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 members of the seven transmembrane domain G-protein-coupled receptor superfamily. 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 Gs, a series of chimeric mutants were constructed by replacing exons of human CCK-B receptor (CCK-BR), from the second to the fifth (last) exon, with human CCK-AR counterparts. 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 exhibited dose-dependent increases in intracellular calcium mobilization similar to both wild-type receptors. However, only the wild-type CCK-AR and chimeric mutants containing the second exon of CCK-AR were able to mediate significantly greater increases in intracellular cAMP content and adenylyl cyclase activity compared with wild-type CCK-BR. A CCK-BR mutant was further constructed by replacing five amino acids, Gly-Leu-Ser-Arg-(Arg)-Leu, in the first intracellular loop with the corresponding five CCK-AR specific amino acids, Ile-Arg-Asn-Lys-(Arg)-Met. The 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 EC50 values of 8.5 +/- 1 nM and 23 +/- 7 nM, respectively. These data suggest that the first intracellular loop of CCK-AR is essential for coupling to Gs and activation of adenylyl cyclase signal transduction cascade.
Wu
V
V
CURE/Digestive Diseases Research Center, Division of Digestive Diseases, Department of Medicine, UCLA School of Medicine, and West Los Angeles Veterans Administration Medical Center, Los Angeles, California 90073, USA.vwu@ucla.edu
Yang
M
M
McRoberts
J A
JA
Ren
J
J
Seensalu
R
R
Zeng
N
N
Dagrag
M
M
Birnbaumer
M
M
Walsh
J H
JH
D000262:Adenylyl Cyclases / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D005755:Gastrins / Q000008:administration & dosage / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D012844:Sincalide / Q000008:administration & dosage / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D005755:Gastrins; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D012844:Sincalide