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• W2113681204 abstract "The cholecystokinin (CCK) receptor-1 (CCK1R) is a G protein-coupled receptor, which mediates important central and peripheral cholecystokinin actions. Our aim was to progress in mapping of the CCK1R binding site by identifying residues that interact with the methionine and phenylalanine residues of the C-terminal moiety of CCK because these are crucial for its binding and biological activity, and to determine whether CCK and the selective non-peptide agonist, SR-146,131, share a common binding site. Identification of putative amino acids of the CCK1R binding site was achieved by dynamics-based docking of the ligand CCK in a refined three-dimensional model of the CCK1R using, as constraints, previous results that identified contact points between residues of CCK and CCK1R (Kennedy, K., Gigoux, V., Escrieut, C., Maigret, B., Martinez, J., Moroder, L., Frehel, D., Gully, D., Vaysse, N., and Fourmy, D. (1997) J. Biol. Chem. 272, 2920–2926 and Gigoux, V., Escrieut, C., Fehrentz, J. A., Poirot, S., Maigret, B., Moroder, L., Gully, D., Martinez, J., Vaysse, N., and Fourmy, D. (1999) J. Biol. Chem. 274, 20457–20464). By this approach, a series of residues forming connected hydrophobic clusters were identified. Pharmacological and functional analysis of mutated receptors indicated that a network of hydrophobic residues including Cys-94, Met-121, Val-125, Phe-218, Ile-329, Phe-330, Trp-326, Ile-352, Leu-356, and Tyr-360, is involved in the binding site for CCK and in the activation process of the CCK1R. Within this hydrophobic network, the physico-chemical nature of residue 121 seems to be essential for CCK1R functioning. Finally, the biological properties of mutants together with dynamic docking of SR-146,131 in the CCK1R binding site demonstrated that SR-146,131 occupies a region of CCK1R binding site which interacts with the C-terminal amidated tripeptide of CCK, i.e. Met-Asp-Phe-NH2. These new and important insights will serve to better understand the activation process of CCK1R and to design or optimize ligands. The cholecystokinin (CCK) receptor-1 (CCK1R) is a G protein-coupled receptor, which mediates important central and peripheral cholecystokinin actions. Our aim was to progress in mapping of the CCK1R binding site by identifying residues that interact with the methionine and phenylalanine residues of the C-terminal moiety of CCK because these are crucial for its binding and biological activity, and to determine whether CCK and the selective non-peptide agonist, SR-146,131, share a common binding site. Identification of putative amino acids of the CCK1R binding site was achieved by dynamics-based docking of the ligand CCK in a refined three-dimensional model of the CCK1R using, as constraints, previous results that identified contact points between residues of CCK and CCK1R (Kennedy, K., Gigoux, V., Escrieut, C., Maigret, B., Martinez, J., Moroder, L., Frehel, D., Gully, D., Vaysse, N., and Fourmy, D. (1997) J. Biol. Chem. 272, 2920–2926 and Gigoux, V., Escrieut, C., Fehrentz, J. A., Poirot, S., Maigret, B., Moroder, L., Gully, D., Martinez, J., Vaysse, N., and Fourmy, D. (1999) J. Biol. Chem. 274, 20457–20464). By this approach, a series of residues forming connected hydrophobic clusters were identified. Pharmacological and functional analysis of mutated receptors indicated that a network of hydrophobic residues including Cys-94, Met-121, Val-125, Phe-218, Ile-329, Phe-330, Trp-326, Ile-352, Leu-356, and Tyr-360, is involved in the binding site for CCK and in the activation process of the CCK1R. Within this hydrophobic network, the physico-chemical nature of residue 121 seems to be essential for CCK1R functioning. Finally, the biological properties of mutants together with dynamic docking of SR-146,131 in the CCK1R binding site demonstrated that SR-146,131 occupies a region of CCK1R binding site which interacts with the C-terminal amidated tripeptide of CCK, i.e. Met-Asp-Phe-NH2. These new and important insights will serve to better understand the activation process of CCK1R and to design or optimize ligands. Cholecystokinin (CCK) 1CCKcholecystokininCCKnRcholecystokinin receptor-nWTwild-typeBHBolton-HunterRPreverse phaseHPLChigh performance liquid chromatographyESI-MSelectrospray ionization-mass spectrometryTMtransmembraneGTPγSguanosine 5′-3-O-(thio)triphosphate 1CCKcholecystokininCCKnRcholecystokinin receptor-nWTwild-typeBHBolton-HunterRPreverse phaseHPLChigh performance liquid chromatographyESI-MSelectrospray ionization-mass spectrometryTMtransmembraneGTPγSguanosine 5′-3-O-(thio)triphosphate is a neuropeptide that has a wide spectrum of biological actions. CCK is composed of several molecular variants, the octapeptide (CCK-8: Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) being the major fully active one (1Villanueva M.L. Collins S.M. Jensen R.T. Gardner J.D. Am. J. Physiol. 1982; 242: G416-G422PubMed Google Scholar, 2Jensen R.T. Lemp G.F. Gardner J.D. J. Biol. Chem. 1982; 257: 5554-5559Abstract Full Text PDF PubMed Google Scholar). Two CCK receptors have been characterized pharmacologically, biologically and subsequently cloned, the CCK1 receptor (abbreviated CCK1R, previously named CCKA receptor) and the CCK2 receptor (abbreviated CCK2R, previously named CCK-B/gastrin receptor), which both belong to the superfamily of G protein-coupled receptors (3de Weerth A. Pisegna J.R. Huppi K. Wank S.A. Biochem. Biophys. Res. Commun. 1993; 194: 811-818Crossref PubMed Scopus (140) Google Scholar, 4Pisegna J.R. de Weerth A. Huppi K. Wank S.A. Biochem. Biophys. Res. Commun. 1992; 189: 296-303Crossref PubMed Scopus (216) Google Scholar). The CCK1R and CCK2R can exist in several conformational states, which bind CCK with high, low, and very low affinities, respectively, and share the functional coupling to phospholipase C, via binding to heterotrimeric GTP-binding protein(s) (5Talkad V.D. Fortune K.P. Pollo D.A. Shah G.N. Wank S.A. Gardner J.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1868-1872Crossref PubMed Scopus (47) Google Scholar, 6Huang S.C. Fortune K.P. Wank S.A. Kopin A.S. Gardner J.D. J. Biol. Chem. 1994; 269: 26121-26126Abstract Full Text PDF PubMed Google Scholar, 7Williams J.A. Blevins G.T., Jr. Physiol. Rev. 1993; 73: 701-723Crossref PubMed Scopus (95) Google Scholar). CCK1R-mediated effects include control of gallbladder contraction, pancreatic exocrine secretion, gastric emptying and gut motility, and satiety (8Silvente-Poirot S. Dufresne M. Vaysse N. Fourmy D. Eur. J. Biochem. 1993; 215: 513-529Crossref PubMed Scopus (127) Google Scholar, 9Wank S.A. Am. J. Physiol. 1998; 274: G607-G613PubMed Google Scholar). The wide spectrum of biological functions regulated by the CCK1R makes it a candidate target for a therapeutic approach in a number of diseases related to nutrient assimilation. This led a number of academic and pharmaceutical research groups to design specific and highly potent agonists and antagonists for this receptor (8Silvente-Poirot S. Dufresne M. Vaysse N. Fourmy D. Eur. J. Biochem. 1993; 215: 513-529Crossref PubMed Scopus (127) Google Scholar, 9Wank S.A. Am. J. Physiol. 1998; 274: G607-G613PubMed Google Scholar). cholecystokinin cholecystokinin receptor-n wild-type Bolton-Hunter reverse phase high performance liquid chromatography electrospray ionization-mass spectrometry transmembrane guanosine 5′-3-O-(thio)triphosphate cholecystokinin cholecystokinin receptor-n wild-type Bolton-Hunter reverse phase high performance liquid chromatography electrospray ionization-mass spectrometry transmembrane guanosine 5′-3-O-(thio)triphosphate Pharmacological studies have shown that chemically distinct molecules such as peptides, peptoids, and non-peptides can bind to the CCK1R with very close affinities (8Silvente-Poirot S. Dufresne M. Vaysse N. Fourmy D. Eur. J. Biochem. 1993; 215: 513-529Crossref PubMed Scopus (127) Google Scholar, 9Wank S.A. Am. J. Physiol. 1998; 274: G607-G613PubMed Google Scholar). On the other hand, within each chemical family of CCK1R ligands, compounds having close structures are agonists, partial agonists, or antagonists, indicating that appropriate modifications within the pharmacophore switches an agonist to an antagonist and vice versa. This can be illustrated with both peptide and non-peptide ligands of CCK1R. For instance, JMV 179, a CCK heptapeptide analogue in which the C-terminal amidated phenylalanine and the l-tryptophan have been replaced by a phenylethyl ester moiety and a d-tryptophan, respectively, is a full CCK1R antagonist (10Lignon 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). This antagonist has been converted to JMV 180, a peptide exhibiting dual agonistic/antagonistic activity, by exchanging the d-tryptophan for anl-tryptophan (7Williams J.A. Blevins G.T., Jr. Physiol. Rev. 1993; 73: 701-723Crossref PubMed Scopus (95) Google Scholar, 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). Another interesting example came from the discovery of the non-peptide CCK1R agonist, SR-146,131, by chemical modification of the CCK1R antagonist, SR-27,897 (12Gully D. Frehel D. Marcy C. Spinazze A. Lespy L. Neliat G. Maffrand J.P. Le Fur G. Eur. J. Pharmacol. 1993; 232: 13-19Crossref PubMed Scopus (79) Google Scholar, 13Bignon E. Alonso R. Arnone M. Boigegrain R. Brodin R. Gueudet C. Heaulme M. Keane P. Landi M. Molimard J.C. Olliero D. Poncelet M. Seban E. Simiand J. Soubrie P. Pascal M. Maffrand J.P. Le Fur G. J. Pharmacol. Exp. Ther. 1999; 289: 752-761PubMed Google Scholar) (Fig. 1). These examples, which could probably be extended to multiple G protein-coupled receptors, raise the important questions of whether closely related ligands having opposite biological activities share the same binding site and of which intrinsic mechanism(s) at the binding site level govern(s) G protein-coupled receptor functioning. One of our recent objectives has been to define the agonist binding site on the CCK1R and to identify interactions between critical residues of that binding site and chemical functions of the pharmacophoric domain of CCK (Figs. 1 and 3). Amino acids within three regions of the CCK1R were identified as belonging to the binding site for CCK. Trp-39 and Gln-40, located at the top of transmembrane helix I, were shown to interact directly with the N-terminal portion of CCK (14Kennedy 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). Met-195 and Arg-197, located in the second extracellular loop, were then shown to interact with the sulfated tyrosine (15Gigoux 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, 16Gigoux V. Maigret B. Escrieut C. Silvente-Poirot S. Bouisson M. Fehrentz J.A. Moroder L. Gully D. Martinez J. Vaysse N. Fourmy A.D. Protein Sci. 1999; 8: 2347-2354PubMed Google Scholar). More recently, Arg-336 and Asn-333, at the top of helix VI, were demonstrated to pair with the Asp carboxylate and the C-terminal amide of CCK, respectively (17Gigoux V. Escrieut C. Fehrentz J.A. Poirot S. Maigret B. Moroder L. Gully D. Martinez J. Vaysse N. Fourmy D. J. Biol. Chem. 1999; 274: 20457-20464Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The first two identified amino acids of the CCK1R (Trp-39 and Gln-40) contribute weakly to CCK1R affinity for and response to CCK, whereas all others play a more critical role because of their interaction with residues of CCK, which are essential for both binding and biological activity of CCK. However, contact points within the CCK1R binding site for other key residues of CCK such as the Trp, Met, and Phe residues have not yet been identified. To progress in the mapping of the CCK1R binding site(s), the three-dimensional model of the CCK1R·CCK complex was optimized, leading to the identification of putative amino acids involved in the interaction with the Met and Phe residues of the ligand CCK. Mutation of candidate residues and extensive characterization of the resulting mutants allowed us to position the C-terminal biological part of CCK in hydrophobic pockets formed by aromatic and nonaromatic amino acids located in the upper half of transmembrane helices III, V, VI, and VII. Our data, therefore, refute the model of the CCK1R·CCK complex proposed by other investigators in which the C terminus of CCK interacts with an amino acid residue (Trp-39) of helix I (18Ding X.Q. Dolu V. Hadac E.M. Holicky E.L. Pinon D.I. Lybrand T.P. Miller L.J. J. Mol. Biol. 2001; 276: 4236-4244Scopus (42) Google Scholar). Furthermore, binding site for the non-peptide agonist SR-146,131 was identified and experimentally validated as overlapping with that of the C-terminal tripeptide of CCK. Finally, the role of Met-121 located on helix III in the activation of the CCK1R by agonists was demonstrated. The C-terminal nonapeptide analogue (Nle)-CCK-9 (Fig. 1) was synthesized as described previously (19Moroder L. Wilschowitz L. Gemeiner M. Gohring W. Knof S. Scharf R. Thamm P. Gardner J.D. Solomon T.E. Wunsch E. Hoppe-Seylers Z. Physiol. Chem. 1981; 362: 929-942Crossref PubMed Scopus (59) Google Scholar). The other analogues of Fig. 1 were synthesized on an ACT 396 synthesizer by applying the Fmoc (N-(9-fluorenyl)methoxycarbonyl)/ter-butyl chemistry and chlorotrityl resin (PepChem). Upon deprotection and resin cleavage with trifluoroacetic acid/H2O/triethylsilane (9:0.5:0.5) at 0 °C, the crude products were precipitated by methyltert-butyl ether/hexane (2:1) and purified by preparative RP-HPLC (250/20 Nucleosil 300/5 C18; Machery & Nagel) and characterized by analytical RP-HPLC (linear gradient acetonitrile/2% H3PO4 from 5 to 90% in 15 min; 125/4 Nucleosil 100/5 C18; Machery & Nagel) and ESI-MS (API 165; PerkinElmer Life Sciences); (Ala-7)-CCK: homogeneous on analytical RP-HPLC (t R = 6,7 min); ESI-MS: m/z: 1209.4 [M + H+]; M r = 1209.5 calculated for C52H69N14O18S1; (Ala-9)-CCK: homogeneous on analytical RP-HPLC (t R = 6,5 min); ESI-MS: m/z: 1176.2 [M + H+]; M r = 1175.5 calculated for C49H71N14O18S1; (Gln-7)-CCK: homogeneous on analytical RP-HPLC (t R = 7,6 min); ESI-MS: m/z: 1266.8 [M + H+]; M r = 1266.5 calculated for C54H72N15O19S1. The non-peptide antagonist of the CCK1R, 1-[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl-indoyl]acetic acid (SR-27,897), and its tritiated derivative, [3H]SR-27,897 (31 Ci/mmol), as well as the non-peptide agonist of the CCK1R, 2-[4-(4-chloro-2-,5-dimethoxyphenyl)-5-(2-cyclohexylethyl)thiazol-2-ylcarbamoyl]-5,7-dimethyl-indol-1-yl-1-acetic acid (SR-146,131), were donated by Sanofi-Synthelabo (Toulouse, France) (12Gully D. Frehel D. Marcy C. Spinazze A. Lespy L. Neliat G. Maffrand J.P. Le Fur G. Eur. J. Pharmacol. 1993; 232: 13-19Crossref PubMed Scopus (79) Google Scholar, 13Bignon E. Alonso R. Arnone M. Boigegrain R. Brodin R. Gueudet C. Heaulme M. Keane P. Landi M. Molimard J.C. Olliero D. Poncelet M. Seban E. Simiand J. Soubrie P. Pascal M. Maffrand J.P. Le Fur G. J. Pharmacol. Exp. Ther. 1999; 289: 752-761PubMed Google Scholar). 125INa andmyo-[3H]inositol (5 μCi/ml) were from Amersham Biosciences, Inc., Les Ulis, France. (Thr,Nle)-CCK-9 was conjugated with Bolton-Hunter reagent, purified, and radioiodinated as described previously (20Fourmy D. Lopez P. Poirot S. Jimenez J. Dufresne M. Moroder L. Powers S.P. Vaysse N. Eur. J. Biochem. 1989; 185: 397-403Crossref PubMed Scopus (43) Google Scholar). The specific activity of the radioiodinated peptide was 1600–2000 Ci/mmol. All other chemicals were obtained from commercial sources. The model of empty CCK1R was built using the transmembrane (TM) helical arrangement found in the bacteriorhodopsin crystal structure as starting point (21Henderson R. Baldwin J.M. Ceska T.A. Zemlin F. Beckmann E. Downing K.H. J. Mol. Biol. 1990; 213: 899-929Crossref PubMed Scopus (2520) Google Scholar). It was then modified according to the rhodopsin crystal structure (22Unger V.M. Hargrave P.A. Baldwin J.M. Schertler G.F. Nature. 1997; 389: 203-206Crossref PubMed Scopus (480) Google Scholar, 23Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A., Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5003) Google Scholar) and to the mutant data base “input/output” information scheme defined in the Viseur program (24Campagne F. Jestin R. Reversat J.L. Bernassau J.M. Maigret B. J. Comput. Aided Mol. Des. 1999; 13: 625-643Crossref PubMed Scopus (30) Google Scholar). Extracellular and intracellular loops connecting the transmembrane helices were then added to the preliminary seven-helix bundle, and the structural model was optimized by the use of simulated annealing procedures. The entire system was finally relaxed and submitted to a 1-ns molecular dynamics with possible translational and rotational movements of individual TM helices taken into account. The final model respects most transmembrane arrangements found in the recent x-ray structure of rhodopsin (23Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A., Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5003) Google Scholar). For docking of CCK ligand into the CCK1R binding site, experimental data that identified contact points between residues Trp-39 and Gln-40 and the N-terminal moiety of CCK served as a first constraint to place CCK within the CCK1R grove (14Kennedy 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 a first step, manual docking was achieved by taking into account molecular electrostatic potentials at the top of the receptor grove. The resulting complex was submitted to annealing molecular dynamics calculations. The resulting theoretical positioning of CCK into the CCK1R binding site was experimentally validated by two-dimensional site-directed mutagenesis. By doing so, Met-195–Arg-197, Arg-336, and Asn-333 were shown to belong the CCK1R binding site and to interact with the sulfated tyrosine of CCK, the Asp-8 carboxylate, and the C-terminal amide, respectively (15Gigoux 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, 16Gigoux V. Maigret B. Escrieut C. Silvente-Poirot S. Bouisson M. Fehrentz J.A. Moroder L. Gully D. Martinez J. Vaysse N. Fourmy A.D. Protein Sci. 1999; 8: 2347-2354PubMed Google Scholar, 17Gigoux V. Escrieut C. Fehrentz J.A. Poirot S. Maigret B. Moroder L. Gully D. Martinez J. Vaysse N. Fourmy D. J. Biol. Chem. 1999; 274: 20457-20464Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The program package (Insight II, Discover, Homology, Biopolymer) from Molecular Simulations Inc. (San Diego, CA) was used for all the calculations. Mutant receptor cDNAs were constructed by oligonucleotide-directed mutagenesis (QuikChange™ site-directed mutagenesis kit, Stratagene, France) using the human CCK1R cDNAs cloned into pRFENeo vector as template (25Kennedy K. Escrieut C. Dufresne M. Clerc P. Vaysse N. Fourmy D. Biochem. Biophys. Res. Commun. 1995; 213: 845-852Crossref PubMed Scopus (37) Google Scholar). Oligonucleotides were designed to include a silent restriction site to facilitate analysis of mutant constructs by restriction endonuclease digestion. The presence of the desired and the absence of undesired mutations were confirmed by automated sequencing of both cDNA strands (Applied Biosystems). COS-7 cells (1.5 × 106) were plated onto 10-cm culture dishes and grown in Dulbecco's modified Eagle's medium containing 5% fetal calf serum (complete medium) in a 5% CO2 atmosphere at 37 °C. After overnight incubation, cells were transfected with 2.5 μg/plate of pRFENeo vectors containing the cDNA for the wild-type or mutated CCK1 receptors, using a modified DEAE-dextran method. Cells were transferred to 24-well plates at a density of 80,000–150,000 cells/well 24 h after transfection. Approximately 24 h after the transfer of transfected cells to 24-well plates, the cells were washed with phosphate-buffered saline, pH 6.95, 0.1% BSA and then incubated for 60 min at 37 °C in 0.5 ml of Dulbecco's modified Eagle's medium, 0.1% BSA with either 71 pm125I-BH-(Thr,Nle)-CCK-9 or 1.83 nm[3H]SR-27,897 in the presence or the absence of competing agonists or antagonists. The cells were washed twice with cold phosphate-buffered saline, pH 6.95, containing 2% BSA, and cell-associated radioligand was collected with 0.1 n NaOH added to each well. The radioactivity was directly counted in a γ counter (Auto-Gamma, Packard, Downers Grove, IL) or added to scintillant and counted for the tritiated radioligand. Approximately 24 h after the transfer to 24-well plates and following overnight incubation in complete medium containing 2 μCi/mlmyo-2-[3H]inositol, the transfected cells were washed with Dulbecco's modified Eagle's medium and then incubated for 30 min in 1 ml/well Dulbecco's modified Eagle's medium containing 20 mm LiCl at 37 °C. The cells were washed with PI buffer at pH 7.45: phosphate-buffered saline containing 135 mmNaCl, 20 mm HEPES, 2 mm CaCl2, 1.2 mm MgSO4, 1 mm EGTA, 10 mm LiCl, 11.1 mm glucose, and 0.5% BSA. The cells were then incubated for 60 min at 37 °C in 0.3 ml of PI buffer with or without ligands at various concentrations. The reaction was stopped by adding 1 ml of methanol/chlorhydric acid to each well, and the content was transferred to a column (Dowex AG 1-X8, formate form, Bio-Rad) for the determination of inositol phosphates. The columns were washed twice with 3 ml of distilled water and twice more with 2 ml of 5 mm sodium tetraborate, 60 mm sodium formate. The content of each column was eluted by addition of 2.5 ml of 1m ammonium formate, 100 mm formic acid. 0.5 ml of the eluted fraction was added to scintillant, and β radioactivity was counted. Approximately 65 h after transfection, the cells were washed three times with phosphate-buffered saline, pH 6.95, scraped from the plate in 10 mm Hepes buffer, pH 7.0, containing 0.01% soybean trypsin inhibitor, 0.1% bacitracin, 0.1 mm phenylmethylsulfonyl fluoride and frozen in liquid N2. After thawing at 37 °C, the cells were subjected to another cycle of freeze/thawing and then centrifuged at 25,000 × g for 20 min. The membrane pellet was resuspended in 50 mm Hepes buffer, pH 7.0, containing 115 mm NaCl, 5 mm MgCl2, 0.01% soybean trypsin inhibitor, 0.1% bacitracin, 1 mm EGTA, 0.1 mm phenylmethylsulfonyl fluoride (binding buffer); aliquoted; and stored at −80 °C until use. Membrane protein concentrations were determined using the Bio-Rad protein assay kit. To assess the effect of GTPγS on CCK binding, membranes from transfected COS-7 cells (0.4–4 μg of proteins) were incubated with 71 pm125I-BH-(Thr,Nle)-CCK-9 in the absence or in the presence of increasing concentrations GTPγS in binding buffer for 120 min at 25 °C. Nonspecific binding was measured in the presence of 1 μm CCK. Previous structure-activity studies using synthetic replicates of CCK and pancreatic acini from rodents, a biological model naturally expressing CCK1R, have clearly shown the importance of both Met and Phe residues for binding and activity of CCK; however, no such study has been reported for human CCK1R in any expression system (26Jensen R.T. Wank S.A. Rowley W.H. Sato S. Gardner J.D. Trends Pharmacol. Sci. 1989; 10: 418-423Abstract Full Text PDF PubMed Scopus (164) Google Scholar). Therefore, we first determined to what extent the Met and Phe side chains contribute to the affinity of CCK for human CCK1R expressed in COS-7 cells and to its capacity to induce production of total inositol phosphates. As shown in Fig.2, replacement of Met-7 in CCK by an Ala residue caused 4000- and 390-fold decrease in affinity and potency, respectively. In contrast, substitution of Met-7 by Nle did not affect affinity and potency of CCK. Furthermore, exchange of Phe for Ala was found to induce a 4900- and 2700-fold decrease in affinity and potency of the analogues, respectively. The efficacies of (Nle-7)-CCK, (Met-7)-CCK, and (Ala-7)-CCK were comparable, whereas that of (Ala-9)-CCK reached only 40% referred to the parent analogue (Nle-7)-CCK. Hence, both Met-7 and Phe-9 side chains contribute significantly to receptor binding and activation potency of CCK, confirming data obtained previously on receptors from rodents and guinea pig (27Marseigne I. Dor A. Begue D. Reibaud M. Zundel J.L. Blanchard J.C. Pelaprat D. Roques B.P. J. Med. Chem. 1988; 31: 966-970Crossref PubMed Scopus (18) Google Scholar, 28Spanarkel M. Martinez J. Briet C. Jensen R.T. Gardner J.D. J. Biol. Chem. 1983; 258: 6746-6749Abstract Full Text PDF PubMed Google Scholar). These results also confirm that replacement of Met-7 with Nle does not affect affinity and activity of CCK. Considering the major anchoring points of CCK inside the receptor discovered in our previous studies, it appears that the Nle/Met-7 residue is located in the vicinity of a hydrophobic pocket constituted by residues Leu-50, Ile-51, Leu-53, Pro-101, Val-125, Met-121, Ileu-352, and Leu-356. In the model of the (WT)-CCK1R, both Nle-7 or Met-7 side chains are positioned in the same way. The Phe-9 aromatic side chain of CCK is also positioned into a well defined cavity delineated by Phe-330 at the bottom, and Pro-177, Val-125, Leu-214, Ile-329 around Phe-330, which itself belongs to a large aromatic cluster constituted by the side chains of Cys-94, Phe-130, Trp-166, Phe-170, Phe-218, Phe-322, Phe-323, Trp-326, and Tyr-360. These two pockets are connected via the Val-125 side chain so that helix movements changing the structure of one of them may have consequences on the other. Examination of the organization of the two clusters in the three-dimensional model of the CCK1R·CCK complex suggests that not only a single, but several hydrophobic residues are contributing to the binding energy between Nle/Met-7 or Phe side chains of CCK and the CCK1R. As a consequence and unlike the charged residues of the binding pocket that were characterized previously, a mutation of only one of these amino acid residues of the CCK1R was not expected to induce changes in affinity and biological potency of the CCK1R to an extent comparable with effects caused by replacements of Nle/Met or Phe in CCK. Among all residues forming hydrophobic clusters around the Nle/Met-7 and Phe-9 side chains, in a first instance those in closest contact were chosen for mutagenesis experiments (Fig. 3). These were exchanged for amino acids lacking the chemical functions thought to be responsible for the interactions. In addition, Met-121 and Ile-329, which appear to be important for the equilibrium within the hydrophobic clusters surrounding Nle/Met-7 and Phe-9 of CCK, were each exchanged for more bulky and hydrophobic residues, namely Val and Phe, respectively. In a first series of experiments, COS-7 cells expressing mutated receptors were assayed for binding of the non-peptide antagonist [3H]SR-27,897. Indeed, binding of this antagonist, unlike that of an agonist, offers the advantage of allowing detection of CCK1R independent of the coupling state to G protein(s), thus yielding accurate expression levels of all mutants (15Gigoux 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, 17Gigoux V. Escrieut C. Fehrentz J.A. Poirot S. Maigret B. Moroder L. Gully D. Martinez J. Vaysse N. Fourmy D. J. Biol. Chem. 1999; 274: 20457-20464Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Ligand binding data are summarized in TableI. No binding of [3H]SR-27,897 was found with the (I329F)-CCK1R mutant, a result that could be interpreted at this stage of the study either by an absence of expression of the mutant or by a direct or indirect effect of the mutation on receptor affinity for the ligand. Results with all other CCK1R mutants confirmed their expression at COS-7 cell surface at levels varying from 0.6 to 10.0 pmol/106 cells, which permitted further characterization. In addition, the binding data clearly revealed that all mutants (except (I329F)-CCK1R) bind the non-peptide antagonist with an affinity very close to that of (WT)-CCK1R. This finding indicates that the mutations did not dramatically disturb the conformational state of the CCK1R and that the mutated residues are not directly involved in the binding pocket of the antagonist SR-27,897.Table IEffects of CCK1R mutations on CCK1R expression and affinity for the non-peptide antagonist, SR-27,897 and the peptide agonist, CCKCCK1RSR 27897 bindingCCK bindingK dFmutB maxHigh affinity sitesLow affinity sitesB maxK d (1)FmutK d (2)FmutHigh affinityLow affinitynmpmol/10 6 cellsnmnmpmol/10 6 cellsWT3.3 ± 0.51.09.2 ± 1.61.20 ± 0.051.071 ± 141.00.18 ± 0.024.9 ± 0.7L50A3.1 ± 0.70.96.3 ± 0.51.1 ± 0.51.051 ± 80.70.05 ± 0.032.2 ± 0.6I51A6.4 ± 3.81.97.9 ± 0.92.0 ± 0.51.744 ± 30.70.30 ± 0.052.2 ± 0.6L53A1.9 ± 0.60.65.6 ± 1.51.4 ± 0.71.252 ± 120.70.14 ± 0.052.7 ± 0.4C94L7.4 ± 2.82.24.9 ± 0.974.0 ± 5.062ND3.1 ± 0.3NDM121V6.2 ± 1.41.96.5 ± 3.419.0 ± 1.316ND7.0 ± 0.6NDM121A8.9 ± 2.62.710.0 ± 3.32.1 ± 0.21.8ND13.2 ± 1.4NDI352A5.4 ± 11.64.7 ± 0.2No bindingL356A4.0 ± 0.31.25.1 ± 0.40.97 ± 0.030.8561 ± 11480.016 ± 0.0044.4 ± 1.9V12" @default.
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• W2113681204 title "The Biologically Crucial C Terminus of Cholecystokinin and the Non-peptide Agonist SR-146,131 Share a Common Binding Site in the Human CCK1 Receptor" @default.
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