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Volume 272, Number 39, Issue of September 26, 1997 pp. 24393-24401
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Direct Identification of a Distinct Site of Interaction between the Carboxyl-terminal Residue of Cholecystokinin and the Type A Cholecystokinin Receptor Using Photoaffinity Labeling

(Received for publication, June 3, 1997, and in revised form, July 18, 1997)

Zongshi Ji , Elizabeth M. Hadac , Randel M. Henne ^§ , Sunil A. Patel ^§ , Terry P. Lybrand ^§ and Laurence J. Miller

From the  Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905 and the ^§ University of Washington, Center for Bioengineering, Seattle, Washington 98195-1750

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-[Nle^28,31,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.

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