Arginine 336 and Asparagine 333 of the Human Cholecystokinin-A Receptor Binding Site Interact with the Penultimate Aspartic Acid and the C-terminal Amide of Cholecystokinin
1999; Elsevier BV; Volume: 274; Issue: 29 Linguagem: Inglês
10.1074/jbc.274.29.20457
ISSN1083-351X
AutoresVéronique Gigoux, Chantal Escrieut, Jean‐Alain Fehrentz, Sandrine Silvente‐Poirot, Bernard Maigret, Luis Moroder, Danielle Gully, Jean Martínez, Nicole Vaysse, Daniel Fourmy,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe cholecystokinin-A receptor (CCK-AR) is a G protein-coupled receptor that mediates important central and peripheral cholecystokinin actions. Residues of the CCK-AR binding site that interact with the C-terminal part of CCK that is endowed with biological activity are still unknown. Here we report on the identification of Arg-336 and Asn-333 of CCK-AR, which interact with the Asp-8 carboxylate and the C-terminal amide of CCK-9, respectively. Identification of the two amino acids was achieved by dynamics-based docking of CCK in a refined three-dimensional model of CCK-AR using, as constraints, previous results that demonstrated that Trp-39/Gln-40 and Met-195/Arg-197 interact with the N terminus and the sulfated tyrosine of CCK, respectively. Arg-336-Asp-8 and Asn-333-amide interactions were pharmacologically assessed by mutational exchange of Arg-336 and Asn-333 in the receptor or reciprocal elimination of the partner chemical functions in CCK. This study also allowed us to demonstrate that (i) the identified interactions are crucial for stabilizing the high affinity phospholipase C-coupled state of the CCK-AR·CCK complex, (ii) Arg-336 and Asn-333 are directly involved in interactions with nonpeptide antagonists SR-27,897 and L-364,718, and (iii) Arg-336 but not Asn-333 is directly involved in the binding of the peptide antagonist JMV 179 and the peptide partial agonist JMV 180. These data will be used to obtain an integrated dynamic view of the molecular processes that link agonist binding to receptor activation. The cholecystokinin-A receptor (CCK-AR) is a G protein-coupled receptor that mediates important central and peripheral cholecystokinin actions. Residues of the CCK-AR binding site that interact with the C-terminal part of CCK that is endowed with biological activity are still unknown. Here we report on the identification of Arg-336 and Asn-333 of CCK-AR, which interact with the Asp-8 carboxylate and the C-terminal amide of CCK-9, respectively. Identification of the two amino acids was achieved by dynamics-based docking of CCK in a refined three-dimensional model of CCK-AR using, as constraints, previous results that demonstrated that Trp-39/Gln-40 and Met-195/Arg-197 interact with the N terminus and the sulfated tyrosine of CCK, respectively. Arg-336-Asp-8 and Asn-333-amide interactions were pharmacologically assessed by mutational exchange of Arg-336 and Asn-333 in the receptor or reciprocal elimination of the partner chemical functions in CCK. This study also allowed us to demonstrate that (i) the identified interactions are crucial for stabilizing the high affinity phospholipase C-coupled state of the CCK-AR·CCK complex, (ii) Arg-336 and Asn-333 are directly involved in interactions with nonpeptide antagonists SR-27,897 and L-364,718, and (iii) Arg-336 but not Asn-333 is directly involved in the binding of the peptide antagonist JMV 179 and the peptide partial agonist JMV 180. These data will be used to obtain an integrated dynamic view of the molecular processes that link agonist binding to receptor activation. The cholecystokinin-A receptor (CCK-AR) 1The abbreviations CCK-ARcholecystokinin receptor-AWTwild-type 1The abbreviations CCK-ARcholecystokinin receptor-AWTwild-typeis a member of the superfamily of G protein-coupled receptors and transduces CCK signals into target cells (1de Weerth A. Pisegna J.R. Huppi K. Wank S.A. Biochem. Biophys. Res. Commun. 1993; 194: 811-818Crossref PubMed Scopus (140) Google Scholar, 2Ulrich C.D. Ferber I. Holicky E. Hadac E. Buell G. Miller L.J. Biochem. Biophys. Res. Commun. 1993; 193: 204-211Crossref PubMed Scopus (91) Google Scholar). CCK-AR has important physiological functions in the central nervous system (stimulation of satiety) and in peripheral organs, particularly in the gut (stimulation of gall bladder contraction, pancreatic secretions, and digestive motility) (3Silvente-Poirot S. Dufresne M. Vaysse N. Fourmy D. Eur. J. Biochem. 1993; 215: 513-529Crossref PubMed Scopus (127) Google Scholar). CCK-AR, like other G protein-coupled receptors, may be involved in the development of certain pathologies in humans and as such is an important target for therapeutic intervention involving the pharmacological blockade or activation of the receptor using appropriate ligands (4Wank S.A. Am. J. Physiol. 1998; 274: G607-G613PubMed Google Scholar, 5Inoue H. Iannotti C.A. Welling C.M. Veile R. Donis-Keller H. Permutt M.A. Genomics. 1997; 42: 331-335Crossref PubMed Scopus (45) Google Scholar).The natural ligand of CCK-AR, CCK, is composed of several molecular variants, the octapeptide (CCK-8) being the major fully active one (6Rehfeld J.F. van Solinge W.W. Adv. Cancer Res. 1994; 63: 295-347Crossref PubMed Google Scholar). Posttranslational processing of CCK involves sulfation of the tyrosine at position seven from the C-terminal and α-amidation of the C-terminal phenylalanine residue (7Beinfeld M.C. Life Sci. 1997; 61: 2359-2366Crossref PubMed Scopus (27) Google Scholar). Studies using synthetic fragments have shown that this sulfation and the amidation of the octapeptide are required for full biological activity and that the C-terminal tetrapeptide Trp-Met-Asp-Phe-NH2 corresponds to the minimal fragment endowed with biological activity (8Jensen R.T. Lemp G.F. Gardner J.D. J. Biol. Chem. 1982; 257: 5554-5559Abstract Full Text PDF PubMed Google Scholar). Elimination of the carboxy-amidated phenylalanine in CCK switches the activity of the molecule from a full agonist on pancreatic amylase secretion to a partial agonist or an antagonist according to the animal species used (9Howard J.M. Knight M. Jensen R.T. Gardner J.D. Am. J. Physiol. 1984; 247: G261-G264PubMed Google Scholar, 10Gardner J.D. Knight M. Sutliff V.E. Tamminga C.A. Jensen R.T. Am. J. Physiol. 1984; 246: G292-G295PubMed Google Scholar). Some antagonistic molecules were synthetized on the basis of the importance of the carboxy-amidated phenylalanine for full biological activity. Two such molecules, JMV 180 and JMV 179, have been used extensively for the characterization of CCK-AR (11Galas M.C. Lignon M.F. Rodriguez M. Mendre C. Fulcrand P. Laur J. Martinez J. Am. J. Physiol. 1988; 254: G176-G182PubMed Google Scholar, 12Lignon M.F. Galas M.C. Rodriguez M. Laur J. Aumelas A. Martinez J. J. Biol. Chem. 1987; 262: 7226-7231Abstract Full Text PDF PubMed Google Scholar, 13Silvente Poirot S. Hadjiivanova C. Escrieut C. Dufresne M. Martinez J. Vaysse N. Fourmy D. Eur. J. Biochem. 1993; 212: 529-538Crossref PubMed Scopus (18) Google Scholar, 14Poirot S.S. Escrieut C. Dufresne M. Martinez J. Bouisson M. Vaysse N. Fourmy D. Mol. Pharmacol. 1994; 45: 599-607PubMed Google Scholar, 15Williams J.A. Blevins Jr., G.T. Physiol. Rev. 1993; 73: 701-723Crossref PubMed Scopus (95) Google Scholar). Evidence exists to show that JMV 180 induces distinct pharmacological effects and signal transduction pathways from those of the natural agonist CCK through binding to CCK-AR (15Williams J.A. Blevins Jr., G.T. Physiol. Rev. 1993; 73: 701-723Crossref PubMed Scopus (95) Google Scholar). These studies suggest that interactions between the C-terminal amidated phenylalanine of CCK and so far unknown amino acid(s) within the CCK-AR agonist binding site are important for full activation of the receptor. The tryptophan and aspartic acid within the C-terminal tetrapeptide of CCK are also key amino acids (16Rolland M. Rodriguez M. Lignon M.F. Galas M.C. Laur J. Aumelas A. Martinez J. Int. J. Pept. Protein Res. 1991; 38: 181-192Crossref PubMed Scopus (10) Google Scholar,17Pan G.Z. Martinez J. Bodanszky M. Jensen R.T. Gardner J.D. Biochim. Biophys. Acta. 1981; 678: 352-357Crossref PubMed Scopus (20) Google Scholar). The structure of several classes of CCK-AR antagonists and agonists contain structural elements that resemble tryptophan and aspartic acid side-chains, suggesting that all ligands may share determinants of the CCK-AR binding site (3Silvente-Poirot S. Dufresne M. Vaysse N. Fourmy D. Eur. J. Biochem. 1993; 215: 513-529Crossref PubMed Scopus (127) Google Scholar, 18Freidinger R.M. Med. Res. Rev. 1989; 9: 271-290Crossref PubMed Scopus (69) Google Scholar).Most of the available data regarding the topography of the agonist and antagonist binding sites of G protein-coupled receptors were derived from studies using site-directed mutagenesis. Data from these studies led to the conclusion that G protein-coupled receptors for neuropeptides have determinants of their agonist binding sites located both in extracellular regions and within the transmembrane domains (19Fong T.M. Strader C.D. Med. Res. Rev. 1994; 14: 387-399Crossref PubMed Scopus (34) Google Scholar, 20Schwartz T.W. Rosenkilde M.M. Trends Pharmacol. Sci. 1996; 17: 213-216Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 21Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). For CCK-AR, several lines of evidence support the view that its binding site for CCK comprises amino acids located in extracellular domains. Indeed, the removal of the first 43 amino acids of CCK-AR, and more recently, the mutation of two residues located at the top of the first transmembrane segment, (i.e. Trp-39 and Gln-40) were found to decrease the affinity of the CCK agonist but not that of nonpeptide antagonists (22Kennedy K. Escrieut C. Dufresne M. Clerc P. Vaysse N. Fourmy D. Biochem. Biophys. Res. Commun. 1995; 213: 845-852Crossref PubMed Scopus (37) Google Scholar, 23Kennedy K. Gigoux V. Escrieut C. Maigret B. Martinez J. Moroder L. Frehel D. Gully D. Vaysse N. Fourmy D. J. Biol. Chem. 1997; 272: 2920-2926Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In latter study, we showed that receptors mutated at positions 39 and 40 exhibited decreased affinities for CCK octa- and nonapeptide but did bind CCK heptapeptide with the same affinity as the wild-type receptor. These data were interpreted as providing a strong indication that Trp-39 and Gln-40 are part of the CCK binding site by interacting directly the N-terminal portion of CCK. Using these data and molecular dynamics-based ligand docking, two additional residues, Met-195 and Arg-197, located in the second extracellular loop, were recently identified as part of the agonist binding site because they interact with the sulfated tyrosine of CCK, which is crucial for binding and activity of CCK (24Gigoux V. Escrieut C. Silvente-Poirot S. Maigret B. Gouilleux L. Fehrentz J.A. Gully D. Moroder L. Vaysse N. Fourmy D. J. Biol. Chem. 1998; 273: 14380-14386Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 25Gigoux V. Escrieut C. Poirot S. Bouisson M. Maigret B. Fehrentz J. Martinez J. Moroder L. Vaysse N. Fourmy D. Dig. Dis. Sci. 1998; 43: 1884Google Scholar).Identification of amino acids of CCK-AR that interact with the C-terminal moiety of CCK represents one of the prerequisites for the understanding of how CCK signal is converted into receptor activation. Here, we present important new data that identify residues Arg-336 and Asn-333 as part of the CCK-AR agonist binding site. These amino acids, which are located at the top of transmembrane segment VI, are interacting with the aspartic acid carboxylate and the C-terminal amide of CCK, respectively, which are crucial for binding and activity. In addition, these amino acids are involved differentially in the binding site of peptide and nonpeptide antagonists of CCK-AR, supporting the existence of binding sites for individual agonists/antagonists, which may or may not overlap.DISCUSSIONIn the current study, we have identified two new residues, Arg-336 and Asn-333 of the human CCK-AR, that are crucial for binding and biological activity of CCK. We have demonstrated that these two amino acids interact with the Asp-8 side-chain and C-terminal amide of CCK, respectively, and that they play a crucial role in both the binding and functional properties of CCK-AR. In addition we have shown that, unlike previously identified amino acids (Trp-39/Gln-40 and Met-195/Arg-197) (23Kennedy K. Gigoux V. Escrieut C. Maigret B. Martinez J. Moroder L. Frehel D. Gully D. Vaysse N. Fourmy D. J. Biol. Chem. 1997; 272: 2920-2926Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 24Gigoux V. Escrieut C. Silvente-Poirot S. Maigret B. Gouilleux L. Fehrentz J.A. Gully D. Moroder L. Vaysse N. Fourmy D. J. Biol. Chem. 1998; 273: 14380-14386Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 25Gigoux V. Escrieut C. Poirot S. Bouisson M. Maigret B. Fehrentz J. Martinez J. Moroder L. Vaysse N. Fourmy D. Dig. Dis. Sci. 1998; 43: 1884Google Scholar), the two newly identified residues are involved differentially in the binding site of peptide and nonpeptide antagonists of CCK-AR.The identification of Arg-336 and Asn-333 was reached after optimization of CCK docking into the three-dimensional model of CCK-AR. For this optimization, we constrained the CCK peptide through interactions between Trp-39 and Gln-40, located at the upper part of the first transmembrane domain and the N-terminal moiety of CCK, and between Met-195 and Arg-197, located within the second extracellular loop and the sulfated tyrosine of CCK (23Kennedy K. Gigoux V. Escrieut C. Maigret B. Martinez J. Moroder L. Frehel D. Gully D. Vaysse N. Fourmy D. J. Biol. Chem. 1997; 272: 2920-2926Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 24Gigoux V. Escrieut C. Silvente-Poirot S. Maigret B. Gouilleux L. Fehrentz J.A. Gully D. Moroder L. Vaysse N. Fourmy D. J. Biol. Chem. 1998; 273: 14380-14386Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). This procedure allowed us to position the Asp-8 carboxylate side-chain of CCK in interaction with the Arg-336 guanidium and the C-terminal amide of CCK in contact with Asn-333. Exchange of Arg-336 and Asn-333 for amino acids lacking the chemical functions expected to interact with CCK resulted in dramatic decreases in both the affinity of CCK-AR for CCK and of its potency to mediate CCK-stimulated production of inositol phosphates. These experimental data suggested that Arg-336 and Asn-333 are involved in the CCK-AR binding site for CCK. Because the observed losses in function are not sufficient to distinguish between direct and indirect effects caused by the mutations, additional experiments were conducted.Experimental data demonstrating the interaction between Arg-336 and the Asp-8 carboxylate of CCK were obtained. First, binding experiments revealed that exchange of Arg-336 for a Met strongly affected the affinity of the CCK-AR mutant for CCK-related peptides containing an Asp carboxylate, whereas affinity for (Ala8)-CCK remained nearly constant. Second, exchange of Arg-336 of CCK-AR for an Asp caused a decrease in the potency of the mutated receptor when stimulated by unmodified CCK that was equal to that observed when the wild-type CCK-AR was stimulated by the CCK analogue having Asp-8 substituted by an Arg. This decrease in potency was larger than those obtained when Arg-336 and Asp-8 were individually exchanged for noncharged residues, clearly indicating that they result from repulsive forces that were introduced in the CCK-AR·CCK complexes. Third, and more importantly, the simultaneous double mutation Arg-336 → Asp in CCK-AR and Asp-8 → Arg in CCK yielded a CCK-AR·CCK complex ((R336D)-CCK-AR·(Arg-8)-CCK), the affinity of which was identical to that resulting from CCK binding to the very low affinity state of the wild-type CCK-AR ((WT)-CCK-AR·CCK). The specific and direct roles of Arg-336 in the CCK-AR binding site was further supported by the differential effect of its mutation on antagonist binding. Indeed, Arg-336 mutation caused stronger changes in receptor affinity for CCK-related peptides and SR-27897 than for L-364,718. Structural differences between these compounds account for such data because only CCK-related peptides and SR-27,897 contain a carboxylate residue (Fig.3).Experimental data also support the fact that Asn-333 is the amino acid of the CCK-AR binding site that interacts with the C-terminal amide of CCK. Indeed, functional analysis of (N333A)-CCK-AR indicated that, in contrast to (WT)-CCK-AR, the mutant poorly discriminated between (PheCH3)-CCK and unmodified CCK. It also responded to (PheCH3)-CCK with only a 2-fold lower potency than did (WT)-CCK-AR, whereas it responded to unmodified CCK with 1350-fold lower potency than (WT)-CCK-AR. Moreover, (N333A)-CCK-AR bound125I-BH-JMV 179, the structure of which is lacking the C-terminal amide as (WT)-CCK-AR (Fig. 3). The binding properties of (N333A)-CCK-AR toward nonpeptide antagonists that possess a carboxyamide moiety are clearly in favor of an interaction between Asn-333 and the carboxyamide of CCK ligands. Indeed, (N333A)-CCK-AR had a decreased affinity for SR-27,897 as well as for L-364,718, suggesting that Asn-333 is a crucial residue of the CCK-AR binding site shared by CCK and the nonpeptide antagonists SR-27,897 and L-364,718.To our knowledge, Arg-336 of CCK-AR has not been previously mutated by others. In contrast, mutation of an Asn residue of the rat CCK-AR that corresponds to Asn-333 in the human CCK-AR was reported to affect receptor sensitivity to both agonist (CCK) and nonpeptide antagonist (L-364,718). The authors suggested that the observed effects were due to receptor expression default (33Smeets R.L. Ap I.J. Hermsen H.P. Ophorst O.J. Van Emst-de Vries S.E. De Pont J.J. Willems P.H. Eur. J. Pharmacol. 1997; 325: 93-99Crossref PubMed Scopus (12) Google Scholar). The results from our study confirmed that exchange of Asn-333 for an Ala diminishes receptor expression at the cell surface. More importantly, they demonstrated direct involvement of Asn-333 in ligand recognition. In the human CCK-B/GR, exchange of Asn-353, homologous to Asn-333 of the human CCK-AR, for an Ala decreased affinity of the receptor for CCK (34Blaker M. Ren Y. Gordon M.C. Hsu J.E. Beinborn M. Kopin A.S. Mol. Pharmacol. 1998; 54: 857-863Crossref PubMed Scopus (32) Google Scholar, 35Kopin A.S. McBride E.W. Quinn S.M. Kolakowski Jr., L.F. Beinborn M. J. Biol. Chem. 1995; 270: 5019-5023Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Mutagenesis data so far available on the CCK-B/GR indicate that the CCK binding site of this receptor probably differs from that of CCK-AR, although it presents some similarities. Other authors who have mutated numerous amino acids in transmembrane domains of the CCK-B/GR have demonstrated that these amino acids did not play a critical role in CCK binding. In contrast, several amino acids within extracellular regions, particularly at the top of the first transmembrane domain and in the first and second extracellular loops, were found to be critical for CCK binding and activity (36Silvente-Poirot S. Escrieut C. Wank S.A. Mol. Pharmacol. 1998; 54: 364-371Crossref PubMed Scopus (58) Google Scholar). According to these different reports, the major energetic contribution to CCK binding to CCK-B/GR is likely to be conferred by extracellular residues. In CCK-AR, in addition to amino acids from extracellular regions (Trp-39/Gln-40 and Met-195/Arg-197), Asn-333 and Arg-336, which are located at the top of the sixth transmembrane domain, strongly contribute to CCK recognition.Pharmacological and functional data showing the importance of Arg-336-Asp-8 and Asn-333-amide interactions are in agreement with the first structure/function studies on CCK peptides using biological models naturally expressing CCK-AR. Indeed, it was shown that substitution of the Asp equivalent to Asp-8 of CCK-9 used in the current work by an Ala, a Glu, or a β-Ala decreases biological potency of CCK on gall bladder and pancreatic acinar cells by several hundred fold (17Pan G.Z. Martinez J. Bodanszky M. Jensen R.T. Gardner J.D. Biochim. Biophys. Acta. 1981; 678: 352-357Crossref PubMed Scopus (20) Google Scholar, 37Gardner J.D. Conlon T.P. Kleveman H.L. Adams T.D. Ondetti M.A. J. Clin. Invest. 1975; 56: 366-375Crossref PubMed Scopus (113) Google Scholar, 38Ondetti M.A. Rubin B. Engel S.L. Pluscec J. Sheehan J.T. Am. J. Dig. Dis. 1970; 15: 149-156Crossref PubMed Scopus (150) Google Scholar). Moreover, the importance of the C-terminal amide for biological activity of CCK has been demonstrated in structure-function studies with a series of CCK peptides having modified C termini. In fact, CCK peptides in which the C-terminal phenylalanine was replaced by a phenylethylamide (Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-NH-CH2CH2-C6H5) stimulated amylase release from rat pancreatic acini with a 1000-fold lower potency than amidated CCK, whereas it had only a 50-fold lower affinity for CCK-AR (11Galas M.C. Lignon M.F. Rodriguez M. Mendre C. Fulcrand P. Laur J. Martinez J. Am. J. Physiol. 1988; 254: G176-G182PubMed Google Scholar). JMV 180 is the most commonly used peptide of this series of CCK analogues. JMV 180 has its C-terminal phenylalanine substituted by a phenylethyl ester (Fig. 3). It was shown that JMV 180 binds to native rat pancreatic CCK-AR with a 10-fold lower affinity than amidated CCK and that it behaves as a partial agonist on amylase release (11Galas M.C. Lignon M.F. Rodriguez M. Mendre C. Fulcrand P. Laur J. Martinez J. Am. J. Physiol. 1988; 254: G176-G182PubMed Google Scholar, 15Williams J.A. Blevins Jr., G.T. Physiol. Rev. 1993; 73: 701-723Crossref PubMed Scopus (95) Google Scholar, 39Lignon M.F. Galas M.C. Rodriguez M. Martinez J. Cell Signal. 1990; 2: 339-346Crossref PubMed Scopus (15) Google Scholar). Although it is well recognized that JMV 180 induces distinct pharmacological effects and intracellular signals from CCK, the underlying molecular mechanisms remain to be precisely determined.Concerning the mechanisms whereby G protein-coupled receptors are activated by their agonist ligands, the ternary complex model and the analysis of constitutively active G protein-coupled receptors led us to consider that receptors spontaneously interchange between several conformations and that the agonist exerts its biological effect either by selecting or by stabilizing and inducing (or both) an active conformation (40Gether U. Kobilka B.K. J. Biol. Chem. 1998; 273: 17979-17982Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). In other words, binding of the agonist allows for a reduction in the energy barrier for a transition from an inactive to an active state of the receptor. In order to better understand the mechanisms that govern CCK-AR activation, additional studies, including those that will identify residues that interact with the aromatic ring of the C-terminal Phe and the Trp of CCK are necessary. However, in the course of our work regarding mapping of CCK-AR agonist binding site, we have found that several residues of CCK, such as the sulfated tyrosine, that are crucial for high affinity binding and biological activity, are much less important for binding to low and very low affinity states of CCK-AR (24Gigoux V. Escrieut C. Silvente-Poirot S. Maigret B. Gouilleux L. Fehrentz J.A. Gully D. Moroder L. Vaysse N. Fourmy D. J. Biol. Chem. 1998; 273: 14380-14386Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The results were interpreted as an indication that these residues of CCK contribute to stabilization of the high affinity state of the receptor-ligand complex. Our previous study, which analyzed the pharmacological and the functional properties of the (M195L)-CCK-AR mutant, extended this conclusion to amino acids of the receptor that interact with these crucial residues of CCK (24Gigoux V. Escrieut C. Silvente-Poirot S. Maigret B. Gouilleux L. Fehrentz J.A. Gully D. Moroder L. Vaysse N. Fourmy D. J. Biol. Chem. 1998; 273: 14380-14386Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Results from the current study, showing that the (R336D)-CCK-AR·(Arg-8)-CCK complex had an affinity identical to that resulting from binding of CCK to the very low affinity state of (WT)-CCK-AR but remained inactive, confirm that correct positioning of pairing amino acids at the binding site is a prerequisite for occurrence of an active CCK-AR·CCK complex. These results illustrate how two-dimensional mutagenesis applied to putative peptide binding site of G protein-coupled receptors is a difficult task in term of gaining function.Direct interactions of peptide ligands with their receptors have only been reported in a limited number of cases. Recently, in the secretin receptor, interactions between two basic residues and secretin-Asp-3 were characterized using a strategy of two-dimensional site-directed mutagenesis (41Di Paolo E. De Neef P. Moguilevsky N. Petry H. Bollen A. Waelbroeck M. Robberecht P. FEBS Lett. 1998; 424: 207-210Crossref PubMed Scopus (49) Google Scholar). In the AT1 angiotensin-II receptor, two extracellular residues, His-183 of the second extracellular loop and Asp-281 of the third extracellular loop were shown to interact with the N-terminal residues Asp-1 and Arg-2 of angiotensin-II (42Feng Y.H. Noda K. Saad Y. Liu X.P. Husain A. Karnik S.S. J. Biol. Chem. 1995; 270: 12846-12850Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). In contrast, amino acids of the receptor that interact with residues of the C-terminal half of angiotensin-II, which is essential for binding and biological activity, were identified within transmembrane domains III, V, and VI (43Noda K. Saad Y. Karnik S.S. J. Biol. Chem. 1995; 270: 28511-28514Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In the NK-1 neurokinin receptor, several extracellular residues in addition to residues from the upper part of transmembrane domains were shown to directly interact with the natural ligand, Substance P (44Fong T.M. Yu H. Huang R.R. Strader C.D. Biochemistry. 1992; 31: 11806-11811Crossref PubMed Scopus (113) Google Scholar, 45Fong T.M. Yu H. Strader C.D. Biophys. J. 1992; 62: 59-60Abstract Full Text PDF PubMed Scopus (9) Google Scholar, 46Huang R.R. Yu H. Strader C.D. Fong T.M. Biochemistry. 1994; 33: 3007-3013Crossref PubMed Scopus (135) Google Scholar). Interestingly, binding sites for nonpeptide antagonists on these two receptors were localized in a pocket within transmembrane domains and appear therefore to be composed almost exclusively of amino acids distinct from those of the binding site for peptide agonists (20Schwartz T.W. Rosenkilde M.M. Trends Pharmacol. Sci. 1996; 17: 213-216Abstract Full Text PDF PubMed Scopus (125) Google Scholar,47Gether U. Nilsson L. Lowe III, J.A. Schwartz T.W. J. Biol. Chem. 1994; 269: 23959-23964Abstract Full Text PDF PubMed Google Scholar, 48Fong T.M. Huang R.R. Strader C.D. J. Biol. Chem. 1992; 267: 25664-25667Abstract Full Text PDF PubMed Google Scholar). Such findings imply that in these receptors, nonpeptide antagonists exclude the binding of agonist by allosteric regulation of the receptor conformation (20Schwartz T.W. Rosenkilde M.M. Trends Pharmacol. Sci. 1996; 17: 213-216Abstract Full Text PDF PubMed Scopus (125) Google Scholar). With respect to this point, our data on CCK-AR showing that Asn-333 and to a lesser extent Arg-336 are likely to be directly involved in the binding of nonpeptide antagonists SR-27897 and L-364,718, as well as in that of CCK, are original and indicate that no simple generalization can be made as to where ligands bind to G protein-coupled receptors.In conclusion, the amino acids identified in this study (Arg-336 and Asn-333) together with those from previous studies (Trp-39, Gln-40, Met-195, and Arg-197) (23Kennedy K. Gigoux V. Escrieut C. Maigret B. Martinez J. Moroder L. Frehel D. Gully D. Vaysse N. Fourmy D. J. Biol. Chem. 1997; 272: 2920-2926Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 24Gigoux V. Escrieut C. Silvente-Poirot S. Maigret B. Gouilleux L. Fehrentz J.A. Gully D. Moroder L. Vaysse N. Fourmy D. J. Biol. Chem. 1998; 273: 14380-14386Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 25Gigoux V. Escrieut C. Poirot S. Bouisson M. Maigret B. Fehrentz J. Martinez J. Moroder L. Vaysse N. Fourmy D. Dig. Dis. Sci. 1998; 43: 1884Google Scholar) indicate that the agonist binding site of CCK-AR is made up of several hydrophilic amino acids. Electrostatic interactions are therefore likely to represent the critical driving force for CCK binding to the high affinity active state of CCK-AR. Our data will be used to obtain an integrated dynamic view of the molecular processes that link agonist binding to receptor activation. The cholecystokinin-A receptor (CCK-AR) 1The abbreviations CCK-ARcholecystokinin receptor-AWTwild-type 1The abbreviations CCK-ARcholecystokinin receptor-AWTwild-typeis a member of the superfamily of G protein-coupled receptors and transduces CCK signals into target cells (1de Weerth A. Pisegna J.R. Huppi K. Wank S.A. Biochem. Biophys. Res. Commun. 1993; 194: 811-818Crossref PubMed Scopus (140) Google Scholar, 2Ulrich C.D. Ferber I. Holicky E. Hadac E. Buell G. Miller L.J. Biochem. Biophys. Res. Commun. 1993; 193: 204-211Crossref PubMed Scopus (91) Google Scholar). CCK-AR has important physiological functions in the central nervous system (stimulation of satiety) and in peripheral organs, particularly in the gut (stimulation of gall bladder contraction, pancreatic secretions, and digestive motility) (3Silvente-Poirot S. Dufresne M. Vaysse N. Fourmy D. Eur. J. Biochem. 1993; 215: 513-529Crossref PubMed Scopus (127) Google Scholar). CCK-AR, like other G protein-coupled receptors, may be involved in the development of certain pathologies in humans and as such is an important target for therapeutic intervention involving the pharmacological blockade or activation of the receptor using appropriate ligands (4Wank S.A. Am. J. Physiol. 1998; 274: G607-G613PubMed Google Scholar, 5Inoue H. Iannotti C.A. Welli
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