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. Guanine nucleotide-binding protein (G protein) 1 -coupled receptors are the largest group of plasma membrane receptors, representing a superfamily with a remarkable diversity of activating ligands. Our best understanding of the molecular basis for ligand binding to members of this superfamily is the binding of the chromophore to rhodopsin and the binding of biogenic amines to adrenergic receptors. These insights come from complementary studies of receptor mutagenesis, photoaffinity labeling, and reciprocal chemical modification of ligand and receptor (1-6). All available data focus the relevant interactions to sites at the core of the coalescence of transmembrane helices, in the outer third of the bilayer. Even with this extensive information, the constrained nature of the ligands, and the relatively confined space for ligand docking, the debate continues regarding the specific siting of the agonist ligands in some of these receptor systems (7,8).Understanding the interactions between peptide ligands and their G protein-coupled receptors represents an even greater challenge. By first principles, these ligands are quite flexible and can achieve many conformations. Whereas some peptides appear to have some preferred conformation in solution (9), there is little information regarding how such structures relate to the receptor-bound states of these ligands. 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). Given the extended size of the pharmacophoric domains and the solubilities of the peptide ligands, these regions of interaction seem plausible. We know, however, that receptor mutagenesis can modify receptor function nonspecifically, interfering with biosynthetic processing or trafficking or having an allosteric effect, rather than necessarily directly interfering with a site of ligand-receptor interaction. For a very limited number of peptide receptors in this family, direct sites of contact have been recently described using photoaffinity labeling approaches (10 -13).Cholecystokinin (CCK) is a peptide hormone and neurotransmitter that has a wide spectrum of physiologic actions (14). These relate largely to control of nutrient assimilation, through regulation of...

Numerous techniques have been used to elucidate the structural basis for interaction of cholecystokinin (CCK)-related peptides with their hormone-binding receptor, the CCK-A receptor (CCK-AR), including structure-activity relationship studies, site-directed mutagenesis, photoaffinity-labeling, and solution NMR analysis of both CCK peptide ligands and peptide fragments derived from the CCK-A receptor. Different structural models have been developed for the peptide-receptor complexes using various subsets of the available experimental data (Giragossian & Mierke 2001;Ding et al. 2002;Escrieut et al. 2002). Here, we review details of the various models and evaluate the impact of selected experimental data sets on model development.