A Truncated Isoform of Human CCK-B/Gastrin Receptor Generated by Alternative Usage of a Novel Exon

doi:10.1006/bbrc.1995.1328

Biochemical and Biophysical Research Communications

Volume 208, Issue 1, 8 March 1995, Pages 230-237

https://doi.org/10.1006/bbrc.1995.1328 Get rights and content

Abstract

An isoform cDNA of CCK-B/gastrin receptor was isolated from human stomach. This cDNA differed from initially cloned cDNA only in the 5′-end region and encoded a truncated isoform (ΔCCK-B) in which the putative N-terminal extracellular domain of the CCK-B/gastrin receptor was completely lost. Isolation of genomic CCK-B/gastrin receptor DNA revealed that this transcript is generated by alternative usage of a novel exon, termed exon 1b. Human stomach expressed both transcripts of ΔCCK-B and entire CCK-B/gastrin receptor (CCK-BR), whereas human stomach cancer cell line AGS exclusively expressed ΔCCK-B transcripts. Transfection of COS-7 with ΔCCK-B cDNA led to the appearance of binding sites for ¹²⁵I-CCK-8. Its ligand selectivity was different from that of CCK-BR. These results suggest the molecular diversity in CCK-B/gastrin receptor subtypes.

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However, these data are still controversial since abundance of variant transcript in colorectal cancers as well as its constitutive activity were not always confirmed (Cheng et al., 2005; Korner et al., 2010). Two other CCK2R transcript variants, isolated from gastric cancer cells and colorectal cancers were described (Miyake, 1995; Schmitz et al., 2001). The demonstration of the clinical relevance of the expression of ΔE2-CCK2R in human tumors will require further investigations on a large series of tumor samples as our current study used a highly heterogeneous series of samples with only few cases per tumor type and thus could not permit a meaningful correlative evaluation with any kind of clinical parameters.
The cholecystokinin-2 receptor (CCK2R), is expressed in cancers where it contributes to tumor progression. The CCK2R is over-expressed in a sub-set of tumors, allowing its use in tumor targeting with a radiolabel ligand. Since discrepancies between mRNA levels and CCK2R binding sites were noticed, we searched for abnormally spliced variants in tumors from various origins having been previously reported to frequently express cholecystokinin receptors, such as medullary thyroid carcinomas, gastrointestinal stromal tumors, leiomyomas and leiomyosarcomas, and gastroenteropancreatic tumors. A variant of the CCK2R coding for a putative five-transmembrane domains receptor has been cloned. This variant represented as much as 6% of CCK2R levels. Ectopic expression in COS-7 cells revealed that this variant lacks biological activity due to its sequestration in endoplasmic reticulum. When co-expressed with the CCK2R, this variant diminished membrane density of the CCK2R and CCK2R-mediated activity (phospholipase-C and ERK activation). In conclusion, a novel splice variant acting as a dominant negative on membrane density of the CCK2R may be of importance for the pathophysiology of certain tumors and for their in vivo CCK2R-targeting.
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To determine the possible structural basis for differential cholesterol sensitivity of these receptors, we began by utilizing a series of chimeric receptors (CCK2R/CCK1R) originally prepared and characterized by Wu et al. (17). These chimeric constructs take advantage of the fact that the genes encoding human CCK1R and CCK2R each contain five exons in a similar organization (17, 32–34). The exons from CCK1R were sequentially replaced by exons from CCK2R, starting from the N terminus.
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In addition, CCK2 receptor expression and/or gastrin binding has been demonstrated in clinical samples from several gastrointestinal cancers including colorectal, gastric and pancreatic cancers [45–47]. However, the identification of additional CCK2 receptor isoforms (truncated CCK2 receptor, [48]; CCK2S and CCK2L; [49] and CCKBri4sv, [50]) has resulted in some speculation over the possibility that the neoplastic effects of gastrin (and its multiple molecular forms) could be mediated by an alternative CCK2 receptor [51]. The clinical pharmacological relevance of the existence of additional CCK2 receptor subtypes remains unclear as only small differences in antagonist affinity values have been reported between the different isoforms studied to date [52].
Gastrin, acting through peripheral cholecystokinin (CCK) 2 receptors, is a major hormonal regulator of gastric acid secretion. The effects of gastrin on acid secretion occur both acutely and chronically because gastrin directly stimulates gastric acid secretion and also exerts trophic effects on the enterochromaffin-like and parietal cells that together constitute the acid secretory apparatus of the stomach. Several antagonists that target the CCK2 receptor have been identified and investigated for the treatment of gastroesophageal reflux disease and pancreatic cancer. In this paper, we discuss the contribution of gastrin to these disease pathologies and the data generated to date from clinical studies investigating CCK2 receptor antagonists.

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Regular ArticleA Truncated Isoform of Human CCK-B/Gastrin Receptor Generated by Alternative Usage of a Novel Exon

Abstract

Regular Article
A Truncated Isoform of Human CCK-B/Gastrin Receptor Generated by Alternative Usage of a Novel Exon