I. Introduction

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The peptide cholecystokinin (CCK)² was originally discovered in the gastrointestinal tract (Ivy and Oldberg, 1928) and has been shown to mediate pancreatic secretion and contraction of gallbladder. Then, CCK was described in the mammalian central nervous system (CNS) as a gastrin-like immunoreactive material (Vanderhaeghen et al., 1975), and it is now generally believed to be the most widespread and abundant neuropeptide in the CNS. This peptide, initially characterized as a 33-amino-acid sequence, is present in a variety of biologically active molecular forms derived from a 115-amino-acid precursor molecule (prepro-CCK; Deschenes et al., 1984), such as CCK-58, CCK-39, CCK-33, CCK-22, sulfated CCK-8 [Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂] and CCK-7, unsulfated CCK-8 and CCK-7, CCK-5, and CCK-4 (Trp-Met-Asp-Phe-NH₂; Fig. 1; Rehfeld and Nielsen, 1995). The presence of CCK in both gut and brain raises the intriguing issue of the evolutionary significance of separate pools of a peptide in two systems originating from different embryonic zones (i.e., endoderm and ectoderm, respectively).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Predicted structure of human preprocholecystokinin. The signal peptide consists of residues 20 to 1. The amino terminal flanking peptide consists of residues 1 to 25. The largest characterized form from brain and intestine, CCK-58, consists of residues 26 to 83. Other active molecular forms are derived from this precursor, such as CCK-39, CCK-33, CCK-22, CCK-7, and CCK-5.

Receptors for CCK have been pharmacologically classified on the basis of their affinity for the endogenous peptide agonists CCK and gastrin, which share the same COOH-terminal pentapeptide amide sequence but differ in sulfation at the sixth (gastrin) or seventh (CCK) tyrosyl residue. Two types of CCK receptors (type A, "alimentary", and type B, "brain") have thus been distinguished. The CCK-A receptor was first characterized using pancreatic acinar cells (Sankaran et al., 1980), whereas the CCK-B receptor, with a different pharmacological profile, was discovered in the brain (CCK-B; Innis and Snyder, 1980b). The gastrin receptor mediating acid secretion in the stomach was initially thought to constitute a third type of high-affinity receptor on the basis of its location and small differences in affinity for CCK and gastrin-like peptides (Song et al., 1993). However, subsequent cloning of gastrin and CCK-B receptors revealed their molecular identity (see later). CCK-A and CCK-B receptor types have been shown to differ by their relative affinity for the natural ligands, their differential distribution, and their molecular structure. The CCK-A receptor binds sulfated CCK with a 500- to 1000-fold higher affinity than sulfated gastrin or nonsulfated CCK (Silvente-Poirot et al., 1993a). The CCK-B/gastrin receptor binds gastrin and CCK with almost the same affinity and discriminates poorly between the sulfated and nonsulfated CCK analogs (Saito et al., 1980). The distribution of CCK-A and CCK-B/gastrin receptors is tissue dependent (see below).

Based on pharmacological and biochemical studies, the existence of subtypes of CCK-A and CCK-B receptors has been postulated. Nevertheless, only two genes have been cloned. The initial nomenclature of the receptors as CCK-A and CCK-B receptors is generally accepted by pharmacologists and molecular biologists. Based on the guidelines defined by the International Union of Pharmacology (IUPHAR) Committee on Receptor Nomenclature and Drug Classification, receptors should be named after their endogenous ligands and identified by a numerical subscript corresponding to the chronological order of the formal demonstration of their existence by cloning and sequencing (Vanhoutte et al., 1996). Because the CCK-A receptor was the first to be cloned, it should be renamed CCK₁, and the CCK-B receptor should become CCK₂. According to these guidelines, new splice variants, if pharmacologically relevant, should be indicated by subscript lowercase letters, in parentheses, such as CCK_1(a), CCK_1(b), CCK_2(a), and CCK_2(b) receptors. This new nomenclature would allow any newly discovered CCK receptor to be logically named according to the same informative guidelines (see Vanhoutte et al., 1996).

This rational nomenclature has been adopted in the present review, which is devoted to the two CCK receptors whose existence has been firmly established through cloning.

	II. Characterization of Cholecystokinin (CCK) Receptors

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A. CCK₁ (CCK-A) Receptors

1. CCK₁ Receptor Clones. The size of the CCK₁ receptor demonstrated by ligand affinity crosslinking studies varied depending on the ligand, the crosslinking reagent, the species, and the tissue expressing the CCK receptor (Svoboda et al., 1982; Rosenzweig et al., 1983; Miller, 1984; Fourmy et al., 1987; Pearson and Miller, 1987; Pearson et al., 1987a,b; Shaw et al., 1987; Schjoldager et al., 1988; Powers et al., 1991). In rat pancreatic acinar cells, the CCK₁ receptor was found to be an 85- to 95-kDa, N-linked glycoprotein with a 42- to 44-kDa protein core.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the rat CCK1 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 ECLs, possibly forming a disulfide bridge, and a possible palmitoylated conserved cysteine in the cytoplasmic tail. NH2---, N terminus; COOH---, C terminus.

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Schematic representation of the rat CCK2 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 ECLs, possibly forming a disulfide bridge, and a possible palmitoylated conserved cysteine in the cytoplasmic tail. NH2---, N terminus; COOH---, C terminus.

B. CCK₂ (CCK-B) Receptors

1. CCK₂ Receptor Clones. Affinity crosslinking studies of the CCK₂ receptor using ¹²⁵I-[Leu or NLeu¹⁵]-gastrin-2-17,disuccinimidyl suberate and either a 60 to 70% pure canine gastric parietal cell preparation or a solubilized porcine gastric mucosal extract identified two glycoproteins of 78 and 74 kDa, respectively (Svoboda et al., 1982; Baldwin et al., 1986; Chiba et al., 1988; Baldwin, 1993).

2. Gastrin Receptors Are CCK₂ Receptors. Gastrin receptors in the stomach and CCK₂ receptors in the brain were historically viewed as distinct types of CCK receptors on the basis of their different relative affinities for CCK and gastrin-like peptides (Menozzi et al., 1989). However, the canine parietal cell gastrin receptor expressed in COS cells exhibits the same relative affinities for CCK-8 and gastrin as those of native human and guinea pig CCK₂ receptors. The canine parietal gastrin receptor was also considered to be a distinct receptor because of a reversal in affinity for L-364,718 versus L-365,260 in comparison with CCK₂ receptors in the brain of other species (Lotti and Chang, 1989). The basis for this reversal has subsequently been ascribed to a species-specific change of a single nucleotide resulting in a single amino acid substitution (Leu355 in canine receptor versus Val319 in the human receptor) in TMVI (Beinborn et al., 1993). Similar to the human, guinea pig, and rat CCK₂ receptors (Pisegna et al., 1992; Wank et al., 1992b), cloning of the CCK₂ receptor from canine brain (Wank, 1995) resulted in a single cDNA identical to that for the canine parietal cell gastrin receptor (Kopin et al., 1992). Clearly, the identification of a single CCK₂ receptor-encoding gene through low- and high-stringency hybridization of cDNA and genomic libraries and Northern and Southern blot analyses in numerous species indicates that gastrin receptors do correspond to CCK₂ receptors located in the gastrointestinal tract and do not constitute a third type of CCK receptor (Wank, 1995).

3. Antagonists of CCK₂ Receptors. Many attempts have been made to develop potent and specific nonpeptide antagonists of CCK₂/gastrin receptor. As a result, several new chemical entities appeared, exhibiting high selectivity for specific populations of CCK₂/gastrin receptors. The various compounds under development belong to the following main chemical classes: amino acid, benzodiazepine, dipeptoid, pyrazolidinone, and ureidoacetamides derivatives (for a review, see Makovec and D'Amato, 1997).

4. Agonists of CCK₂ Receptors. Different strategies have been followed to design potent and selective agonists of CCK₂ receptors. One of these was to protect CCK-8 [Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂] from degrading enzymes such as aminopeptidase A (Migaud et al., 1996) and a thiol/serine protease cleaving this peptide at the Met-Gly bond (Camus et al., 1989; Rose et al., 1996). The biologically active Boc[Nle^28,31]CCK27-33 (BDNL; Ruiz-Gayo et al., 1985) was used as the parent compound to design enzyme-resistant analogs. In this compound, the major sites of cleavage are at the Trp30/Nle31 and Nle28/Gly29 bonds. BDNL is potentially resistant to aminopeptidase cleavage due to its tert-butyloxycarbonyl N-terminal-protecting group (Ruiz-Gayo et al., 1985; Durieux et al., 1986a). Thus, several enzyme-resistant BDNL analogs containing either a retro-inversion of the Nle28-Gly amide bond, an (NMe)Nle31 residue, or a combination of these two modifications have been synthesized (Charpentier et al., 1988a). This led to BC 264 (Tables 8 and 9), a highly potent CCK₂ receptor agonist that exhibits about the same affinity (K_i = 0.1-0.5 nM) in all species (guinea pig, rat, mouse, monkey, humans) and was at that time the only systemically active CCK₂ receptor agonist (Charpentier et al., 1988a; Durieux et al., 1991). The peptidase-resistant bioactive analog [³H]pBC264 was also developed (Durieux et al., 1989) by replacing the Boc group with a tritiated propionyl residue. The radioactivity present in the mouse brain 15 min after i.v. injection of the tritiated compound represented 1.6/10,000 of the total radioactivity injected. Moreover, as shown by HPLC, [³H]pBC264 was very resistant to metabolism, because more than 85% of the radioactivity present in the brain corresponded to the intact molecule (Ruiz-Gayo et al., 1990). On the other hand, despite its intrinsic flexibility, CCK-8 was found through NMR to exist preferentially under a folded form in aqueous solution (Fournié-Zaluski et al., 1986) with a proximity between Asp1 and Gly4. This property was used to synthesize cyclic peptides through amide bond formation between Asp1 or between - or -carboxyl group of Glu1 and Lys4 side chains, such as BC 254 and BC 197 (Tables 8 and 9), which were found highly potent and selective CCK₂ receptor agonists (Charpentier et al., 1988b, 1989). Another nonsulfated CCK-8 analog, [N-methyl-Nle^28,31]CCK26-33 (SNF-8702; Tables 8 and 9), has also been described, which has about 4000-fold greater affinity for CCK₂ than for CCK₁ receptors (Knapp et al., 1990).

	III. Molecular Biology of CCK Receptors

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The genes encoding the CCK₁ receptor (Miller et al., 1995; Wank, 1995; Inoue et al., 1997) and the CCK₂ receptor (Song et al., 1993) in humans are organized in a similar manner consisting of five exons and four introns. The receptor genes have homologous exon/intron splice sites with exon 1 coding for the extracellular N-terminal sequence, exon 2 coding for the sequence from the beginning of TMI to the first part of TMIII, exon 3 coding for the sequence from TMIII to the beginning of TMV, exon 4 coding for the sequence from TMV to the first fourth of the third intracellular loop, and exon 5 coding for the remainder of the receptor (Fig. 4). The genes for the rat (Takata et al., 1995) and mouse (Lacourse et al., 1997) CCK₁ receptors and rabbit (Blandizzi et al., 1994) and mouse (Nagata et al., 1996) CCK₂ receptors are organized similarly to those for humans. This high degree of conservation of the sequence and organization between CCK₁ and CCK₂ receptor genes and the fact that the brain and pancreas of the bullfrog Rana catesbeiana and Xenopus laevis express only one CCK receptor (Vigna et al., 1984, 1986) suggest that the CCK₁ and CCK₂ receptor genes evolved sometime after amphibia from duplication of a common ancestral gene (as for the gene encoding the receptor ligands, CCK and gastrin). This concept is further supported by the cloning of a gene encoding a CCK receptor from a X. laevis brain cDNA library. This receptor is expressed in brain and stomach but is undetectable in pancreas. The deduced amino acid sequence from this gene has 55 and 56% amino acid identity with the human CCK₁ and CCK₂ receptors, respectively. This receptor expressed in COS-7 cells has a CCK₁ receptor type pharmacological profile (sulfated CCK > gastrin-17 > nonsulfated CCK-8 > CCK-4) like that of the native receptor in X. laevis brain and pancreas (Vigna et al., 1986; Schmitz et al., 1996) but with a relatively high affinity for sulfated gastrin, as expected for a CCK₂ receptor. Nevertheless, like typical CCK₁ receptors, the CCK receptor obtained from the X. laevis brain cDNA library has a higher affinity for L-364,718 than for L-365,260, and it is not recognized by CAM 1714 or CAM 1028 (Schmitz et al., 1996).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Schematic representation of genes encoding human CCK1 and CCK2 receptors. Shown are position and size of the exons (shaded boxes) and introns (lines) comprising the genes for the CCK1 and the CCK2 receptors; smaller arabic numbers represent size of each exon and intron in base pairs. Roman numerals refer to putative transmembrane-spanning regions encoded within each exon. ATG and TGA, putative start and stop codons, respectively. CCK2 receptor gene: the second splice variant (short form) differs only in the size of exon 4, in which a sequence is absent compared with long form, corresponding to a block of five amino acids within the third intracellular loop. The third splice variant encodes an N-terminally truncated receptor. The gene structure is similar, except that there is an alternative first exon (exon 1b) that makes up the 5' untranslated region of this truncated receptor.

Alternative splicing of exon 4 of the human CCK₂ receptor gene results in two CCK₂ receptor transcripts that differ by a block of five amino acids within the third intracellular loop (Song et al., 1993; Fig. 4). The shorter transcript is largely predominant in stomach, although its relative distribution in individual cell types has not been examined. To date, the physiological relevance of the two isoforms of the human CCK₂ receptor is not known. A comparison of the shorter and longer isoforms revealed no significant differences in agonist affinity and signal transduction (Ito et al., 1993, 1994b; Wank et al., 1994b).

Another splice variant of the human CCK₂ receptor transcript, designated CCK₂ receptor, which differs at the 5' end from the CCK₂ receptor transcript described earlier, was discovered using a polymerase chain reaction-based cloning strategy (Miyake, 1995). CCK₂ receptor encodes an N-terminally truncated receptor that starts with the methionine Met67 in TMI and is otherwise identical in the remaining sequence. The gene structure is similar to that previously reported for the human CCK₂ receptor (Song et al., 1993) except that the first intron was of ~10 kb (compared with 1.177 kb) and contained the sequence for the alternative first exon that makes up the 5' untranslated region of CCK₂ receptor (Fig. 4). The first methionine of exon 2, which is common to both CCK₂ and CCK₂ receptors, serves as the translational initiation site for the CCK₂ receptor. CCK₂ receptor transiently expressed in COS-7 cells has a ~3-fold lower affinity for CCK-8 and a ~30-fold lower affinity for gastrin compared with the CCK₂ receptor, but its affinity for the antagonists L-365,260 and L-364,718 is unchanged. Both CCK₂ and CCK₂ receptor transcripts have been detected in brain, stomach, and pancreas through the use of reverse transcription-polymerase chain reaction (Miyake, 1995). According to the guidelines defined by the IUPHAR committee, because these splice variants do not appear to be major variants, they are not indicated by subscript lowercase letters.

On the other hand, Jagerschmidt et al. (1994) isolated several CCK₂ receptor mRNA isoforms from rat brain tissue, including a truncated mRNA species. 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. Thus, although five precursor mRNAs were found in the cerebral cortex and the hypothalamus, only one fully processed messenger was detected in the hippocampus. In the case of 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 the rat (Pélaprat et al., 1987).

The human CCK₁ receptor gene has been localized to chromosome 4 using a panel of human/hamster hybrid DNAs (Huppi et al., 1995). The mouse CCK₁ receptor gene has been mapped to a syntenic region on chromosome 5 using a wild × inbred backcross panel of mice [(BALB/cAN × Mus spretus) F₁ × BALB/cAN] (Huppi et al., 1995). This region of mouse chromosome 5 is syntenic with human chromosome 4p16.2-p15.1 (Huppi et al., 1995). The human CCK₁ receptor was further mapped to 4p15.1-p15.2 using fluorescence in situ hybridization and physically mapped between the markers AFMb355ya5 and AFMa283yh5 (Inoue et al., 1997). The rat CCK₁ receptor gene has been localized to a syntenic region on chromosome 14 by fluorescence in situ hybridization (Takiguchi et al., 1997).

The human CCK₂ receptor has been localized to chromosome 11 in humans and a syntenic region on chromosome 7 in the mouse using a panel of human/hamster hybrid DNAs (Huppi et al., 1995). Fluorescence in situ hybridization of human metaphase chromosomal spreads has further localized the human CCK₂ receptor gene to the distal short arm of chromosome 11 (11p15.4; Song et al., 1993; Zimonjic et al., 1994). The colocalization of the CCK₁ receptor gene with the dopamine D₅ receptor gene at 4p15.1-p15.3 (Sherrington et al., 1993) and of the CCK₂ receptor gene with the gene encoding the dopamine D₄ receptor at 11p15.4-p15.5 (Gelernter et al., 1992; Pisegna et al., 1992) is especially interesting in view of the coexistence of CCK and dopamine in midbrain neurons and the regulation of mesolimbic dopaminergic pathways by both CCK₁ and CCK₂ receptors (Crawley and Corwin, 1994).

An inbred strain of Long Evans rats, the Otsuka Long-Evans Tokushima Fatty rats, that is considered to be a model for late-onset non-insulin-dependent diabetes mellitus, was discovered to have no detectable levels of CCK₁ receptor gene expression. Subsequent cloning of their CCK₁ receptor gene revealed a deletion of 6847 bp encompassing the promoter region and first and second exons (Takiguchi et al., 1997). Although these rats are known to have polygenic abnormalities, the presence of several metabolic and behavioral abnormalities has been attributed to the loss of CCK₁ receptor expression.

Targeted disruption of the CCK₂ receptor gene has been achieved in mice (Nagata et al., 1996). Homozygous mutant mice were viable and fertile and appeared to be grossly normal into adulthood (Langhans et al., 1997). CCK₂^/ mutant mice have much fewer gastric parietal and ECL cells than so wild-type animals, which is in line with the growth-promoting effects of gastrin at the CCK₂ receptor previously seen in patients with hypergastrinemia due to the Zollinger-Ellison syndrome. Also, as expected, these mice were hypochlorhydric and hypergastrinemic (Nagata et al., 1996). Together, these results demonstrate the importance of the CCK₂ receptor in maintaining the normal cellular composition and function of the gastric mucosa.

Moreover, the physiological implication of CCK₂ receptor can now be further investigated in CCK₂ receptor-deficient mice obtained through gene targeting. The first experiments reported with this interesting model show a critical role of CCK₂ receptors in memory process. CCK₂ receptor-deficient mice have an impairment of performance in the memory task (Sebret et al., 1999; for more details, see VIIB4. CCK and Memory Processes).

	IV. Receptor Structure/Function Studies

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A. Signal Transduction

1. CCK₁ Receptors. The modulation of CCK₁ receptor affinity by guanine nucleotides in early studies suggested that they belong to the GPCR superfamily. This has been confirmed through the cloning of CCK₁ receptors (Wank et al., 1992a), which revealed their seven-transmembrane receptor structure.

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2. CCK₂ Receptors. Molecular cloning of CCK₂ receptors has shown that this receptor is a member of the seven-transmembrane domain GPCR superfamily (Wank et al., 1992b). This confirmed previous results showing that nonhydrolyzable GTP analogs reduced the binding of selective CCK₂ receptor agonists, as expected of the coupling of these receptors with G proteins (Knapp et al., 1990; Durieux et al., 1992).