Purification, molecular cloning, and functional expression of the cholecystokinin receptor from rat pancreas.

Abstract

The cholecystokinin (CCK) family of peptides and their receptors are widely distributed throughout the gastrointestinal and central nervous systems where they regulate secretion, motility, growth, anxiety, and satiety. The CCK receptors can be subdivided into at least two subtypes, CCKA and CCKB on the basis of pharmacological studies. We report here the purification of the CCKA receptor to homogeneity from rat pancreas by using ion-exchange and multiple affinity chromatographic separations. This allowed partial peptide sequencing after chemical/enzymatic ceavage and synthesis of degenerate oligonucleotide primers. These primers were used for initial cloning of the cDNA from rat pancreas by PCR. The predicted protein sequence ofthe cDNA clone contained the five partial peptide sequences obtained from the purified protein.Seven putative transmembrane domains suggest its membership in the guanine nucleotide-binding regulatory proteincoupled receptor superfamily. In vitro transcripts of the cDNA clone were functionally expressed in Xenopus oocytes and displayed the expected agonist and antagonist specificity.The cholecystokinin (CCK) family of peptides was originally isolated from the mammalian gastrointestinal tract (1) and was one of the first gastrointestinal peptides to be discovered in the brain (2). Their receptors appear throughout the gastrointestinal and nervous systems and can be pharmacologically classified into two subtypes, CCKA and CCKB (3). CCKA receptors mediate physiologic gallbladder contraction, pancreatic growth and enzyme secretion, delayed gastric emptying, relaxation of the sphincter of Oddi, and potentiation of insulin secretion (3). The CCKA type receptor also appears in the anterior pituitary, the myenteric plexus, and areas of the central nervous system (midbrain) where CCK-containing dopaminergic neurons have been implicated in the pathogenesis of schizophrenia, Parkinson disease, drug addiction, and feeding disorders (4). Experimental rat pancreatic carcinogenesis is promoted by CCK through the CCKA type receptor (5). CCK acting at peripheral CCKA receptors and at central CCKA and CCKB gastrin receptors plays a significant role in the nervous system control of appetite (6).Recently, antagonists highly selective and potent for each of the CCK receptor subtypes have been developed. The two most potent and selective antagonists are L-364,718 (7) and PD134308 (8) for CCKA and CCKB receptors, respectively.In pancreatic acinar cells, CCKA receptors are coupled to a guanine nucleotide-binding regulatory protein (G protein), which activates phospholipase C, breakdown of inositol phospholipids, mobilization of intracellular calcium, and activation of protein kinase C (3). Activation of this pathway in Xenopus oocytes by CCK receptors that have been functionally expressed in the plasma membrane after injection of either rat brain total RNA (9) or the rat pancreatic acinar carcinoma cell line, AR42J (10), mRNA (11) results in a depolarizing current due to Ca2+-dependent chloride channels o...

Study of the states and populations of the rat pancreatic cholecystokinin receptor using the full peptide antagonist JMV 179

“…Furthermore, the primary structure of cloned CCK receptor suggests its membership in the family of guanine-nucleotidebinding regulatory protein coupled receptors [6]. Nevertheless, the question of whether the same receptor molecule or two distinct molecules account for the two affinity states for agonist ligands has not been clearly resolved in the case of plasma membrane pancreatic CCK receptor [5,91.…”

“…Pancreatic acinar cells from rat possess only Asubtype CCK receptors [2-41, the occupation of which by CCK agonists stimulates enzyme exocytosis [ 5 ] . The recent cloning and sequencing of the CCK receptor cDNA has provided important information [6]. However, many questions…”

The peripheral cholecystokinin receptors

“…In 1992, two groups succeeded in the molecular cloning of the cDNA encoding the rat pancreatic CCK-A receptor [215] and the canine parietal cell CCK-B receptor [238].…”

“…Wank et al [215] purified to homogeneity sufficient receptor protein to obtain partial sequences of five receptor fragments. From these peptides, degenerate oligonucleotides were synthesized and used as primers to amplify, by the polymerase chain reaction, a cDNA fragment which was then utilized as a probe to screen a rat pancreatic cDNA library.…”

“…Most importantly, CNBr cleavage and Lys-C digestions of one tenth of the purified material, followed by o-phthalaldehyde blocking, allowed five peptide fragments to be sequenced. Based on the sequence of two of these peptides, degenerate oligonucleotides were synthesized in order to clone the receptor cDNA [215].…”