The human gastrin/cholecystokinin type B receptor gene: alternative splice donor site in exon 4 generates two variant mRNAs.

Abstract

Gastrin and its carboxyl-terminal homolog cholecystokinin (CCK) exert a variety of biological actions in the brain and gastrointestinal tract that are mediated in part through one or more G protein-coupled receptors which exhibit similar affinity for both peptides. Genomic clones encoding a human gastrin/CCKB receptor were isolated by screening a human EMBL phage library with a partial-length DNA fragment which was based on the nucleotide sequence of the canine gastrin receptor. The gene contained a 1356-bp open reading frame consisting of five exons interrupted by 4 introns and was assigned to human chromosome llpl5.4. A region of exon 4, which encodes a portion of the putative third intracellular loop, appears to be alternatively spliced to yield two different mRNAs, one containing (452 amino acids; long isoform) and the other lacking (447 amino acids; short isoform) the pentapeptide sequence Gly-Gly-Ala-Gly-Pro. The two receptor isoforms may contribute to functional differences in gastrin-and CCK-mediated signal transduction. (12,13). They encode homologous proteins of similar length (453 and 450 aa, respectively) with seven deduced transmembrane regions, comparable to other G protein-coupled receptors. The recombinant receptors, when transfected into COS-7 cells, manifest high (nanomolar) affinities for both gastrin and CCK and are coupled to an increase in intracellular calcium (12,13). Structurally, the canine gastrin receptor displays nearly 90% amino acid identity with the putative CCKB receptors from human and rat (9-11).The sequence and organization of the full-length genes encoding members of the gastrin/CCK receptor family have not been identified. Such information is essential for analysis of their transcriptional regulation and structural relationships to other receptor genes. We have cloned and sequenced a gastrin/CCKB receptor gene from a human genomic library**. The gene appears to be alternatively spliced to yield two different receptor isoforms which differ by 5 aa in the putative third intracellular loop. The gastrin/CCKB receptor gene was assigned to human chromosome 11p15.4 by fluorescence in situ hybridization. METHODSGenomic Library Screening and DNA Sequencing. A human genomic library in the bacteriophage AEMBL-3 (Clontech) was screened with a 32P-labeled cDNA fragment generated by the polymerase chain reaction (PCR) with primers based on the cDNA sequence of the canine gastrin receptor (see below). Two positive clones were digested with Sst I and the resulting restriction fragments of length 0.7-3.2 kb (Fig. 1) were subcloned into phage M13mpl8 or -mpl9 and sequenced in both directions by the dideoxy method (14). In Abbreviation: CCK, cholecystokinin. tPresent address:

Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol

“…These chimeric constructs take advantage of the fact that the genes encoding human CCK1R and CCK2R each contain fi ve exons in a similar organization ( 17,(32)(33)(34). The exons from CCK1R were sequentially replaced by exons from CCK2R, starting from the N terminus.…”

Frameshift Mutations of Human Gastrin Receptor Gene (hGARE) in Gastrointestinal Cancers with Microsatellite Instability

“…Direct sequencing of Laghi et al the product we obtained from LoVo cells demonstrated the presence of only one of the two isoforms that are known to be generated by exon 4 alternative splicing ( Fig. 4c) (Song et al, 1993). Interestingly, this isoform corresponded with the hGARE frameshiftmutated allele and has been characterized by a (T) 7 repeat in exon 5 (Fig.…”

Potential role of endocrine gastrin in the colonic adenoma carcinoma sequence

“…Furthermore, gastrin stimulation of CCK-2 receptors expressed by AR42J cells was shown in an in vitro system to reverse apoptosis induced by serum withdrawal through the induction/ activation of protein kinase BAkt (Todisco et al, 2001). A number of isoforms of the CCK-2 receptor have now been discovered (Song et al, 1993;Miyake, 1995;Ohtaki et al, 1996). These include the 'long isoform' which has, due to alternate splicing, an additional five amino acids located within the third intracellular domain (Song et al, 1993;Ohtaki et al, 1996).…”

“…A number of isoforms of the CCK-2 receptor have now been discovered (Song et al, 1993;Miyake, 1995;Ohtaki et al, 1996). These include the 'long isoform' which has, due to alternate splicing, an additional five amino acids located within the third intracellular domain (Song et al, 1993;Ohtaki et al, 1996). Use of an alternative first exon, 1b, which remains untranslated, leads to a receptor truncated at the amino-terminus, known as the DCCK-2.…”