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• W2052667701 abstract "Affinity labeling is a powerful tool to establish spatial approximations between photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenylalanine (Bpa) sited in position 24, adjacent to the pharmacophoric domain of this hormone (positions 27–33). This probe was a fully efficacious agonist that bound to the CCK receptor saturably and with high affinity (K i = 8.9 ± 1.1 nm). It covalently labeled the CCK receptor either within the amino terminus (between Asn10 and Lys37) or within the third extracellular loop (Glu345), as demonstrated by proteolytic peptide mapping, deglycosylation, micropurification, and Edman degradation sequencing. Truncation of the receptor to eliminate residues 1–30 had no detrimental effect on CCK binding, stimulated signaling, or affinity labeling through a residue within the pharmacophore (Bpa29) but resulted in elimination of the covalent attachment of the Bpa24 probe to the receptor. Thus, the distal amino terminus of the CCK receptor resides above the docked ligand, compressing the portion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellular trypsin damaged the truncated construct but not the wild type receptor, suggesting that this domain also may play a protective role. Use of these additional insights into molecular approximations provided key constraints for molecular modeling of the peptide-receptor complex, supporting the counterclockwise organization of the transmembrane helical domains. Affinity labeling is a powerful tool to establish spatial approximations between photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenylalanine (Bpa) sited in position 24, adjacent to the pharmacophoric domain of this hormone (positions 27–33). This probe was a fully efficacious agonist that bound to the CCK receptor saturably and with high affinity (K i = 8.9 ± 1.1 nm). It covalently labeled the CCK receptor either within the amino terminus (between Asn10 and Lys37) or within the third extracellular loop (Glu345), as demonstrated by proteolytic peptide mapping, deglycosylation, micropurification, and Edman degradation sequencing. Truncation of the receptor to eliminate residues 1–30 had no detrimental effect on CCK binding, stimulated signaling, or affinity labeling through a residue within the pharmacophore (Bpa29) but resulted in elimination of the covalent attachment of the Bpa24 probe to the receptor. Thus, the distal amino terminus of the CCK receptor resides above the docked ligand, compressing the portion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellular trypsin damaged the truncated construct but not the wild type receptor, suggesting that this domain also may play a protective role. Use of these additional insights into molecular approximations provided key constraints for molecular modeling of the peptide-receptor complex, supporting the counterclockwise organization of the transmembrane helical domains. guanine nucleotide-binding protein cholecystokinin benzoylphenylalanine cyanogen bromide staphylococcal V8 protease CCK receptor high pressure liquid chromatography 4-morpholineethanesulfonic acid Chinese hamster ovary Guanine nucleotide-binding protein (G protein)1-coupled receptors represent a remarkable group of structurally homologous membrane proteins that can bind and be activated by widely diverse ligands. The molecular details of how ligands as structurally dissimilar as photons, biogenic amines, peptides, and glycoproteins can elicit similar conformational changes in the cytosolic face of their receptors (where G protein-coupling occurs) are far from clear. Our best understanding of this process relates to the smallest ligands that appear to bind within the confluence of helices within the lipid bilayer (1Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar), where we have analogous low resolution crystal structures on which to rely (2Grigorieff N. Ceska T.A. Downing K.H. Baldwin J.M. Henderson R. J. Mol. Biol. 1996; 259: 393-421Crossref PubMed Scopus (868) Google Scholar,3Unger V.M. Hargrave P.A. Baldwin J.M. Schertler G.F. Nature. 1997; 389: 203-206Crossref PubMed Scopus (480) Google Scholar). As the ligands get larger and more structurally complex, the binding domains tend to move toward the extracellular face of the membrane, with extracellular loop and amino-terminal tail domains becoming more important (1Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 4Schwartz T.W. Curr. Opin. Biotechnol. 1994; 5: 434-444Crossref PubMed Scopus (280) Google Scholar). These are receptor domains for which we have minimal meaningful structural data.We have been quite interested in the molecular basis of ligand binding to the type A cholecystokinin (CCK) receptor (5Pearson R.K. Miller L.J. J. Biol. Chem. 1987; 262: 869-876Abstract Full Text PDF PubMed Google Scholar, 6Powers S.P. Fourmy D. Gaisano H. Miller L.J. J. Biol. Chem. 1988; 263: 5295-5300Abstract Full Text PDF PubMed Google Scholar, 7Klueppelberg U.G. Gaisano H.Y. Powers S.P. Miller L.J. Biochemistry. 1989; 28: 3463-3468Crossref PubMed Scopus (23) Google Scholar, 8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R. Lybrand T. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 10Hadac E.M. Ji Z.S. Pinon D.I. Henne R.M. Lybrand T.P. Miller L.J. J. Med. Chem. 1999; 42: 2105-2111Crossref PubMed Scopus (49) Google Scholar). This receptor is a member of the class I family of G protein-coupled receptors, along with rhodopsin and the β-adrenergic receptor (11Kolakowski L.F. Receptors and Channels. 1994; 2: 1-7PubMed Google Scholar). CCK occurs as a series of linear peptides, having lengths ranging from 8 to 58 residues (12Mutt V. Glass G.B.J. Cholecystokinin: Isolation, Structure, and Functions. Raven Press, Ltd., New York1980: 169-221Google Scholar). These all share their carboxyl-terminal domain, with the carboxyl-terminal heptapeptide-amide representing the minimal region that has full potency and efficacy for stimulating targets of this hormone. Two molecular approximations have been experimentally determined for residues within this region of CCK and residues within the ligand-binding domain of this receptor (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R. Lybrand T. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 10Hadac E.M. Ji Z.S. Pinon D.I. Henne R.M. Lybrand T.P. Miller L.J. J. Med. Chem. 1999; 42: 2105-2111Crossref PubMed Scopus (49) Google Scholar). Extension or structural modification of the amino terminus of CCK-8 has been well tolerated, without interfering with receptor binding or activation (13Ondetti 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,14Pearson R.K. Powers S.P. Hadac E.M. Gaisano H. Miller L.J. Biochem. Biophys. Res. Commun. 1987; 147: 346-353Crossref PubMed Scopus (25) Google Scholar). One provocative report has recently suggested that the naturally occurring amino-terminal extension that is present in CCK-58 can affect the conformation of the biologically active carboxyl-terminal octapeptide and can have a positive effect on the action of this hormone (15Keire D.A. Solomon T.E. Reeve Jr., J.R. Biochem. Biophys. Res. Commun. 1999; 266: 400-404Crossref PubMed Scopus (15) Google Scholar).With these observations in mind, we initiated the current group of studies. We were particularly interested in the structural details of how an amino-terminal extension from the CCK pharmacophore might be positioned relative to the CCK receptor. This work has given us new insights into the structure and function of the amino-terminal tail of this receptor. Unlike affinity labeling through photolabile residues that are positioned within the pharmacophore of CCK that have yielded only focused sites of covalent attachment to the receptor (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R. Lybrand T. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), the present work with a photolabile benzophenone residue positioned outside of this domain has resulted in demonstration of the ability to label either of two sites in distinct regions of the CCK receptor. This suggests that this position within the receptor-bound ligand may retain substantial mobility and not be held tightly in a single position relative to the receptor.One of the sites of covalent labeling was in a position in the amino-terminal tail of the CCK receptor that can be eliminated without having any negative impact on receptor binding or signaling. The other site of labeling was within the third extracellular loop domain. However, by truncation of the region of the receptor amino terminus in which the first contact was present, this photolabile residue no longer came in contact with or labeled the CCK receptor. These observations suggest that the amino-terminal domain of the receptor might provide a protective cover for the peptide-binding domain within the receptor. Indeed, when comparing the sensitivity of wild type and truncated receptor constructs to extracellular protease treatment, we found the latter to be much more amenable to damage by tryptic protease.The insights coming from the molecular approximations with a photolabile residue sited in position 24 of a CCK-like ligand, when combined with our previous ligand binding and cross-linking data (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R. Lybrand T. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 10Hadac E.M. Ji Z.S. Pinon D.I. Henne R.M. Lybrand T.P. Miller L.J. J. Med. Chem. 1999; 42: 2105-2111Crossref PubMed Scopus (49) Google Scholar), also provided us with the opportunity to propose a distinct topological model for CCK binding to its receptor, having a counterclockwise helix bundle topology. This refined molecular model of the ligand-receptor complex is quite distinct from models recently proposed in the literature based on less direct receptor mutagenesis studies (16Kennedy K. Gigoux V. Escrieut C. Martinez J. Moroder L. Frehel D. Gully D. Vayse N. Fourmy D. J. Biol. Chem. 1997; 272: 2920-2926Abstract Full Text Full Text PDF PubMed Scopus (71) 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, 18Gigoux V. Maigret B. Escrieut C. Silvente-Poirot S. Bouisson M. Fehrentz J.A. Moroder L. Gully D. Martinez J. Vaysse N. Fourmy D. Protein Sci. 1999; 8: 2347-2354PubMed Google Scholar). The current model is fully consistent with all existing experimental data and will continue to spawn experimentally testable predictions as it is further refined.DISCUSSIONG protein-coupled receptors can provide pockets for binding small ligands within the confluence of their seven intramembranous helical segments and for binding large glycoprotein ligands in specialized structural domains configured within large amino-terminal domains (1Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar,4Schwartz T.W. Curr. Opin. Biotechnol. 1994; 5: 434-444Crossref PubMed Scopus (280) Google Scholar). These receptors can also bind and be activated by peptide ligands, such as CCK. The emerging themes for such binding suggest critical contributions by extracellular loop and amino-terminal tail domains that are near the external face of the membrane, as well as including examples of portions of ligands dipping down into the confluence of helices.Our current understanding of the molecular basis of CCK binding is based on receptor mutagenesis and affinity labeling studies. Both support the importance for peptide binding of regions just outside of the first transmembrane segment and in the extracellular loop domains (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R. Lybrand T. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 16Kennedy K. Gigoux V. Escrieut C. Martinez J. Moroder L. Frehel D. Gully D. Vayse N. Fourmy D. J. Biol. Chem. 1997; 272: 2920-2926Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). There has, however, been substantial controversy related to the details of CCK peptide docking to these domains. Very distinct models of the peptide-occupied receptor have been proposed (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 16Kennedy K. Gigoux V. Escrieut C. Martinez J. Moroder L. Frehel D. Gully D. Vayse N. Fourmy D. J. Biol. Chem. 1997; 272: 2920-2926Abstract Full Text Full Text PDF PubMed Scopus (71) 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). Nonpeptidyl ligand binding to this receptor seems to occur deeper in the membrane within the confluence of helices (34Smeets R.L.L. IJzerman A.P. Hermsen H.P.H. Ophorst O.J.A.E. Vries S.E.V. De Pont J.J.H.H.M. Willems P.H.G.M. Eur. J. Pharmacol. 1997; 325: 93-99Crossref PubMed Scopus (12) Google Scholar).Established structure-activity relationships for CCK have localized the pharmacophoric domain to the carboxyl-terminal heptapeptide-amide (12Mutt V. Glass G.B.J. Cholecystokinin: Isolation, Structure, and Functions. Raven Press, Ltd., New York1980: 169-221Google Scholar,13Ondetti 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). Almost every residue within this domain makes an important contribution to binding and activity. In contrast, almost any modification to the amino terminus of CCK-8 that has been attempted has been well tolerated, without modifying receptor binding or signaling. It was this feature that encouraged our positioning of the amino terminus of CCK-8 above the ligand-binding domain of the CCK receptor in our evolving model, such that an extension would not make contact with the regions of this receptor that are known to be important for function (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 10Hadac E.M. Ji Z.S. Pinon D.I. Henne R.M. Lybrand T.P. Miller L.J. J. Med. Chem. 1999; 42: 2105-2111Crossref PubMed Scopus (49) Google Scholar). It is noteworthy that another molecular model has placed the amino terminus of CCK much closer to the membrane and directed toward the other side of the helical bundle (16Kennedy K. Gigoux V. Escrieut C. Martinez J. Moroder L. Frehel D. Gully D. Vayse N. Fourmy D. J. Biol. Chem. 1997; 272: 2920-2926Abstract Full Text Full Text PDF PubMed Scopus (71) 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). Such a model is inconsistent with the residue-residue approximations that have been directly established by photoaffinity labeling studies (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar,10Hadac E.M. Ji Z.S. Pinon D.I. Henne R.M. Lybrand T.P. Miller L.J. J. Med. Chem. 1999; 42: 2105-2111Crossref PubMed Scopus (49) Google Scholar).To date, there has been the successful photoaffinity labeling of distinct spatially approximated residues within the CCK receptor through two positions within the pharmacophoric domain of CCK (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R. Lybrand T. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 10Hadac E.M. Ji Z.S. Pinon D.I. Henne R.M. Lybrand T.P. Miller L.J. J. Med. Chem. 1999; 42: 2105-2111Crossref PubMed Scopus (49) Google Scholar). This has been accomplished with two different photoreactive moieties in position 33 of CCK (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 10Hadac E.M. Ji Z.S. Pinon D.I. Henne R.M. Lybrand T.P. Miller L.J. J. Med. Chem. 1999; 42: 2105-2111Crossref PubMed Scopus (49) Google Scholar) and with a benzophenone moiety in position 29 of CCK (9Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R. Lybrand T. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The position 33 photoprobes covalently labeled receptor residue Trp39 just above the first transmembrane segment. The position 29 photoprobe established a covalent bond with receptor residues His347 and Leu348 just above the seventh transmembrane segment. It is noteworthy that both of these represented focused contacts with a single receptor domain, as might be expected from the high affinity tight interaction between native agonist ligand and the ligand-binding domain of the receptor.In contrast, in the present work, the Bpa residue in position 24 of CCK established covalent bonds to either of two distinct domains of the CCK receptor. This probe was fully characterized as a high affinity ligand that had full efficacy relative to natural CCK. While one of the covalently labeled receptor domains was within a region that is known to be important, the third extracellular loop, it is notable that this contact was lost when the amino terminus of the receptor was shortened by truncation. The second labeled domain was a portion of the amino-terminal tail of the CCK receptor that can be eliminated by truncation, without any detrimental effects on CCK binding or agonist-stimulated signaling. This contact is therefore not critical for ligand affinity or function.These spatial approximations, however, provide the basis to postulate that the docked peptide is tucked under the protective cover of the amino terminus of the CCK receptor. This function was further supported by the demonstration that this domain protected the receptor from extracellular proteolytic attack. Eliminating this region of the receptor by truncation had no detrimental effect on ligand binding affinity or agonist-stimulated signaling, but resulted in a more labile receptor that was much more sensitive to proteolysis. The glycosylation of the amino-terminal domain of the CCK receptor is probably most responsible for this resistance to proteolysis. This is a recognized function of the glycosylation of membrane proteins (35Pang R.T.K. Ng S.S.M. Cheng C.H.K. Holtmann M.H. Miller L.J. Chow B.K.C. Endocrinology. 1999; 140: 5102-5111Crossref PubMed Scopus (37) Google Scholar, 36Ho H.H. Gilbert M.T. Nussenzveig D.R. Gershengorn M.C. Biochemistry. 1999; 38: 1866-1872Crossref PubMed Scopus (43) Google Scholar). Another established function for membrane protein glycosylation relates to assisting in solubility and folding during biosynthesis that is also likely relevant to the CCK receptor.Although position 24 in the CCK ligands is external to the established pharmacophore, cross-linking data generated by substitution of a photolabile residue at this position provides extremely useful constraint data for three-dimensional model building studies. The results reported here provide clear evidence that our earlier decision to position the amino terminus of the ligand facing away from receptor binding site (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 10Hadac E.M. Ji Z.S. Pinon D.I. Henne R.M. Lybrand T.P. Miller L.J. J. Med. Chem. 1999; 42: 2105-2111Crossref PubMed Scopus (49) Google Scholar) was appropriate. The photoaffinity labeling data for the ligand amino terminus, combined with ligand binding studies and cross-linking data for positions within the peptide pharmacophore, also enable us for the first time to propose a distinct topological model for the peptide ligand-CCK receptor complex. Based on the full set of photoaffinity labeling and ligand binding data now available, it appears likely that the CCK receptor possesses a counterclockwise helix bundle topology. More definitive biophysical data will be needed to ultimately verify this prediction.With this refinement in our working model, the tyrosine-sulfate residue in position 27 of CCK that has been shown to be so important in structure-activity studies (37Huang S.C., Yu, D.H. Wank S.A. Mantey S. Gardner J.D. Jensen R.T. Peptides. 1989; 10: 785-789Crossref PubMed Scopus (63) Google Scholar) is now in direct contact with Arg197. These residues, therefore, represent potential partners for charge-charge interaction. Indeed, Arg197 is one of only three basic residues in the extracellular domain of the CCK receptor that has been reported to have a marked negative impact on CCK binding when replaced with an Ala residue (38Gouldson P. Legoux P. Carillon C. Dumont X. Le Fur G. Ferrara P. Shire D. Eur. J. Pharmacol. 2000; 389: 115-124Crossref PubMed Scopus (19) Google Scholar).Thus, using an analogue of CCK with a photolabile Bpa moiety in position 24, we have learned much about the binding domain for this hormone within its receptor. While the pharmacophoric domain of the peptide is held in constant approximation with the regions of the CCK receptor close to the membrane, the amino-terminal extension from the pharmacophore likely moves further away from the bilayer and is overlaid by a region of the receptor amino terminus that has no direct effect on binding affinity or on biological activity. Instead, this region of the receptor serves a protective function over the more critical domain below. The distinct positions of covalent labeling with this probe also provide additional detail to refine the molecular model of the docked peptide agonist. This evolving model now best supports a counterclockwise helical bundle topology for the receptor and residue approximations that are fully consistent with all existing experimental data.AddendumSince submission and review of this manuscript, a preliminary crystal structure for bovine rhodopsin has appeared Palczewski, 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., and Miyano, M. (2000) Science 289,739–745 that exhibits a counterclockwise helix bundle topology. Since the CCK receptor is also a member of the rhodopsin β-adrenergic receptor family, it is quite likely that the helix bundle topology will be similar. Guanine nucleotide-binding protein (G protein)1-coupled receptors represent a remarkable group of structurally homologous membrane proteins that can bind and be activated by widely diverse ligands. The molecular details of how ligands as structurally dissimilar as photons, biogenic amines, peptides, and glycoproteins can elicit similar conformational changes in the cytosolic face of their receptors (where G protein-coupling occurs) are far from clear. Our best understanding of this process relates to the smallest ligands that appear to bind within the confluence of helices within the lipid bilayer (1Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar), where we have analogous low resolution crystal structures on which to rely (2Grigorieff N. Ceska T.A. Downing K.H. Baldwin J.M. Henderson R. J. Mol. Biol. 1996; 259: 393-421Crossref PubMed Scopus (868) Google Scholar,3Unger V.M. Hargrave P.A. Baldwin J.M. Schertler G.F. Nature. 1997; 389: 203-206Crossref PubMed Scopus (480) Google Scholar). As the ligands get larger and more structurally complex, the binding domains tend to move toward the extracellular face of the membrane, with extracellular loop and amino-terminal tail domains becoming more important (1Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 4Schwartz T.W. Curr. Opin. Biotechnol. 1994; 5: 434-444Crossref PubMed Scopus (280) Google Scholar). These are receptor domains for which we have minimal meaningful structural data. We have been quite interested in the molecular basis of ligand binding to the type A cholecystokinin (CCK) receptor (5Pearson R.K. Miller L.J. J. Biol. Chem. 1987; 262: 869-876Abstract Full Text PDF PubMed Google Scholar, 6Powers S.P. Fourmy D. Gaisano H. Miller L.J. J. Biol. Chem. 1988; 263: 5295-5300Abstract Full Text PDF PubMed Google Scholar, 7Klueppelberg U.G. Gaisano H.Y. Powers S.P. Miller L.J. Biochemistry. 1989; 28: 3463-3468Crossref PubMed Scopus (23) Google Scholar, 8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R. Lybrand T. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 10Hadac E.M. Ji Z.S. Pinon D.I. Henne R.M. Lybrand T.P. Miller L.J. J. Med. Chem. 1999; 42: 2105-2111Crossref PubMed Scopus (49) Google Scholar). This receptor is a member of the class I family of G protein-coupled receptors, along with rhodopsin and the β-adrenergic receptor (11Kolakowski L.F. Receptors and Channels. 1994; 2: 1-7PubMed Google Scholar). CCK occurs as a series of linear peptides, having lengths ranging from 8 to 58 residues (12Mutt V. Glass G.B.J. Cholecystokinin: Isolation, Structure, and Functions. Raven Press, Ltd., New York1980: 169-221Google Scholar). These all share their carboxyl-terminal domain, with the carboxyl-terminal heptapeptide-amide representing the minimal region that has full potency and efficacy for stimulating targets of this hormone. Two molecular approximations have been experimentally determined for residues within this region of CCK and residues within the ligand-binding domain of this receptor (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Chem. 1997; 272: 24393-24401Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Hadac E.M. Pinon D.I. Ji Z. Holicky E.L. Henne R. Lybrand T. Miller L.J. J. Biol. Chem. 1998; 273: 12988-12993Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 10Hadac E.M. Ji Z.S. Pinon D.I. Henne R.M. Lybrand T.P. Miller L.J. J. Med. Chem. 1999; 42: 2105-2111Crossref PubMed Scopus (49) Google Scholar). Extension or structural modification of the amino terminus of CCK-8 has been well tolerated, without interfering with receptor binding or activation (13Ondetti 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,14Pearson R.K. Powers S.P. Hadac E.M. Gaisano H. Miller L.J. Biochem. Biophys. Res. Commun. 1987; 147: 346-353Crossref PubMed Scopus (25) Google Scholar). One provocative report has recently suggested that the naturally occurring amino-terminal extension that is present in CCK-58 can affect the conformation of the biologically active carboxyl-terminal octapeptide and can have a positive effect on the action of this hormone (15Keire D.A. Solomon T.E. Reeve Jr., J.R. Biochem. Biophys. Res. Commun. 1999; 266: 400-404Crossref PubMed Scopus (15) Google Scholar). With these observations in mind, we initiated the current group of studies. We were particularly interested in the structural details of how an amino-terminal extension from the CCK pharmacophore might be positioned relative to the CCK receptor. This work has given us new insights into the structure and function of the amino-terminal tail of this receptor. Unlike affinity labeling through photolabile residues that are positioned within the pharmacophore of CCK that have yielded only focused sites of covalent attachment to the receptor (8Ji Z.S. Hadac E.M. Henne R.M. Patel S.A. Lybrand T.P. Miller L.J. J. Biol. Ch" @default.
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• W2052667701 title "Refinement of the Structure of the Ligand-occupied Cholecystokinin Receptor Using a Photolabile Amino-terminal Probe" @default.
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