Mouse cholecystokinin type-A receptor gene and its structural analysis

Abstract

The mouse cholecystokinin type-A receptor (CCK_AR) gene was cloned and sequenced, and the exon/intron boundaries were determined by cDNA cloning. The gene, approximately 10 kb in length, contains the entire coding region, and consists of five exons. The deduced amino acid sequence was homologous with that of other species, with the exception of an additional DNA sequence encoding 7 amino acids in exon 5. A region of the 5′ end of exon 2 appeared to be alternatively spliced, and generated an isoform shorter by 52 bases. The shorter isoform may encode an 48 amino acid open reading frame due to frameshift of translation. These two mRNA isoforms were expressed equally in the mouse gallbladders.

Introduction

The signals of cholecystokinin (CCK) are transduced through receptors which are present throughout the gastrointestinal tract and the central nervous system (CNS). The receptors can be subdivided pharmacologically by their binding affinities for CCK/gastrin family peptides into two subtypes: CCK_A receptors (CCK_AR) and CCK_B receptors (CCK_BR). Molecular cloning analyses of both types of receptors also support these physiological observations (Wank et al., 1992a, Wank et al., 1992b; Pisegna et al., 1992; Ulrich et al., 1993; De Weerth et al., 1993a, De Weerth et al., 1993b; Wank, 1995). CCK_AR have been demonstrated to be present in the gastrointestinal system, vagus nerve and localized areas of the central nervous system. In the gastrointestinal system, it mediates smooth muscle contraction of the gallbladder, pancreatic growth and enzyme secretion (Crawley, 1991). Abnormalities in gallbladder contraction in response to exogenously administered CCK have been observed in patients with gallstones, suggesting a generalized abnormality of smooth muscle function (Kellow et al., 1987). CCK_AR are distributed to certain regions of the CNS, such as the nucleus tractus solitarius, area postrema, interpenduncular nucleus, posterior hypothalamus and nucleus accumbens (Hill et al., 1990; Crawley, 1991). It has been demonstrated that one function of CCK_AR in the hypothalamus is the regulation of food intake (Silver and Morley, 1991; Hirosue et al., 1993; Moran et al., 1993; Silvente-Poirot et al., 1993). We intensively analyzed the structure and expression of the CCK_AR gene in rats (Funakoshi et al., 1994, Funakoshi et al., 1995; Takata et al., 1995), then we suggested that some of the pathological disorders may depend on lack of the expression of the CCK_AR gene (Funakoshi et al., 1996). It is believed that the study of mice is also useful for genetic analysis, such as gene targeting experiments, however, little is known about the structure and function of the mouse CCK_AR gene. In the present study, we cloned and sequenced the mouse CCK_AR gene and its cDNA, then demonstrated alternative splicing of the gene to yield two different receptor mRNA isoforms. This is the first report showing the gene structure as well as alternative splicing of mouse CCK_AR mRNA.