Arginine 336 and asparagine 333 of the human cholecystokinin-A receptor binding site interact with the penultimate aspartic acid and the C-terminal amide of cholecystokinin

The cholecystokinin-A receptor (CCK-AR) is a G protein-coupled receptor tha t mediates important central and peripheral cholecystokinin actions. Residu es of the CCK-AR binding site that interact with the C-terminal part of CCK that is endowed with biological activity are still unknown. Here we report on the identification of Arg-336 and Asn-333 of CCK-AR, which interact wit h the Asp-8 carboxylate and the C-terminal amide of CCK-9, respectively. Id entification of the two amino acids was achieved by dynamics-based docking of CCK in a refined three-dimensional model of CCK-AR using, as constraints , previous results that demonstrated that Trp-39/Gln-40 and Met-195/Arg-197 interact with the N terminus and the sulfated tyrosine of CCK, respectivel y. Arg-336-Asp-8 and Asn-333-amide interactions were pharmacologically asse ssed by mutational exchange of Arg-336 and Asn-333 in the receptor or recip rocal elimination of the partner chemical functions in CCK, This study also allowed us to demonstrate that (i) the identified interactions are crucial for stabilizing the high affinity phospholipase C-coupled state of the CCK -AR CCK complex, (ii) Arg-336 and Asn-333 are directly involved in interact ions with nonpeptide antagonists SR-27,897 and L-364,718, and (iii) Arg-336 but not Asn-333 is directly involved in the binding of the peptide antagon ist JMV 179 and the peptide partial agonist JMV 180, These data will be use d to obtain an integrated dynamic view of the molecular processes that link agonist binding to receptor activation.