Key amino acids of the cholecystokinin (CCK) peptide for receptor binding are sulfated Y27, W30, D32, and F33-NH2. Three-dimensional modeling showed that the CCK-A receptor (CCK-AR) antagonist devazepide penetrated into the transmembrane (TM) domains, whereas CCK was placed on the surface of the CCK-AR. Four types of rat CCK-AR cDNAs were transfected into CHO-K1 and COS-7 cells: normal CCK-AR cDNA transfected cells (wild type, WT); K120 substituted with V; K130V; and R352V. Binding of [H-3]CCK-8 was observed in WT and K130V, but not in K120V and R352V. CCK caused Ca2+ spiking in WT and K130V, whereas K120V and R352V had no effect. Three chimeras including the CCK-AR/3beta2 adrenergic receptor (beta2AR), 3Nibeta2AR, and 3Cibeta2AR were constructed. Two groups of point mutations in the CCK-AR3i were also made: Y252V, S274V, S281V, and S289V (non-phospho-acceptor Y or S); S260V, S264V, S271V, and S275V (phospho-acceptor S). WT and CCK-AR/ 3Cibeta2AR increased [Ca2+](i) in response to CCK; 3Nibeta2AR was vice versa. CCK failed to increase [IP3] in phospho-acceptor S to V without affecting binding. Non-phospho-acceptor S or Y to V showed normal response. Thus, Lys 120 outside the TM2 and Arg352 outside the TM6 of the CCK-AR are amino acids interacting with Tyr[SO3H]27 and Asp32 of the CCK peptide for binding. Phospho-acceptor Ser groups in the CCK-AR 3Ni are amino acids for initiating cell signaling. (C) 2003 Elsevier Inc. All rights reserved.