mm Petri dishes for ligand binding assay, or on glass slides in 24-well trays for immunofluorescence studies. The cells were used 48 h after transfection. Transfected COS-7 cells were grown in 12-well trays as described above. The culture medium was discarded, and adherent cells were gently rinsed with phosphate-buffered saline. They were then incubated without or with peptides under continuous agitation in 0.5 ml of phosphate-buffered saline containing 2% (w/v) bovine serum albumin, 0.1% (w/v) bacitracin, 0.01 mg/ml aprotinin, and 1 mm3-isobutyl-1-methylxanthine as described (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Couvineau A. Rouyer-Fessard C. Darmoul D. Maoret J.J. Carrero I. Ogier-Denis E. Laburthe M. Biochem. Biophys. Res. Commun. 1994; 200: 769-776Crossref PubMed Scopus (150) Google Scholar). At the end of incubation (30 min at 25 °C) the medium was removed and cells were lysed with 1 m perchloric acid. The cAMP present in the lysate was measured by radioimmunoassay as described (12Laburthe M. Rousset M. Rouyer-Fessard C. Couvineau A. Chantret I. Chevalier G. Zweibaum A. J. Biol. Chem. 1987; 262: 10180-10184Abstract Full Text PDF PubMed Google Scholar). Cell number was determined in parallel wells, and data were calculated as picomoles of cAMP/106 cells and shown as percentages of maximum stimulation elicited by 10 μm VIP or secretin. Assays were performed in triplicate and repeated in at least three independent experiments. The concentration of peptides eliciting half-maximal stimulation of cAMP production (EC50) was determined by computer analysis using the Prism software suite (GraphPad Software, San Diego, CA), and the ratio EC50 for secretin/EC50 for VIP was calculated. Membrane homogenate (200 μg of protein/ml) prepared from transfected cells was incubated for 40 min at 30 °C in 20 mm HEPES buffer, pH 7.4, containing 2% (w/v) bovine serum albumin, 0.1% (w/v) bacitracin, 0.05 nm125I-VIP or 125I-secretin in the presence of increasing concentrations of unlabeled VIP or secretin. Bound and free peptides were separated as described (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Couvineau A. Rouyer-Fessard C. Darmoul D. Maoret J.J. Carrero I. Ogier-Denis E. Laburthe M. Biochem. Biophys. Res. Commun. 1994; 200: 769-776Crossref PubMed Scopus (150) Google Scholar), and the radioactivity was then measured in a γ-counter. Nonspecific binding was determined in the presence of excess of unlabeled VIP or secretin (10 μm). The specific binding was calculated as the difference between 125I-peptide totally bound and the nonspecific binding. The Ligand computer program (13Munson P.J. Rodbard D. Anal. Biochem. 1980; 197: 220-239Crossref Scopus (8073) Google Scholar) was used to analyze binding data and to determine the concentrations of VIP or secretin that elicited half-maximal inhibition of specific125I-peptide binding (IC50). Cellular expression of all chimeras was determined by indirect-immunofluorescence experiments of tagged receptor constructs after transfection of COS-7 as previously reported (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). All chimeras described in this study were expressed at the plasma membrane of transfected cells. As previously reported for native receptors (14Salomon R. Couvineau A. Rouyer-Fessard C. Voisin T. Lavallee D. Blais A. Darmoul D. Laburthe M. Am. J. Physiol. 1993; 264: E294-E300PubMed Google Scholar, 15Robberecht P., De Neef P. Waelbroeck M. Camus J.C. Scemama J.L. Fourmy D. Pradayrol L. Vaysse N. Christophe J. Pancreas. 1988; 3: 529-535Crossref PubMed Scopus (35) Google Scholar), the recombinant human VPAC1 receptor displays high affinity for VIP and low affinity for secretin, whereas the recombinant human secretin receptor has a reversed selectivity with high affinity for secretin and low affinity for VIP (Fig. 1). This was clear from binding experiments using 125I-VIP or125I-secretin as tracers and also from experiments in which cAMP production upon VIP or secretin challenges was measured (Fig. 1). To further characterize the phenotype of receptors, we measured the EC50 of VIP and secretin in stimulating cAMP production and calculated the ratio EC50 for secretin/EC50 for VIP, which is referred to as S/V (TableI). The ratios for VPAC1 receptor and secretin receptor were 500 and 7.1 × 10−4, respectively. A first set of receptor chimeras consisted of exchanging the entire N-terminal extracellular domain. The secretin receptor having the N-terminal domain of the VPAC1 receptor (V144S construct) construct was still able to discriminate between secretin and VIP [S/V ratio = 34], though to a lower extent than the wild-type secretin receptor (Fig. 1 and Table I). In contrast, the VPAC1 receptor having the N-terminal domain of the secretin receptor (S144V construct) no longer discriminated between VIP and secretin (Fig. 1) with a S/V ratio of 1.2 (Table I). We therefore decided to determine which motif in the N-terminal domain of the VPAC1 receptor ensures the discrimination of secretin versus VIP. In this context a very informative chimera was obtained by replacing only the first 123 amino acids of the VPAC1 receptor by the corresponding sequence of the secretin receptor. Indeed, this chimera (S123V) discriminated very well between VIP and secretin (Fig. 1) with a S/V ratio of 300, very close to that of the wild-type VPAC1 receptor (Table I). This data suggested that the relevant motif is present in the 123–144 sequence that connects the N-terminal ectodomain to the first transmembrane domain of the VPAC1 receptor. This sequence was further explored by introducing small blocks of secretin receptor amino acids in the 123–144 sequence of the S123V chimera (Fig. 2). This resulted in the construction of seven new chimeras between S123V and S144V (Fig.2). It appeared that three sets of chimeric receptors could be identified. One set consisted of receptor chimeras, S123V to S139V, having S/V ratios >100, which were very similar to the S/V ratio of the S123V chimera (Table II) or to the wild-type VPAC1 receptor (Table I). A second set consisted of the S140V chimeric receptor having a S/V ratio of 18. Finally, a third set consisted of the receptor chimeras S141V and S142V having a S/V ratio of 1.6 and 2.8, respectively, i.e. very close to the S/V ratio of the S144V chimera (Table II). These data supported the idea that a structural determinant responsible for impeding a high affinity of secretin consisted of the Gly140Ser141 pair of amino acids in the VPAC1 receptor, which are replaced by LK in the secretin receptor (Fig. 2).Table IParameters of VIP- or secretin-stimulated cAMP production for wild-type VPAC1, secretin, or chimeric receptors S144V, V144S, and S123V after transfection of cDNAs into COS cellsEC50VIP(nM)1-aEC50 was determined as described under Experimental Procedures section.EC50Sec(nM)1-aEC50 was determined as described under Experimental Procedures section.S/V1-bS/V was the ratio of EC50 for secretin/EC50for VIP.VPAC10.80 ± 0.06400 ± 250500SecretinR121 ± 450.28 ± 0.130.02S144V5.69 ± 1.096.73 ± 1.091.18V144S12 ± 5400 ± 22034S123V3.02 ± 0.80900 ± 473300Results are means ± S.E. of three experiments.1-a EC50 was determined as described under Experimental Procedures section.1-b S/V was the ratio of EC50 for secretin/EC50for VIP. Open table in a new tab Table IIParameters of VIP- and secretin-stimulated cAMP production for chimeric receptors after transfection of cDNAs into COS cellsConstruction2-aSee Fig. 2 for description of constructions.EC50VIP(nM)2-bEC50 was determined as described under Experimental Procedures section.EC50Sec(nM)2-bEC50 was determined as described under Experimental Procedures section.S/V2-cS/V was the ratio of EC50 for secretin/ EC50for VIP.S123V3.0 ± 0.8900 ± 473300S127V5.7 ± 0.21000 ± 290175S131V5.5 ± 0.12000 ± 569363S134V5.6 ± 0.21025 ± 282182S139V10.3 ± 1.41366 ± 109132S140V7.2 ± 2.1128 ± 1018S141V9.4 ± 3.514.9 ± 3.81.6S142V2.8 ± 0.47.8 ± 2.12.8S144V5.7 ± 1.16.7 ± 1.81.2Results are means ± S.E. of three experiments.2-a See Fig. 2 for description of constructions.2-b EC50 was determined as described under Experimental Procedures section.2-c S/V was the ratio of EC50 for secretin/ EC50for VIP. Open table in a new tab Results are means ± S.E. of three experiments. Results are means ± S.E. of three experiments. Further experiments were conducted in order to determine whether or not the LK pair of amino acids is sufficient to ensure high affinity of secretin to the VPAC1 receptor. The S123V chimera in which the Gly140 Ser141 pair of amino acids was replaced with LK (S123V-LK construct) has a S/V ratio of 23.4 (Fig.3), suggesting that an additional motif is present in the 123–144 sequence. The S134V chimera in which the Gly140 Ser141 pair of amino acids was replaced with LK (S134V-LK construct) has a S/V ratio <10 (Fig. 3), suggesting that such an additional site may lie within the 134–139 sequence. To further explore this sequence, the phenotype of two other constructs was determined, e.g. S123V-SYLLK and S123V-KRHSYLLK (Fig.3). The S/V ratios were 18.4 and 5.2, respectively. Altogether, these data supported that the Gln135 Thr136 pair associated with the Gly140 Ser141 pair in the VPAC1 receptor sequence is important for impeding a high affinity of secretin. This was clearly confirmed by the fact that the S123V-RH-LK construct had a S/V ratio of 1.7 (Fig. 3). Two further constructs S123V-R-LK and S123V-H-LK had S/V ratios >10 (Fig. 3), supporting the idea that two nonadjacent pairs of amino acids Gln135Thr136 and Gly140 Ser141 impeded the binding of secretin with high affinity to the S123V chimeric receptor. Their respective substitution by the corresponding RH and LK pairs of amino acids of the secretin receptor shifted the S/V ratio from 300 to 1.7 (Fig. 3). To further validate our data, all receptor constructs were analyzed by radioligand binding studies, providing evidence for the following: (i) a similar density of all receptors in transfected COS cell membranes in the range between 400 and 570 fmol/mg protein; (ii) a good correlation between EC50 values for VIP or secretin for stimulating cAMP production and their K d or IC50values in binding studies (Fig. 4). In this paper we demonstrate that a motif consisting of two nonadjacent pairs of amino acids Gln135 Thr136and Gly140 Ser141 in the C-terminal end of the N-terminal ectodomain of the human VPAC1 receptor is involved in the high selectivity of the receptor toward secretin. This motif constitutes a selectivity filter that enables the human VPAC1 receptor to restrict access of the inappropriate ligand secretin even though secretin exhibits >70% sequence homology with VIP. This is a highly relevant task for members of the class II family of G protein-coupled receptors for VIP-related peptides, because these peptides exhibit considerable sequence homologies (16Laburthe M. Kitabgi P. Couvineau A. Amiranoff B. Handb. Exp. Pharmacol. 1993; 106: 133-176Crossref Google Scholar). In a previous report (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), we showed that the human VPAC1 receptor is equipped with another selectivity filter toward the VIP-related peptide PHI (see Fig.5), which exhibits 90% homology with VIP. A few studies have reported the construction of chimeras between VIP receptor and secretin receptors from rats (17Holtmann M.H. Ganguli S. Hadac E.M. Dolu V. Miller L.J. J. Biol. Chem. 1996; 271: 14944-14949Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 18Holtmann M.H. Hadac E.M. Miller L.J. J. Biol. Chem. 1995; 270: 14394-14398Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 19Vilardaga J.P., De Neef P., Di Paolo E. Bollen A. Waelbroeck M. Robberecht P. Biochem. Biophys. Res. Commun. 1995; 211: 885-891Crossref PubMed Scopus (77) Google Scholar, 20Park C.G. Ganguli S.C. Pinon D.I. Hadac E.M. Miller L.J. J. Pharmacol. Exp. Ther. 2000; 295: 682-688PubMed Google Scholar). The rat secretin-VIP chimeric receptor approach indicated that 10 residues at the N terminus of the secretin receptor together with two residues in the first extracellular loop and four residues in the second extracellular loop are critical determinants for secretin binding (17Holtmann M.H. Ganguli S. Hadac E.M. Dolu V. Miller L.J. J. Biol. Chem. 1996; 271: 14944-14949Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Conversely, chimeras between rat VIP and secretin receptors provided evidence indicating that the N-terminal extracellular domain of the rat VPAC1 receptor plays a key role in agonist recognition (18Holtmann M.H. Hadac E.M. Miller L.J. J. Biol. Chem. 1995; 270: 14394-14398Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and potent cyclic AMP response to VIP (20Park C.G. Ganguli S.C. Pinon D.I. Hadac E.M. Miller L.J. J. Pharmacol. Exp. Ther. 2000; 295: 682-688PubMed Google Scholar), this domain being the key element for discrimination between VIP and secretin (19Vilardaga J.P., De Neef P., Di Paolo E. Bollen A. Waelbroeck M. Robberecht P. Biochem. Biophys. Res. Commun. 1995; 211: 885-891Crossref PubMed Scopus (77) Google Scholar). Our data with human receptors are consistent with the idea that the N-terminal ectodomain of the VPAC1 receptor is critical for discrimination between VIP and secretin. Thanks to site-directed mutagenesis and the construction of a large series of chimeric receptors within the N-terminal ectodomain (see under “Results”), a clue to the relevant motif could be found within this large 144-residue domain. The selective filter for restricting access of secretin to the human VPAC1 receptor is present at the C-terminal end of the N-terminal ectodomain, which connects this domain to the first TM (see Fig. 5), and consists in two nonadjacent amino acid pairs, e.g. Gln135Thr136 and Gly140 Ser141. Both the juxtamembrane location of this filter and its amino acid composition made of nonadjacent residues within a small sequence are reminiscent of the properties of a selectivity filter toward PHI, a low affinity VIP agonist previously characterized in the human VPAC1 receptor (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Indeed, based on the observation that PHI has a much higher affinity for rat than for human VPAC1 receptors (21Turner P.R. Bambino T. Nissenson R.A. J. Biol. Chem. 1996; 271: 9205-9208Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), the construction of chimeras between rat and human VPAC1 receptors showed that the critical domain for PHI recognition is present within a sequence comprising part of the first extracellular loop and third TM (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Site-directed mutagenesis of this sequence further indicated that three nonadjacent amino acids are responsible for restricting access of PHI to the human VPAC1 receptor, e.g. Gln207, Gly211, and Met219 (see Fig. 5). Although they share some properties, the selectivity filters for secretin and PHI are nonetheless quite distinct motifs. Whether the view of distinct selectivity filters, which more or less restrict the access of natural VIP agonists to the human VPAC1 receptor, can be extended to other VIP-related peptides remains to be determined. This is important because discrimination between highly homologous natural peptides is clearly a biologically relevant issue as the human VPAC1 receptor binds several natural peptides with the following order of affinity: VIP = PACAP-27 > PACAP-38 > helodermin > GRF = PHM > secretin (see Ref. 4Laburthe M. Couvineau A. Marie J.C. Recept. Channel. 2002; (in press)PubMed Google Scholar for a review). On the other hand, it is worth pointing out that the molecular motifs ensuring the function of selectivity filters are not responsible for the high affinity binding of the cognate peptide ligands. For instance, the S123V chimera and the S123V-RH-LK construct have similar affinity for VIP, whereas they display dramatic differences in their affinity for secretin (Fig. 3). Similarly, the native human VPAC1 receptor and the triple mutant (in which Gln207, Gly211, and Met219 have been respectively substituted for His, Ala, and Val) have identical affinity for VIP, whereas they display important differences in their affinity for PHI (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). These observations are in good agreement with the current view of VIP binding to the human VPAC1 receptor, which implies the interaction of the peptide with two receptor domains: the N-terminal ectodomain where a putative electronegative binding groove with an outspanning tryptophan shell at one end has been identified (7Lins L. Couvineau A. Rouyer-Fessard C. Nicole P. Maoret J.J. Benhamed M. Brasseur R. Thomas A. Laburthe M. J. Biol. Chem. 2001; 276: 10153-10160Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and a still poorly characterized binding domain on the core of the receptor that may consist of at least the first and second extracellular loops and the upper part of the second transmembrane helix as viewed from the outside of the cell (see Ref. 4Laburthe M. Couvineau A. Marie J.C. Recept. Channel. 2002; (in press)PubMed Google Scholar for a review). In conclusion, this work characterizes the molecular motif ensuring the function of a selectivity filter toward secretin in the human VPAC1 receptor. It further substantiates the notion of selectivity filters in human VPAC1 receptor that restrict access of inappropriate ligands, a notion that was previously documented for discrimination between VIP and its related peptide PHI (9Couvineau A. Rouyer-Fessard C. Maoret J.J. Gaudin P. Nicole P. Laburthe M. J. Biol. Chem. 1996; 271: 12795-12800Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). This concept of selective filters also received experimental evidence in studies of PTH and secretin receptors (21Turner P.R. Bambino T. Nissenson R.A. J. Biol. Chem. 1996; 271: 9205-9208Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), two other class II G protein-coupled receptors (1Laburthe M. Couvineau A. Gaudin P. Rouyer-Fessard C. Maoret J.J. Nicole P. Ann. N. Y. Acad. Sci. 1996; 805: 94-111Crossref PubMed Scopus (123) Google Scholar). Indeed, PTH and secretin display no sequence homology with one another, and neither ligand cross-reacts with the other's receptor. However, mutation of a single amino acid in the second transmembrane helix of the secretin receptor to the corresponding residue in the PTH receptor results in a receptor that binds and signals in response to PTH (21Turner P.R. Bambino T. Nissenson R.A. J. Biol. Chem. 1996; 271: 9205-9208Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Quite interestingly, the reciprocal mutation in the PTH receptor likewise unmasks responsiveness to secretin (21Turner P.R. Bambino T. Nissenson R.A. J. Biol. Chem. 1996; 271: 9205-9208Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To understand the physicochemical basis whereby selectivity filters in class II G protein-coupled receptors restrict access to inappropriate ligands will be of great interest for further development in the design of selective ligands of the members of this class of receptors. We thank Dr. C. Ulrich for providing the human secretin cDNA and Drs. Jean-Jacques Lacapère and Jean-Claude Marie for the critical reading of the manuscript.