TY - JOUR T1 - Development of a Highly Selective Allosteric Antagonist Radioligand for the Type 1 Cholecystokinin Receptor and Elucidation of its Molecular Basis of Binding JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.114.095430 SP - mol.114.095430 AU - Maoqing Dong AU - Ashton M. Vattelana AU - Polo C.H. Lam AU - Andrew Orry AU - Ruben Abagyan AU - Arthur Christopoulos AU - Patrick M. Sexton AU - David R. Haines AU - Laurence J. Miller Y1 - 2014/10/15 UR - http://molpharm.aspetjournals.org/content/early/2014/10/15/mol.114.095430.abstract N2 - Understanding the molecular basis of ligand binding to receptors provides insights useful for rational drug design. This work describes development of a new antagonist radioligand (T-0632) of the type 1 cholecystokinin receptor (CCK1R) and exploration of the molecular basis of its binding. This radioligand bound specifically with high affinity within an allosteric pocket of CCK1R. T-0632 fully inhibited binding and action of CCK at this receptor, while exhibiting no saturable binding to closely-related CCK2R. Chimeric CCK1R/CCK2R constructs were used to explore the molecular basis of T-0632 binding. Exchanging exonic regions revealed functional importance of CCK1R exon 3, extending from bottom of TM3 to top of TM5, including portions of the intramembranous pocket as well as ECL2. However, CCK1R mutants in which each residue facing the pocket was changed to that present in CCK2R had no negative impact on T-0632 binding. Extending the chimeric approach to ECL2 established importance of its carboxyl-terminal region, and site-directed mutagenesis of each non-conserved residue in this region revealed importance of Ser208 at the top of TM5. A molecular model of T-0632-occupied CCK1R was consistent with these experimental determinants, also identifying Met121 in TM3 and Arg336 in TM6 as important. While these residues are conserved in CCK2R, mutating them had distinct impact on the two closely-related receptors, suggesting differential orientation. This establishes the molecular basis of binding a highly selective non-peptidyl allosteric antagonist of CCK1R, illustrating differences in docking that extend beyond determinants attributable to distinct residues lining the intramembranous pocket in the two receptor subtypes. ER -