TY - JOUR
T1 - Direct identification of a distinct site of interaction between the carboxyl-terminal residue of cholecystokinin and the type A cholecystokinin receptor using photoaffinity labeling
AU - Ji, Zongshi
AU - Hadac, Elizabeth M.
AU - Henne, Randel M.
AU - Patel, Sunil A.
AU - Lybrand, Terry P.
AU - Miller, Laurence J.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1997/9/26
Y1 - 1997/9/26
N2 - Mechanisms of ligand binding and activation of G protein-coupled receptors are particularly important, due to their ubiquitous expression and potential as drug targets. Molecular interactions between ligands and these receptors are best defined for small molecule ligands that bind within the transmembrane helices. Extracellular domains seem to be more important for peptide ligands, based largely on effects of receptor mutagenesis, where interference with binding or activity can reflect allosteric as well as direct effects. We now take the more direct approach of photoaffinity labeling the active site of the cholecystokinin (CCK) receptor, using a photolabile analogue of CCK having a blocked amino terminus. This probe, 125I-desaminotyrosyl-Gly-[Nle28,31,pNO2-Phe33]CCK-(26-33), binds specifically, saturably, and with high affinity (K(i) = 3.3 nM) and has full agonist activity. This makes likely its being sited in a natural position within the receptor. As substrate, we used CHO-CCK receptor cells overexpressing functional recombinant rat type A CCK receptor. Covalent labeling of the appropriate M(r) = 85,000-95,000 plasma membrane glycoprotein with core of M(r) = 42,000 was established by SDS-polyacrylamide gel electrophoresis and autoradiography. A single domain adjacent to transmembrane 1 was labeled, as established by cyanogen bromide cleavage and separation by gel and/or high pressure liquid chromatography. The site of interaction was further defined by additional proteolysis with trypsin, with purification of the labeled fragment, followed by manual Edman degradation and radiochemical sequencing. This demonstrated that Trp39 was specifically labeled and likely resides proximate to the carboxyl-terminal pNO2-Phe33 residue of the probe. A model of this ligand-bound receptor has been constructed and will be used to plan future experiments to refine our understanding of this interaction.
AB - Mechanisms of ligand binding and activation of G protein-coupled receptors are particularly important, due to their ubiquitous expression and potential as drug targets. Molecular interactions between ligands and these receptors are best defined for small molecule ligands that bind within the transmembrane helices. Extracellular domains seem to be more important for peptide ligands, based largely on effects of receptor mutagenesis, where interference with binding or activity can reflect allosteric as well as direct effects. We now take the more direct approach of photoaffinity labeling the active site of the cholecystokinin (CCK) receptor, using a photolabile analogue of CCK having a blocked amino terminus. This probe, 125I-desaminotyrosyl-Gly-[Nle28,31,pNO2-Phe33]CCK-(26-33), binds specifically, saturably, and with high affinity (K(i) = 3.3 nM) and has full agonist activity. This makes likely its being sited in a natural position within the receptor. As substrate, we used CHO-CCK receptor cells overexpressing functional recombinant rat type A CCK receptor. Covalent labeling of the appropriate M(r) = 85,000-95,000 plasma membrane glycoprotein with core of M(r) = 42,000 was established by SDS-polyacrylamide gel electrophoresis and autoradiography. A single domain adjacent to transmembrane 1 was labeled, as established by cyanogen bromide cleavage and separation by gel and/or high pressure liquid chromatography. The site of interaction was further defined by additional proteolysis with trypsin, with purification of the labeled fragment, followed by manual Edman degradation and radiochemical sequencing. This demonstrated that Trp39 was specifically labeled and likely resides proximate to the carboxyl-terminal pNO2-Phe33 residue of the probe. A model of this ligand-bound receptor has been constructed and will be used to plan future experiments to refine our understanding of this interaction.
UR - http://www.scopus.com/inward/record.url?scp=0030846864&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030846864&partnerID=8YFLogxK
U2 - 10.1074/jbc.272.39.24393
DO - 10.1074/jbc.272.39.24393
M3 - Article
C2 - 9305898
AN - SCOPUS:0030846864
SN - 0021-9258
VL - 272
SP - 24393
EP - 24401
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 39
ER -