Abstract
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 (Ki = 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 Mr = 85,000-95,000 plasma membrane glycoprotein with core of Mr = 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.
Highlights
¶ To whom correspondence and reprint requests should be addressed: Center for Basic Research in Digestive Diseases, Guggenheim 17, Mayo Clinic, Rochester, MN 55905
Most of our insights into binding domains for peptide ligands have come from receptor mutagenesis studies, which have focused attention on receptor domains predicted to be outside the membrane [7, 8]
This comes from both receptor mutagenesis studies and from direct photoaffinity labeling of receptor domains and residues [1,2,3,4,5,6]
Summary
Contributions by an unusual tyrosine sulfate and the carboxylterminal phenylalanine-amide, and important contributions by almost every residue within this domain. We have utilized photoaffinity labeling with a CCK analogue probe that incorporates a photolabile residue intrinsic to its pharmacophoric domain to identify a distinct site of interaction with the receptor. This was achieved by the application of chemical and enzymatic cleavage of the affinity labeled receptor, with the subsequent manual Edman degradation and radiochemical sequencing of the labeled fragment. A domain within this receptor, which was previously shown to be important by receptor truncation and mutagenesis studies, does contain the residue we identify as interacting with the carboxyl terminus of CCK. We present a new model that incorporates this new contact data and is consistent with a large body of agonist structure-activity relationship data for CCK A receptors
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