title: Direct demonstration of unique mode of natural peptide binding to the type 2 cholecystokinin receptor using photoaffinity labeling.
abstract: Direct analysis of mode of peptide docking using intrinsic photoaffinity labeling has provided detailed insights for the molecular basis of cholecystokinin (CCK) interaction with the type 1 CCK receptor. In the current work, this technique has been applied to the closely related type 2 CCK receptor that also binds the natural full agonist peptide, CCK, with high affinity. A series of photolabile CCK analog probes with sites of covalent attachment extending from position 26 through 32 were characterized, with the highest affinity analogs that possessed full biological activity utilized in photoaffinity labeling. The position 29 probe, incorporating a photolabile benzoyl-phenylalanine in that position, was shown to bind with high affinity and to be a full agonist, with potency not different from that of natural CCK, and to covalently label the type 2 CCK receptor in a saturable, specific and efficient manner. Using proteolytic peptide mapping, mutagenesis, and radiochemical Edman degradation sequencing, this probe was shown to establish a covalent bond with type 2 CCK receptor residue Phe¹²⁰ in the first extracellular loop. This was in contrast to its covalent attachment to Glu³⁴⁵ in the third extracellular loop of the type 1 CCK receptor, directly documenting differences in mode of docking this peptide to these receptors.
authors.affiliation: Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Scottsdale, Scottsdale, AZ 85259, USA.
authors.forename: Maoqing
authors.initials: M
authors.lastname: Dong
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D002766:Cholecystokinin / Q000737:chemistry / Q000378:metabolism*; D003412:Cricetulus; D006801:Humans; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D019920:Photoaffinity Labels; D011485:Protein Binding; D043887:Receptor, Cholecystokinin B / Q000378:metabolism*
chemical_list: D019920:Photoaffinity Labels; D043887:Receptor, Cholecystokinin B; D010649:Phenylalanine; D002766:Cholecystokinin; C053764:benzoylphenylalanine
keywords: Cholecystokinin; G protein-coupled receptors; Ligand binding; Photoaffinity labeling; Type 2 cholecystokinin receptor