Single amino acid substitution of serine82 to asparagine in first intracellular loop of human cholecystokinin (CCK)-B receptor confers full cyclic AMP responses to CCK and gastrin

To understand molecular basis of Gs coupling to cholecystokinin (CCK)-A and CCK-B receptor subtypes, we examined cAMP responses in three sets of human CCK receptor mutants expressed in human embryonic kidney (HEK)293 cells. S ingle or double substitutions of the four nonconserved amino acids in the f irst intracellular loop of the CCK-BR were made with their CCK-AR counterpa rts to determine which residues are critical in Gs coupling. Single substit ution of Ser82 to Asn, produced maximal cAMP responses comparable with the chimeric CCK-BR containing the entire first intracellular loop of the CCK-A R. Two other single substitutions, Leu81 to Arg and Leu85 to Met, produced significant but smaller cAMP responses. Ser82 was further changed into Asp, Thr, or Ala to determine the specificity of this position in Gs coupling b y the CCK-BR. Replacements of Ser to Asp or Thr showed significant cAMP inc reases but the stimulatory effects were smaller than Ser to Asn, whereas Se r to Ala did not enhance any cAMP response to either CCK or gastrin. Finall y, CCK-AR reverse mutants were studied to compare them with their correspon ding CCK-BR mutants that showed increased cAMP responses. Substitution of C CK-AR residue Arg68 to Leu resulted in a complete loss of cAMP response, wh ereas Asn69 to Ser or Met72 to Leu showed markedly diminished cAMP response s. These data identify that specific residues in the first intracellular lo op of both CCK receptor subtypes are critical for Gs coupling. Substitution of a single residue Ser82 to Asn in the CCK-BR is sufficient to confer ful l cAMP responses to agonist stimulation.