FIRST INTRACELLULAR LOOP OF THE HUMAN CHOLECYSTOKININ-A RECEPTOR IS ESSENTIAL FOR CYCLIC-AMP SIGNALING IN TRANSFECTED HEK-293 CELLS

Cholecystokinin (CCK)-A and CCK-B receptors are highly homologous memb ers of the seven transmembrane domain G-protein-coupled receptor super family. Genes of both receptors contain five exons and share a similar exon-intron organization. To determine the structural basis of CCK-A receptor (CCK-AR) functionally coupled to G(s), a series of chimeric m utants were constructed by replacing exons of human CCK-B receptor (CC R-BR), from the second to the fifth (last) exon, with human CCK-AR cou nterparts. Binding and signal transduction properties of wild-type and chimeric receptors were examined in stably transfected HEK-293 cells, Chimeric receptors that maintained high affinity binding to CCK exhib ited dose-dependent increases in intracellular calcium mobilization si milar to both wildtype receptors. However; only the wild-type CCK-AR a nd chimeric mutants containing the second exon of CCK-AR were able to mediate significantly greater in: creases in intracellular cAMP conten t and adenylyl cyclase activity compared with wild-type CCK-BR, A CCK- BR mutant was further constructed by replacing five amino acids, Gly-L eu-Ser-Arg-(Arg)-Leu, in the first intracellular loop with the corresp onding five CCK-AR specific amino acids, Ile-Arg-Asn-Lys-(Arg)-Met. Th e resultant receptor maintained high affinity binding to both CCK and gastrin and dose-dependent calcium responses similar to wild-type CCK- BR. However, this first intracellular loop mutant also gained positive cAMP responses to both sulfated CCK-8 and gastrin-17 with EC(50) valu es of 8.5 +/- 1 nm and 23 +/- 7 nnn, respectively. These data suggest that the first intracellular loop of CCK-AR is essential for coupling to G(s) and activation of adenylyl cyclase signal transduction cascade .