Molecular characterization

Receptors for CCK were first characterized on pancreatic acinar cells and identified as CCK1 receptors (Sankaran et al. 1980). Subsequently, a second receptor, the CCK2 receptor, was identified in brain which exhibits a distinct pharmacology (Innis and Snyder 1980).

The human genes for the CCK1 and CCK2 receptors are organized in a similar manner consisting of five exons and four introns. Exon one encodes the putative extracellular amino terminus of the receptor. Exon 2 and 3 encode TM regions I-IV and exon 4 encodes the fifth TM region and an initial portion of the third intracellular loop. Exon 5 encodes the C-terminal part of this intracellular loop, the remaining TM VI and VII and the intracellular C-terminal domain (Song etal. 1993).

A splice variant of the CCK2 receptor was isolated from human stomach (Miyake 1995). This cDNA differed from initially cloned cDNA in the 5'-end region and encoded a truncated isoform in which the putative N-terminal extracellular domain was completely lost. Isolation of genomic CCK2 receptor DNA revealed that the gene structure was similar to that previously reported except that the first intron contained the sequence for an alternative first exon. The alternative usage of this exon causes no change in translated receptor protein. Another splice variant was characterized in exon 4, resulting in the presence of two CCK2 receptor transcripts differing by a block of five amino acids (GGAGP) within the third intracellular loop. No significant difference in agonist affinity or signal transduction was measured between the shorter and longer isoforms (Wank 1995). The shorter transcript is predominant in the stomach. Several CCK2 receptor mRNA isoforms from rat brain tissues and fundus glands have also been isolated, including truncated mRNA species (Jagerschmidt etal. 1994). Unspliced precursor mRNA and the mature form were identified in the cerebral cortex, hypothalamus, and hippocampus in apparently differing proportions according to the region examined, suggesting that the expression of the CCK2 receptor could be modulated at a post-transcriptional level. In the cerebellum, only a completely unspliced mRNA form was found, which is in agreement with previous studies showing that CCK2 receptor-binding sites are not expressed in this structure in rat (Pelaprat et al. 1987).

CCK1 and CCK2 receptors have been cloned from several species and have approximately 50 per cent homology to each other (Wank etal. 1992; see Noble etal. 1999; Fig. 14.1). At the amino acid level, the CCK1 receptor is highly conserved, with an overall sequence homology of 80 per cent and a pairwise amino acid sequence identity of 87-92 per cent in humans, guinea pig, rat, and rabbit. Similarly, the CCK2 receptor is highly conserved in humans, canine, guinea pig, calf, rabbit, and rat, with an overall identity of 72 per cent and pairwise amino acid sequence identities of 84-93 per cent (see Wank 1995). The deduced sequences of the rat CCK1 and CCK2 receptors correspond to 429 and 452 amino acid proteins, respectively. Hydropathy analysis of the primary sequence of CCK1 and CCK2 receptors predicts seven transmembrane-spanning domains (TM) as expected for a member of GPCR superfamily (Dohlman et al. 1991). In agreement with the heavy and variable degrees of glycosylation reported using ligand affinity cross linking techniques (de Weerth et al. 1993), at least three consensus sequence sites for N-linked glycosylation (Asn-X-Ser/Thr) have been identified in the CCK1 and CCK2 receptors sequences. There are multiple potential serine and threonine phosphorylation sites in the CCK2 receptor, one for protein kinase C (PKC) (serine 82, in the first intracellular loop), and two for protein kinase A (PKA) (serine 154 in the second intracellular loop and serine 442 in the cytoplasmic tail). Similar to the CCK2 receptor, CCK1 receptor has three consensus sequences for PKC phosphorylation in the third intracellular loop, and one site in the cytoplasmic tail of the rat pancreatic CCK1 receptor (Ozcelebi and Miller 1995). Moreover, in both receptors there are two cysteines in the first and second extracellular loops, which may form a disulfide bridge required for stabilization of the tertiary structure as demonstrated for other receptors belonging to the GPCR superfamily (Silvente-Poirot et al. 1998), and a cysteine in the C-terminus of the receptor which may serve as a membrane-anchoring palmitoylation site as demonstrated for rhodopsin and the (2-adrenergic receptors (O'Dowd et al. 1988; Ovchinikov et al. 1988).

Finally, based on pharmacological and biochemical studies, the existence of further subtypes of CCK1 and CCK2 receptors has been postulated (Durieux et al. 1986; Knapp et al. 1990; Talkad et al. 1994). Nevertheless, at this time only two genes have been cloned. Gastrin receptors in the stomach and CCK2 receptors in the brain were initially viewed as distinct CCK receptors on the basis of their difference in affinity for CCK- and gastrin-like peptides (Menozzi et al. 1989). Endogenous peptide agonists CCK8 [Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and gastrin [H2N-Gln-Gly-Pro-Trp-Met-Glu-Glu-Glu-Glu-Glu-Ala-Tyr(SO3H)-Gly-Trp-Met-Asp-Phe-NH2] share the same COOH-terminal pentapeptide amide sequence but differ in sulfation at the sixth (gastrin) or seventh (CCK) tyrosyl residue. Agonist binding studies on brain membranes and parietal cells show a six to ten fold and one to two fold higher affinity for CCK than for gastrin, respectively (Jensen et al. 1990). These small differences in agonist binding have generated controversy regarding the existence of subtypes within this receptor class. The identification of a single CCK2 receptor encoding

Fig. 14.1 Schematic representation of the rat CCK1 (a) and CCK2 (b) receptor showing the postulated transmembrane topology, sites for putative NH2-linked glycosylation (tridents), serine and threonine phosphorylation by PKC and protein kinase A (PO3) and conserved cysteines in the first and second extracellular loops, possibly forming a disulfide bridge, and a possible palmitoylated conserved cysteine in the cytoplasmic tail.

Fig. 14.1 Schematic representation of the rat CCK1 (a) and CCK2 (b) receptor showing the postulated transmembrane topology, sites for putative NH2-linked glycosylation (tridents), serine and threonine phosphorylation by PKC and protein kinase A (PO3) and conserved cysteines in the first and second extracellular loops, possibly forming a disulfide bridge, and a possible palmitoylated conserved cysteine in the cytoplasmic tail.

gene through low- and high-stringency hybridization of cDNA and genomic libraries and Northern and Southern blot analysis in numerous species indicates that gastrin receptors do indeed correspond to the CCK2 receptors located in the gastrointestinal tract and do not constitute a third type of CCK receptor (Wank 1995). However, a third receptor subtype has been pharmacologically identified, although not yet cloned. This receptor is a gastrin preferring receptor, described for the first time in an immortalized fibroblast cell line (Swiss 3T3 cells), and which discriminates between ionidated and glycine-extended gastrins (Singh et al. 1995). Based on the guidelines defined by the International Union of Pharmacology (IUPHAR), Committee on receptor Nomenclature and Drug Classification, this receptor cannot be registered, as the formal demonstration of its existence by cloning and sequencing has not been done.

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