Cloning and expression of the rabbit gastric CCK-A receptor.
Cholecystokinin stimulates pancreatic zymogen secretion by binding with high affinity to a receptor on the pancreatic acinar cell. This receptor has been cloned and shown to be a CCK-A subtype. CCK also stimulates pepsinogen secretion from the gastric chief cell with high affinity. Using polymerase chain reaction with primers from the known sequence of the rat pancreatic CCK-A receptor cDNA, we prepared a 600 bp product from rat and rabbit stomach cDNA. From Southern analysis these represented a fragment of a gastric CCK-A receptor. PCR was then used to amplify a rabbit lambda ZAP II gastric epithelial cDNA library with the same primers, and the product was identified by sequencing as representing a CCK-A receptor fragment. When this PCR product was used to screen the library, ten positive clones were identified in a screening of 4.10(5) plaques, and several of these were sequenced. All had essentially the same sequence contained within 2 of these clones consisted of 427 amino acids and was 92% homologous (87% identity) to the known rat pancreatic CCK-A sequence but only 43% homologous to the gastric CCK-B sequence. The cDNA was subcloned into a pcDNA1 expression vector and transiently expressed in the human embryonic kidney cell line, HK 293. The responses of intracellular Ca2+ in these transfected cells to CCK and gastrin were monitored using video imaging. On the average 40% of the cells responded to CCK-8 by a transient elevation of [Ca2+]i followed by a steady state plateau. CCK was a high and gastrin a low affinity ligand for this signal, corresponding to the actions of these ligands on pepsinogen secretion from chief cells and somatostatin release from D cells. Hence from sequence and second messenger responses, the clone represents the CCK-A receptor presumably responsible for pepsinogen secretion by gastric chief cells and somatostatin release from gastric D cells.
Reuben
M
M
Department of Medicine, Wadsworth VA Hospital, Los Angeles, CA 90073.
Rising
L
L
Prinz
C
C
Hersey
S
S
Sachs
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D005755:Gastrins / Q000494:pharmacology; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012844:Sincalide / Q000494:pharmacology; D013270:Stomach / Q000737:chemistry; D014162:Transfection
D017931:DNA Primers; D005755:Gastrins; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin; D012844:Sincalide