Molecular Cloning and Structural Analysis of the Rabbit Gastrin/CCKB Receptor Gene

doi:10.1006/bbrc.1994.2021

Biochemical and Biophysical Research Communications

Volume 202, Issue 2, 29 July 1994, Pages 947-953

https://doi.org/10.1006/bbrc.1994.2021 Get rights and content

Abstract

Gastrin and cholecystokinin exert a variety of physiological actions in the central nervous system and digestive tract that are mediated through one or more receptors exhibiting similar affinity for the two peptides. We isolated genomic clones encoding the rabbit gastrin/CCK_B receptor by screening a rabbit EMBL phage library with a cDNA probe based on the nucleotide sequence of the human gastrin/CCK_B receptor. The gene contained a putative 1356-bp open reading frame consisting of five exons interrupted by 4 introns and encoded a protein of 452 amino acids. The putative protein-coding region of the gene exhibits 93 to 97% amino acid similarity with corresponding cDNAs previously identified in human, canine and rodent brain or gastric tissues.

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Cited by (14)

Molecular cloning of the orphanin FQ receptor gene and differential tissue expression of splice variants in rat
2001, Gene
Citation Excerpt :
These findings prompted us to further characterize the OFQ receptor, and to that end, we attempted to isolate the gene encoding the receptor and to determine whether any isoforms were expressed in rat. A rat genomic library in the bacteriophage EMBL-3 (Clontech) was screened as previously reported (Blandizzi et al., 1994), using a 32P-labeled cDNA fragment generated by the polymerase chain reaction (PCR) with primers based on the cDNA sequences of the rat OFQR (U01913, Bunzow et al., 1994; and L29419, Wick et al., 1994). Positive clones were digested with Pst I, Acc I, and Sac I.
Pharmacological and receptor-ligand binding studies of the cloned orphanin FQ (OFQ) receptor suggest that multiple forms of this receptor may exist. To further characterize the OFQ receptor (OFQR), we attempted to isolate the gene encoding this receptor in rat. The OFQR gene exceeds 10 kb in length and contains six exons ranging from 34 to 524 bp that are interrupted by five introns. The ATG translation initiation codon is located in exon 2, and the open reading frame consists of 1283 bp. Primer extension analysis of the gene revealed two major transcription initiation sites: one in the 5′ flanking region and the other in intron 1. The rat OFQR gene appeared to be alternatively spliced to yield multiple mRNAs. Four splice variants deleted for exon 1 were expressed only in brain. In contrast, five isoforms containing exon 1 were expressed in various tissues, such as brain, testes, and gastrointestinal tract. These data suggest that unique regions in intron 1 and in the 5′ flanking region of the OFQR gene contribute to the regulation of its expression in different tissues.
Structure and developmental expression of the mouse CCK-B receptor gene
2000, Biochemical and Biophysical Research Communications
Cholecystokinin (CCK) and gastrin exert their effects through two receptors, the CCK-A and CCK-B receptors. We have cloned the mouse CCK-B receptor gene (Cckbr) and determined its complete genomic structure, nucleotide sequence, and tissue-specific expression pattern. Cckbr is divided into five exons spanning 11 kb. A primer extension assay was used to map the transcription initiation site to 199 bp upstream of the translational start site. Rapid amplification of cDNA ends was used to localize the 3′ end downstream of an atypical polyadenylation site (GATAAA). Mouse Cckbr transcripts were most abundant in brain and stomach, but were also detected in colon, kidney, ovary, and pancreas. Prenatal expression of both CCK-A and CCK-B receptors in various tissues was analyzed by RT-PCR. The expression pattern was similar to the adult pattern, suggesting that receptor transcription is an early event in gastrointestinal development.
CCK<inf>1</inf> and CCK<inf>2</inf> receptors regulate gastric pepsinogen secretion
1999, European Journal of Pharmacology
The present study investigated (1) the pharmacological profile of cholecystokinin (CCK) receptor subtypes involved in the regulation of gastric pepsinogen secretion, (2) the influence of gastric acidity on peptic responses induced by CCK-8-sulfate (CCK-8S) or gastrin-I; and (3) the mechanisms accounting for the effects of CCK-like peptides on pepsinogen secretion. In anaesthetized rats, i.v. injection of CCK-8S or gastrin-I increased both pepsinogen and acid secretion. The pepsigogue effect of CCK-8S was higher than that of gastrin-I, whereas acid hypersecretion after CCK-8S was lower than that induced by gastrin-I. Peptic output following CCK-8S was partly blocked by i.v. injection of the CCK₁ receptor antagonist, devazepide (−75.3%), or the CCK₂ receptor antagonist, l-365,260 [3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3yl)-N′-(3-methyl-phenyl)urea; −27.9%], but was fully prevented by combined administration of devazepide and l-365,260. The gastric acid hypersecretory effect of CCK-8S was enhanced by devazepide (+84.5%) and blocked by l-365,260. In contrast, the gastric secretory actions of gastrin-I were insensitive to devazepide, but abolished by l-365,260. Excitatory effects of CCK-8S and gastrin-I were not modified by vagotomy or atropine, whereas cimetidine or α-fluoromethylhistidine (irreversible blocker of histidine decarboxylase) partly prevented acid hypersecretion induced by both peptides without affecting their pepsigogue effects. After pretreatment with omeprazole, both CCK-8S and gastrin-I failed to stimulate acid secretion, while they increased pepsinogen output. In rats with gastric perfusion of acid solutions, CCK-8S or gastrin-I increased peptic output in a pH-independent manner either with or without pretreatment with omeprazole. Ablation of capsaicin-sensitive sensory nerves as well as application of lidocaine to the gastric mucosa failed to modify the excitatory effects of CCK-8S or gastrin-I on pepsinogen and acid secretion. Blockade of the nitric oxide (NO) synthase pathway by N^G-nitro-l-arginine-methyl ester prevented the pepsigogue actions of both CCK-8S and gastrin-I (−61.8% and −71.7%, respectively), without affecting the concomitant increase in acid output. In addition, both these peptides significantly increased the release of NO breakdown products into the gastric lumen. The present results suggest that: (1) both CCK₁ and CCK₂ receptors mediate the peptic secretory responses induced by CCK-like peptides; (2) the excitatory inputs of CCK-8S and gastrin-I to chief cells are not driven through acid-dependent mechanisms or capsaicin-sensitive afferent sensory nerves; and (3) under in vivo conditions, the stimulant actions of CCK-like peptides on pepsinogen secretion are mediated, at least in part, by an increase in NO generation.
Molecular structure of the mouse CCK-A receptor gene
1997, Biochemical and Biophysical Research Communications
We have cloned the mouse CCK-A receptor gene (Cckar), determined its nucleotide sequence, and analyzed its expression. The receptor protein is encoded in five exons distributed over 9 kb of genomic DNA. Intron/exon borders were determined by comparing the genomic nucleotide sequence with the mouse cDNA sequence obtained by reverse transcriptase polymerase chain reaction. RNase protection analysis ofCckartranscripts revealed the presence of a splice acceptor site 200 bp upstream of the translational start codon, indicating that the promoter is associated with a non-translated exon at an upstream site. The second coding exon contains a rarely used alternative splice site that would result in the production of a truncated, 48 amino acid protein.Cckaris widely expressed in the gastrointestinal system (pancreas, gallbladder, intestine, colon and stomach), as well as in brain and kidney.
Conserved cholecystokinin receptor transmembrane domain IV amino acids confer peptide affinity
2003, Journal of Molecular Neuroscience
Molecular characterization and organ distribution of type A and B cholecystokinin receptors in cynomolgus monkey
2001, American Journal of Physiology - Gastrointestinal and Liver Physiology

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Regular ArticleMolecular Cloning and Structural Analysis of the Rabbit Gastrin/CCKB Receptor Gene

Abstract

Regular Article
Molecular Cloning and Structural Analysis of the Rabbit Gastrin/CCK_B Receptor Gene