MET-195 OF THE CHOLECYSTOKININ-A RECEPTOR INTERACTS WITH THE SULFATEDTYROSINE OF CHOLECYSTOKININ AND IS CRUCIAL FOR RECEPTOR TRANSITION TOHIGH-AFFINITY STATE

Sulfation of the tyrosine at the seventh position from the C terminus of cholecystokinin (CCK) is crucial for CCK binding to the CCK A recep tor. Using three-dimensional modeling, we identified methionine 195 of the CCK-A receptor as a putative amino acid in interaction with the a romatic ring of the sulfated tyrosine of CCK. We analyzed the role pla yed by the two partners of this interaction, The exchange of Met-195 f or a leucine caused a minor decrease (2.8-fold) on the affinity of the high affinity sites for sulfated CCK-9, a strong drop (73%) of their number, and a 30-fold decrease on the affinity of the Bow and very low affinity sites for sulfated CCK-9, with no change in their number, Th e mutation also caused a 54-fold decrease of the potency of the recept or to induce inositol phosphates production The high affinity sites of the wild-type CCK-A receptor were highly selective (800-fold) toward sulfated versus nonsulfated CCK, whereas low and very low affinity sit es were poorly selective (PO-and 18-fold). in addition, the M195L muta nt bound, and responded to, sulfated CCK analogues with decreased affi nities and potencies, whereas it bound and responded to nonsulfated CC K identically to the wild-type receptor. Thus, Met-195 interacts with the aromatic ring of the sulfated tyrosine to correctly position the s ulfated group of CCK in the binding site of the receptor. This interac tion is essential for CCK-dependent transition of the CCK-A receptor t o a high affinity state. Our data should represent an important step t oward the identification of the residue(s) of the receptor in interact ion with the sulfate moiety of CCK and the understanding of the molecu lar mechanisms that govern CCK-A receptor activation.