Role of the extracellular domains of the cholecystokinin receptor in agonist binding.
The cholecystokinin (CCK) receptor types A and B (CCKAR and CCKBR) are G protein-coupled receptors with approximately 50% amino acid identity; both have high affinity for the sulfated CCK octapeptide (CCK-8), whereas only the CCKBR has high affinity for gastrin. Previously, we identified five amino acids in the second extracellular loop (ECL) of the CCKBR that were essential for gastrin selectivity. Subsequent mutagenesis of one of these five amino acids (H207F) resulted in the loss of radiolabeled CCK-8 binding. CCK-8 stimulated total inositol phosphate accumulation in COS-1 cells transiently expressing the CCKBR-H207F with full efficacy and a 3044-fold reduced potency, which suggests that the loss of radioligand binding was caused by a loss in affinity. Alanine scanning mutagenesis was performed on the amino terminus near the top of transmembrane domain I (TMI) and on ECL1, two extracellular domains implicated in ligand binding by previous mutagenesis studies. 125I-Bolton-Hunter-CCK-8 binding to mutant receptors transiently expressed in COS-1 identified one nonconserved amino acid, R57A, at the top of TMI that caused a 21-fold reduction in CCK-8 affinity and four conserved amino acids, N115A, L116A, F120A and F122A, in the ECL1 that caused a 15.6-, 6-, 440-, and 8-fold reduction in affinity or efficacy. Alanine substitution of the equivalent amino acids in the CCKAR corresponding to each of the five amino acids in ECL1 and ECL2 affecting CCK-8 affinity for the CCKBR revealed only two mutations, L103A and F107A, that decreased CCK-8 affinity (68- and 2885-fold, respectively). These data suggest that CCK-8 interacts at multiple contact points in the extracellular domains of CCK receptors and that the CCKAR and CCKBR have distinct binding sites despite their shared high affinity for CCK-8.