Arginine 197 of the cholecystokinin-A receptor binding site interacts withthe sulfate of the peptide agonist cholecystokinin

The knowledge of the binding sites of G protein-coupled cholecystokinin rec eptors represents important insights that may serve to understand their act ivation processes and to design or optimize ligands. Our aim was to identif y the amino acid of the cholecystokinin-A receptor (CCK-AR) binding site in an interaction with the sulfate of CCK, which is crucial for CCK binding a nd activity. A three-dimensional model of the [CCK-AR-CCK] complex was buil t. In this model, Arg197 was the best candidate residue for a ionic interac tion with the sulfate of CCK, Arg197 was exchanged for a methionine by site -directed mutagenesis. Wild-type and mutated CCK-AR were transiently expres sed in COS-7 cells for pharmacological and functional analysis. The mutated receptor on Arg197 did not bind the agonist radioligand I-125- BH-[Thr, Nl e]-CCK-9: however, it bound the nonpeptide antagonist [H-3]-SR27,897 as the wild-type receptor. The mutant was congruent to 1,470- and 3,200-fold less potent than the wild-type CCK-AR to activate G proteins and to induce inos itol phosphate production, respectively. This is consistent with the 500-fo ld lower potency and 800-fold lower affinity of nonsulfated CCK relative to sulfated CCK on the wild-type receptor. These data, together with those sh owing that the mutated receptor failed to discriminate nonsulfated and sulf ated CCK while it retained other pharmacological features of the CCK-ARI st rongly support an interaction between Arg197 of the CCK-AR binding site and the sulfate of CCK. In addition, the mutated CCK-AR resembled the low affi nity state of the wild-type CCK-AR, suggesting that Arg197-sulfate interact ion regulates conformational changes of the CCK-AR that are required for it s physiological activation.