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Vol. 51, Issue 4, 745-781, December 1999

International Union of Pharmacology. XXI. Structure, Distribution, and Functions of Cholecystokinin Receptors

Florence Noble, Stephen A. Wank, Jacqueline N. Crawley, Jacques Bradwejn, Kim B. Seroogy, Michel Hamon and Bernard P. Roques1

Institut National de la Santé et de la Recherche Médicale U266, Centre National de la Recherche Scientifique UMR 8600, Université René Descartes, Paris, France (B.P.R., F.N.); Royal Ottawa Hospital, University of Ottawa, Ottawa, Ontario, Canada (J.B.); Section on Behavioral Neuropharmacology, Experimental Therapeutics Branch, National Institute of Mental Health, Bethesda, Maryland (J.N.C.); National Institutes of Health, Digestive Diseases Branch, Bethesda, Maryland (S.A.W.); Department of Anatomy and Neurobiology, Chandler Medical Center, University of Kentucky, Lexington, Kentucky (K.B.S.); and Institut National de la Santé et de la Recherche Médicale U288, Faculté de Médecine Pitié-Salpêtrière, Paris, France (M.H.)

I. Introduction
II. Characterization of Cholecystokinin (CCK) Receptors
    A. CCK1 (CCK-A) Receptors
        1. CCK1 Receptor Clones.
        2. Antagonists of CCK1 Receptors.
        3. Agonists of CCK1 Receptors.
    B. CCK2 (CCK-B) Receptors
        1. CCK2 Receptor Clones.
        2. Gastrin Receptors Are CCK2 Receptors.
        3. Antagonists of CCK2 Receptors.
        4. Agonists of CCK2 Receptors.
III. Molecular Biology of CCK Receptors
    A. CCK Receptor Gene Structure
    B. Chromosomal Localization of CCK Receptor Genes
    C. Animal Models without Detectable Levels of CCK Receptors
IV. Receptor Structure/Function Studies
    A. Signal Transduction
        1. CCK1 Receptors.
        2. CCK2 Receptors.
    B. Ligand-Receptor Interaction
        1. Agonists.
        2. Antagonists.
        C. Receptor Regulation.
V. Radioligands and Binding Assays: Heterogeneity of CCK1 and CCK2 Receptors
    A. Radioligands at CCK1 Receptors
    B. Radioligands at CCK2 Receptors
    C. Heterogeneity of CCK2 Receptor-Binding Sites
VI. Distribution of CCK Receptors
    A. Distribution in Central Nervous System
        1. CCK1 Receptors.
        2. CCK2 Receptors.
        3. Regulation of CCK Receptors.
    B. Distribution in Gastrointestinal and Other Systems
VII. Physiological Implications of CCK Receptors
    A. Peripheral Functions
    B. Central Functions
        1. CCK in Panic Attacks and Anxiety.
        2. CCK and Schizophrenia.
        3. CCK and Depression.
        4. CCK and Memory Processes.
        5. Interactions between CCK and Enkephalin
Systems.

VIII. Conclusion
Acknowledgments
References


    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).



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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 CCK1(a), CCK1(b), CCK2(a), and CCK2(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.

The CCK1 receptor was purified to homogeneity from rat pancreas. The purified receptor had a molecular mass of 85 to 95 kDa consistent with previous crosslinking studies (Wank et al., 1992a). Microsequencing of five peptide products derived from either enzymatic digestion or chemical cleavage of the protein receptor allowed the design of degenerate oligonucleotide primers for cloning the cDNA of the CCK1 receptor from a rat pancreatic cDNA library. The deduced sequence of the rat CCK1 receptor corresponds to a 429-amino-acid protein with a calculated molecular mass of 48 kDa. Hydropathy analysis predicts seven transmembrane-spanning domains (TM) as expected for a member of the G protein-coupled receptor (GPCR) superfamily (Dohlman et al., 1991; Fig. 2). The sequence contains at least three consensus sites for N-linked glycosylation (Asn-X-Ser/Thr), consistent with the heavy and variable degree of glycosylation reported using ligand-affinity crosslinking techniques (de Weerth et al., 1993b). The CCK1 receptor has three consensus sequence sites for protein kinase C (PKC) phosphorylation in the third intracellular loop (Graff et al., 1989), consistent with previous data showing that CCK-8- and 12-O-tetradecanoylphorbol-13-acetate-stimulated phosphorylation of serine and threonine residues involves predominantly the third intracellular loop and to a minor extent the cytoplasmic tail of the rat pancreatic CCK1 receptor (Kawano et al., 1992; Ozcelebi and Miller, 1995). In addition, there are conserved cysteines in the first and second extracellular loops (ECLs) of both CCK1 and CCK2 receptors (Figs. 2 and 3), which may form a disulfide bridge required for stabilization of their tertiary structure (Silvente-Poirot et al., 1998), and another cysteine in the C terminus may serve as a membrane-anchoring palmitoylation site (O'Dowd et al., 1988; Ovchinikov et al., 1988).



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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.



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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.

The CCK1 receptor cDNA has subsequently been cloned from guinea pig gallbladder, pancreas, and gastric chief cell (de Weerth et al., 1993b), human gallbladder (de Weerth et al., 1993a; Ulrich et al., 1993), and rabbit gastric (Reuben et al., 1994) cDNA libraries using either low-stringency hybridization or polymerase chain reaction methods. The CCK1 receptor is highly conserved among these species with an overall amino acid homology of 80% and a pairwise amino acid sequence identity of 87 to 92% in humans, guinea pig, rat, and rabbit (Table 1).


                              
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TABLE 1
SwissProt accession numbers for the cloned receptors from various species

2. Antagonists of CCK1 Receptors. Several structurally different CCK1 receptor antagonists have been synthesized. They belong to various series of chemicals, including dipeptoid, benzodiazepine, pyrazolidinine, and amino acid derivatives, and have both excellent selectivity and high affinity for CCK1 receptors.

The first CCK antagonists were derived from a naturally occurring benzodiazepine, asperlicin (Table
2), which has been isolated from the fungus Aspergillus alliaceus (Chang et al., 1985). The demonstrated high in vitro and in vivo potency of asperlicin at CCK1 receptors conferred clear advantages over previously reported CCK antagonists as a tool for investigation of the physiological and pharmacological actions of CCK. The first analogs of asperlicin were designed to assess which structural features of asperlicin could be modified to further enhance its CCK inhibitory potency without compromising its CCK1 selectivity. Unfortunately, this approach failed to overcome the key defects of asperlicin (Bock et al., 1986). Interestingly, asperlicin contains elements of the 1,4-benzodiazepine ring system found in antianxiety agents such as diazepam. On the other hand, several studies support the concept that the natural ligand for the antianxiety benzodiazepine receptor is a peptide (Guidotti et al., 1983; Alho et al., 1985), suggesting that the 5-phenyl-1,4-benzodiazepine ring is in fact a chemical structure that recognizes a peptide receptor. This explains why the 5-phenyl-1,4-benzodiazepine ring was proposed as the basis for the design of improved CCK receptor antagonists (Evans et al., 1986). Indeed, the 3-amino-5-phenyl-1,4-benzodiazepin-2-one derivatives, typified by L-364,718 (MK-329, devazepide; Tables 2 and 3), remained for several years the most potent CCK antagonists described with a good selectivity for CCK1 receptors (IC50 CCK2/CCK1 = 3750).


                              
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TABLE 2
CCK1 receptor antagonists    


                              
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TABLE 3
Affinities of CCK1 receptor antagonists in brain and pancreas membranes

Various tricyclic 1,4-benzodiazepine derivatives were also developed. On the basis of structure-activity relationship studies, as well as the stability and availability of the starting materials of those compounds, (S)-N-[1-(2-fluorophenyl)-3,4,6,7-tetrahydro-4-oxo-pyrrolo[3,2,1-jk][1,4]benzodiazepin-3-yl]-1H-indole-2-carboxamide (FK-480; Satoh et al., 1994; Tables 2 and 3) was selected as a candidate for further evaluation. The results obtained showed that FK-480 is a highly selective and potent CCK1 receptor antagonist (Akiyama and Otsuki, 1994; Ito et al., 1994a).

Several other potent and selective antagonists of the CCK1 receptor have been described, including glutamic acid derivatives such as loxiglumide (CR-1505) or lorglumide (CR-1409; Makovec et al., 1985; Table 2), and partial sequences of the C-terminal region of CCK. The dipeptide, N-tert-butyloxycarbonyl-aspartyl-phenylalaninamide (Boc-Asp-Phe-NH2), representing the two-amino-acid C-terminal fragment common to both CCK and gastrin, is a low-affinity partial agonist at CCK2 receptors but has no activity at CCK1 receptors. This selectivity is abolished by removal of the C-terminal amide. Replacement of the N-tert-butyloxycarbonyl group in this dipeptide with an analog, the 2-naphthalene sulfonyl group, gave 2-naphthalenesulfonyl 1-aspartyl-(2-phenethyl)amide (2-NAP; Tables 2 and 3), which behaves as a competitive antagonist at CCK1 receptors. Interestingly, this compound has a 300-fold greater affinity for CCK1 than CCK2 receptors (Hull et al., 1993).

On the other hand, further development of "dipeptoids", initially characterized as CCK2 receptor antagonists (see below), led to a molecule that has a 100-fold selectivity for the CCK1 receptor, where it acts as a potent competitive antagonist (PD-140,548; Boden et al., 1993).

Several years ago, synthetic peptides with CCK1 receptor antagonist properties were described (Lignon et al., 1987). One of these compounds, designated JMV-179 [Tyr(SO3H)-Ahx-Gly-D-Trp-Ahx-Asp-phenylethylester], corresponds to the C-terminal heptapeptide of CCK in which the phenylalamide and the L-tryptophan residues were substituted by a phenylethyl ester and a D-tryptophan, respectively. In addition, to protect the peptide against oxidation, the two methionines were replaced by a 6-aminohexanoic acid (Ahx) residue. The pharmacological results obtained demonstrated that JMV-179 is a full CCK1 receptor antagonist. In contrast, JMV-180 [Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-phenylethylester] appeared to be an agonist of the stimulatory phase of the amylase release by pancreatic acini (low concentration range) and an antagonist of the inhibitory phase (high concentrations; Galas et al., 1988).

A new serine derivative, (R)-1-[3-(3-carboxypyridine-2-yl)-thio-2-(indol-2-yl)carbonylamino]propionyl-4-diphenylmethylpiperazine (TP-680)] has been recently developed (Akiyama et al., 1996; Tables 2 and 3). This compound showed approximately 2 and 22 times greater selectivity for CCK1 receptors relative to CCK2 receptors than L-364,718 and loxiglumide, respectively. Pharmacological data showed that TP-680 is a selective and irreversible antagonist of CCK1 receptors (Akiyama et al., 1996).

Other CCK1 receptor antagonists have been developed, such as T-0632 (Tables 2 and 3), which is a novel nonpeptide and water-soluble compound that inhibits the specific binding of 125I-CCK-8 to rat CCK1 receptor in a concentration-dependent and competitive manner. The Ki value of T-0632 for the CCK1 receptor, 0.24 nM, is 23,000-fold less than its Ki value (5,600 nM) for the CCK2 receptor (Taniguchi et al., 1996).

Interest in nonpeptide CCK receptor-selective ligands has directed efforts toward the incorporation of conformationally restricted structures as spacers between Trp and Phe residues in the sequence of the CCK2 receptor endogenous ligand CCK-4 (Trp-Met-Asp-Phe-NH2). Thus, recently, a new series of CCK-4 restricted analogs with a 3-oxoindolizidine ring were synthesized. The most remarkable results were obtained with IQM-95,333 (Tables 2 and 3), which displays a CCK1 receptor affinity (Ki = 0.62 nM) similar to that of L-364,718, but with a much higher selectivity (Ki CCK2/Ki CCK1 > 8000; Martin-Martinez et al., 1997).

Another CCK1 receptor antagonist, SR-27,897 (Tables 2 and 3), which is chemically unrelated to peptoids, benzodiazepines, or glutamic acid derivatives, has been developed. This compound was obtained by optimization of a lead compound discovered through the random screening of a large chemical library. SR-27,897 is a highly potent (Ki = 0.2 nM) and selective (CCK2/CCK1 IC50 = 800) antagonist of CCK1 receptors (Gully et al., 1993).

3. Agonists of CCK1 Receptors. Only a few compounds have been reported to be CCK1-selective agonists; most of them are tetrapeptides, hexapeptides, and benzodiazepine derivatives.

Two series of CCK analogs have been developed. One series, exemplified by A-71378 [des-NH2-Tyr(SO3H)-Nle-Gly-Trp-Nle-(NMe)Asp-Phe-NH2], contains an (NMe)Asp residue that is critical for CCK1 receptor selectivity (Holladay et al., 1992
). The other series derived by replacement of the methionine residue of Boc-CCK-4 (Boc-Trp-Met-Asp-Phe-NH2) with side chain-substituted Lys derivatives: Boc-Trp-Lys(X)-Asp-(NMe)Phe-NH2, such as A-71623 (X = o-toluylaminocarbonyl [Tac]) and A-70874 (X = p-hydroxycinnamoyl [Hyc]; Lin et al., 1991; Tables 4 and 5). Exploration of this tetrapeptide series continued through the examination of the effects of N-methylation at the Asp residue. The results obtained showed that analogs containing either (NMe)Asp or (NMe)Asp-(NMe)Phe are highly potent (IC50 values in the nanomolar range) and selective CCK1 receptor agonists (Holladay et al., 1992).


                              
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TABLE 4
CCK1 receptor agonists
des-NH2-Tyr(SO3H)-Nle-Gly-Trp-Nle-(NMe)Asp-Phe-NH2 A-71378 Boc-Trp-Lys(o-tolylaminocarbonyl)-Asp-MePhe-NH2 A-71623 Boc-Trp-Lys(p-hydroxycinnamoyl)-Asp-(NMe)Phe-NH2 A-70874 4-hydroxyphenylacetyl(SO3H)-Nle-Gly-Trp-Nle-(Me)Asp-Phe-NH2 ARL-15849 GW-5823 GW-7854    


                              
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TABLE 5
Affinities of CCK1 receptor agonists in brain and pancreas membranes

The sulfate ester of CCK-8 borne by the tyrosine residue is a critical determinant of the biological activity of this peptide. To increase the stability of this molecule, the sulfated tyrosine has been replaced by a synthetic amino acid (LD)-Phe(p-CH2SO3Na) in which the OSO3H group was replaced by the nonhydrolyzable CH2SO3H group. The biological activity of the new derivative (LD)-Phe(p-CH2SO3Na)-Nle-Gly-Trp-Nle-Asp-Phe-NH2 displays high affinity for CCK1 and CCK2 receptors (nanomolar range; Marseigne et al., 1989).

In the hexapeptide series, it has also been reported that replacement of Asp-Tyr(SO3H) of CCK-8 with Hpa(SO3H) (Hpa is 4-hydroxyphenylacetyl) and N-methylation of Phe do not diminish the affinity for CCK1 or CCK2 receptors (Pierson et al., 1997). Inversion of the chirality of Asp7 in conjunction with N-methylation of Phe8 produces a compound [Hpa(SO3H)-Met-Gly-Trp-Met-D-Asp-MePhe-NH2] that exhibits high affinity and 2100-fold selectivity for CCK1 receptors. Moreover, moving the N-methyl group from Phe to Asp decreased the affinity for CCK2 receptors without affecting that for CCK1 receptors, giving a compound Hpa(SO3H)-Nle-Gly-Trp-Nle-MeAsp-Phe-NH2 (ARL-15849; Tables 4 and 5) with a 6600-fold higher selectivity for the latter receptors (Pierson et al., 1997).

Recently, a series of 1,5-benzodiazepines acting as CCK1 receptor agonists in vitro and in vivo were discovered. Potency within this series was modulated by substituents on the N1-anilinoacetamide moiety (Aquino et al., 1996), with substitution and/or replacement of the C3-position phenylurea moiety (GW5823, GW7854; Hirst et al., 1996; Willson et al., 1996; Henke et al., 1997; Tables 4 and 5).

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).

Using low-stringency hybridization methods, the CCK2 receptor cDNA was cloned from a rat pancreatic acinar carcinoma cell line (AR4-2J) cDNA library known to express CCK2/gastrin receptors. This cDNA was shown to be identical with the CCK2 receptor cDNA cloned from a rat brain cDNA library (Wank et al., 1992b). At the same time, the gastrin receptor cDNA was also cloned from a canine parietal cell cDNA library using a COS cell plasmid expression approach (Kopin et al., 1992). The rat and canine CCK2 receptors are 452 and 453 amino acids long, respectively, and share an 84% amino acid identity. This degree of homology is consistent with interspecies variations of the same receptor and has been considered as an early indication that the gastrin receptor is simply the CCK2 expressed in the stomach (see below). Similar to the CCK1 receptor, hydropathy analysis predicts seven TM domains as expected of a member of the GPCR superfamily (Dohlman et al., 1991). The sequence contains at least three consensus sites for N-linked glycosylation (Asn-X-Ser/Thr), consistent with the heavy and variable degree of glycosylation reported using ligand affinity crosslinking techniques (Baldwin et al., 1986; Chiba et al., 1988; Baldwin, 1993). Similar to the CCK1 receptor, there are conserved cysteines in the first and second ECLs that may form a disulfide bridge required for stabilization of the tertiary structure (Silvente-Poirot et al., 1998), and a cysteine in the C terminus of the receptor may serve as a membrane-anchoring palmitoylation site (O'Dowd et al., 1988; Ovchinikov et al., 1988; Fig. 3).

To date, the CCK2 receptor has been cloned through low-stringency hybridization of cDNA libraries from various sources: rat brain and stomach, the pancreatic tumoral cell line AR4-2J (Wank et al., 1992b), human brain (Pisegna et al., 1992; Ito et al., 1993; Lee et al., 1993; Denyer et al., 1994) and stomach (Pisegna et al., 1992), and guinea pig gallbladder and stomach (de Weerth et al., 1993b). In addition, CCK2 receptor cloning has been achieved from gastric enterochromaffin and parietal cells and brain of Mastomys natalensis (Nakata et al., 1992), calf pancreas (Dufresne et al., 1996), and a rabbit genomic library (Blandizzi et al., 1994; Table 1). The CCK2 receptor is highly conserved in humans, canine, guinea pig, calf, rabbit, M. natalensis, and rat, with an overall amino acid identity of 72% and pairwise amino acid sequence identities of 84 to 93%.

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).

Efforts were notably devoted to the design of an optimized asperlicin structure. Because the asperlicin structure is composed of several heterocyclic domains, it was hypothesized that alternative substructures embedded within the molecular framework of this natural product may provide a rational starting point for the design of novel nonpeptide CCK receptor ligands. On this basis, scientists at Eli Lilly Corp. developed a series of quinazoline derivatives by using a bond disconnection approach (Yu et al., 1991). A combination of the key fragments of the Lilly and Merck series led to the development of novel nonpeptide CCK2 receptor antagonists with substitution on the quinazolinone and phenyl rings. Binding data for this class of compounds suggest that the linker between these rings is a critical determinant for CCK2 receptor-binding affinity. However, these new compounds have a low selectivity for CCK2 receptor (Padia et al., 1997). Indeed, the spatial arrangement of the two moieties appears to be critical for both potency and selectivity. The introduction of ---NH--- as a linker significantly enhanced CCK2 receptor-binding affinity and selectivity, providing compounds with nanomolar binding affinity and good selectivity (Ki CCK1/Ki CCK2 > 500). Moreover, these compounds are active when administered per os (Padia et al., 1998).

On the other hand, the moderate affinity of L-364,718 for CCK2 receptors suggested that the benzodiazepine nucleus might also hold a key to selective ligands for these receptors. The first compound of interest developed using this strategy was L-365,260 (Tables 6 and 7), which revealed to be the first potent and selective non- peptide CCK2 receptor antagonist (Bock et al., 1989). One factor that determined CCK receptor selectivity in this series was the C3-stereochemistry of the benzodiazepine ring system, with the (3R)-enantiomer generally providing CCK2 receptor selectivity. Moreover, recent studies have shown that the C5-phenyl moiety of the core benzodiazepine structure could be replaced by C5-cycloalkyl groups, a modification that retained CCK2 receptor affinity and selectivity. In particular, the C5-cyclohexyl analog displayed subnanomolar affinity for CCK2 receptors (IC50 = 0.28 nM), with improved selectivity (Ki CCK1/Ki CCK2 = 6500) compared with L-365,260 (Chambers et al., 1993).


                              
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TABLE 6
CCK2 receptor antagonists    


                              
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TABLE 7
Affinities of CCK2 receptor antagonists in brain and pancreas membranes

A major drawback associated with these early benzodiazepine-derived CCK2 antagonists was their limited bioavailability and inactivity via the oral route of administration. The incorporation of a (tert-butylcarbonyl) methyl group at the 1-position (Semple et al., 1996a) or a 2-pyridyl group at the 5-position (Semple et al., 1996b) of the parent benzodiazepine structure provides a significant increase in absorption. Similar results have been achieved through the incorporation of an amine-based cationic solubilizing group within the benzodiazepine framework, with a cyclic amine to form an amidino functionality in the 5-position (L-740,093; Showell et al., 1994; Tables 6 and 7). Other attempts to improve aqueous solubility included the introduction of acidic groups (L-368,935 and L-369,466; Bock et al., 1994) or lipophilic surrogates (Chambers et al., 1995) into the 3-position of the aryl urea component of either the 1,4-benzodiazepin-2-one parent system or closely related structures (CP-212,454; Lowe et al., 1995; Tables 6 and 7). The opposite strategy has also been used with the introduction of basic amino substituents into the same region. YM022 is the optimal structure of this new series, with subnanomolar affinity for CCK2 receptors (Nishida et al., 1994). Moreover, when these modifications are combined within the same molecule, the resulting improvements in the in vivo effects appear to be essentially additive, as shown by the compound YF476 (Tables 6 and 7), which has a good oral bioavailability in dogs (Semple et al., 1997).

Other nonpeptide CCK2 receptor antagonists have been developed, derived through rational design from the CCK tetrapeptide (Hughes et al., 1990). This led to tryptophan dipeptoid derivatives such as PD-134,308 (CI-988; Tables 6 and 7) with nanomolar affinity for CCK2 receptors (Horwell, 1991; Horwell et al., 1991). PD-134,308 exhibits a 1600-fold selectivity for CCK2 over CCK1 receptors. C-terminal modifications of this compound led to molecules with subnanomolar affinity for CCK2 receptors. For example, further attempts to optimize the substitution on the phenyl ring led to a compound 19, which has an extraordinarily high affinity for the CCK2 receptor (IC50 = 0.08 nM) and a high degree of selectivity (Ki CCK1/Ki CCK2 = 940; Augelli-Szafran et al., 1996). A direct comparison of the structure of the dipeptoid derivatives showed that the size of these molecules could be reduced to increase their lipophilicity. Such compounds have been synthesized, and some of them have been found to be potent and selective CCK2 receptor antagonists. Moreover, as expected, one of them (RB 211) was shown to be more efficient in crossing the blood-brain barrier than the parent compounds (Blommaert et al., 1993) and devoid of the weak CCK1 receptor agonist properties of dipeptoids (Höcker et al., 1993; Ding et al., 1995). On the other hand, to improve the properties of PD-134,308, numerous conformational restrictions were introduced in its structure. Unfortunately, neither N-terminal cyclization (Fincham et al., 1992b), macrocyclization (Didier et al., 1992; Bolton et al., 1993), nor rigidification of the amide bond (Fincham et al., 1992a) led to any positive result. Only a C-terminal cyclization of PD-134,308 derivatives, by means of a tetrahydronaphtyl group, has been reported to increase the affinity for CCK2 receptors (Higginbottom et al., 1993). This approach has also been used for compounds such as RB 210 (Tables 6 and 7), in which C-terminal constraints can be easily introduced. Thus, the beta -carbon of the phenethyl side chain of RB 210 was linked to the alpha -carbon bearing the carbonyl function, by means of a methylene bridge. This resulted in the formation of a proline ring (Bellier et al., 1997). The most potent compounds of this new series had similar affinities for CCK2 receptors as RB 210. Structure-affinity relationships of this series indicated that lengthening of the distance between the amide nitrogen atom and the phenyl ring was of little importance, whereas the position of the carboxylate group could not be modified. Therefore, the pyrrolidine ring was replaced by piperidine to slightly modify the possible orientation of the aromatic moiety toward the carboxylate without violating any of the requirements previously established in both linear and constrained series for the recognition of CCK2 receptors. However, the resulting compounds behave as moderately potent CCK2 receptor antagonists (Bellier et al., 1998).

As previously mentioned, the clinical development of PD-134,308 (CI-988) was limited due to its poor bioavailability, which was attributed to poor absorption and efficient hepatic extraction. Scientists at Parke-Davis also envisaged that reducing the molecular weight of the parent compound would lead to better absorption. Thus, they synthesized a series of analogs in which the key alpha -methyltryptophan and adamantyloxycarbonyl moieties, required for receptor binding, were kept intact and the C terminus was extensively modified. These modifications led to compounds such as CI-1015 (Tables 6 and 7) for which the oral bioavailability in rat was improved nearly 10-fold and the blood-brain barrier permeability was also enhanced relative to CI-988 (Trivedi et al., 1998).

Two other series have been described, leading to the synthesis of derivatives that have both excellent selectivity and high affinity for CCK2 receptors: the ureidoacetamide class of CCK2 receptor antagonists (RP-73,870; Pendley et al., 1995) and the pyrazolidinones (LY-288,513; Howbert et al., 1992; Tables 6 and 7). Development of the latter series has been discontinued because of adverse effects in preclinical toxicological studies. The nonpeptide ureidoacetamides are potent and selective ligands with nanomolar or subnanomolar affinities for CCK2 receptors and a 100- to 1000-fold selectivity for these receptors over CCK1 receptors. Despite its relatively poor oral bioavailability, RP-73,870 was as potent as other antiulcer compounds after oral administration in a duodenal ulceration model (Pendley et al., 1995).

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[Nle28,31]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 (Ki = 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 alpha - or beta -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-Nle28,31]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).


                              
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TABLE 8
CCK2 receptor agonists


                              
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TABLE 9
Affinities of CCK2 receptor agonists in brain and pancreas membranes

The role of the amino acid in position 31 of CCK-8 in the recognition of CCK1 and CCK2 receptors was investigated through the replacement of Met31 by amino acids with side chains of varying chemical nature. Thus, the introduction of a Phe residue in position 31 in Boc[Nle28,31]CCK27-33 slightly modified the affinity for CCK2 receptor (Ki = 3.7 nM) but led to a larger decrease (Ki = 220 nM) in the affinity for CCK1 receptors. A similar discrimination was observed when the amino acid in position 31 is an alanine residue (Marseigne et al., 1988).

Because nonpeptide ligands have historically offered greater opportunity for manipulation of both pharmacodynamic (selectivity and efficacy) and pharmacokinetic (oral bioavailability, duration) parameters, the development of nonpeptidic CCK2 receptor selective agonists endowed with good stability and bioavailability should provide useful pharmacological tools and possibly therapeutic agents. To design such derivatives, the C-terminal tetrapeptide CCK-4 appeared to be a good molecule to start with, because of its significant CCK2 receptor affinity and selectivity, although it has been shown to trigger panic attacks in humans (de Montigny, 1989; Bradwejn et al., 1991b). Several modifications were made to CCK-4, such as the N-terminal protection of the tetrapeptide in Boc-CCK4 (Harhammer et al., 1991) or modifications of the different amino acids such as the replacement of Met by Nle or (NMe)Nle (Corringer et al., 1993). Recent NMR and molecular dynamics studies indicated that the CCK2 receptor-selective CCK-4 analogs adopt an S-shaped conformation with a relatively well-defined orientation of the side chains (Goudreau et al., 1994). The same type of folded structures has been reported for several potent agonists derived from CCK-4 and containing a [trans-3-propyl-L-proline] (Nadzan et al., 1991), a diketopiperazine skeleton (Shiosaki et al., 1990), or a [(alkylthio)proline] residue (Kolodziej et al., 1995). With this template, other cyclic CCK-4 analogs have been synthesized in which the Trp-Met dipeptide was changed to a diketopiperazine moiety resulting from a cyclization between Nle and N-substituted (D)Trp residues and coupled with a small linker to Asp-Phe-NH2 (Weng et al., 1996a). Moreover, the side chain of Nle in the compound Boc-Trp-(NMe)Nle-Asp-Phe-NH2 together with the N terminus of Trp appeared to be good candidates for another possible cyclization. Thus, cyclic compounds were designed through molecular modeling to mimic the proposed biologically active conformation of these CCK-4 analogs. The goal of this study was to stabilize the bioactive conformation of CCK2 receptor agonists to aid in the design of nonpeptide ligands. This led to the development of macrocyclic constrained CCK-4 analogs that are endowed with agonist properties and able to cross the blood-brain barrier (Blommaert et al., 1997).

Selective and peptidase-resistant CCK2 receptor ligands that derive from Boc-[Nle31]CCK30-33 through the incorporation of non-natural hydrophobic amino acids have also been developed (Weng et al., 1996b). Among these compounds, Boc-[Phg31,Nal33]CCK30-33 proved to be a full agonist at rat hippocampal CCK2 receptors. Moreover, it appeared that modifications of the hydrophobic and steric character of either the C- or N-terminal amino acid substituents of CCK-4 derivatives could affect the agonist or antagonist profile of these peptides. This was shown by the fact that the agonist Boc-[Phg31,Nal33]CCK30-33 could be chemically converted to an antagonist through the addition of two alkyl groups on the terminal CONH2 (Weng et al., 1996b).

Very recently, a new series of highly potent and selective CCK2 receptor agonists were developed (Million et al., 1997). Boc-Trp-(NMe)Nle-Asp-Phe-NH2, the C-terminal tetrapeptide of BC 264, was shown to have a high affinity and to behave as a specific agonist at CCK2 receptors and to adopt the S-shaped preferential conformation. To determine the essential structural components of specific CCK2 receptor agonists, a step-by-step lengthening of the C-terminal tetrapeptide of BC 264 was carried out. Various diacidic moieties, such as malonate or succinate residues, were coupled to the N-terminal portion of the tetrapeptide, leading to RB 400 [HOOC-CH2-CO-Trp-(NMe)Nle-Asp-Phe-NH2] and RB 403 (Tables 8 and 9). RB 400 was also derivatized under its benzylamide and methyl ester forms. Compounds that belong to the RB 400 series possess high affinities for the CCK2 receptor, with a subnanomolar affinity (Ki = 0.42 nM) being obtained in case of RB 400 itself (Million et al., 1997).


    III. Molecular Biology of CCK Receptors
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A. CCK Receptor Gene Structure

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).



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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 Delta 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). Delta 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 Delta CCK2 receptor (Fig. 4). The first methionine of exon 2, which is common to both CCK2 and Delta CCK2 receptors, serves as the translational initiation site for the Delta CCK2 receptor. Delta 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 Delta 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).

B. Chromosomal Localization of CCK Receptor Genes

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).

C. Animal Models without Detectable Levels of CCK Receptors

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.

In the pancreas, CCK is well known to be a major regulatory peptide that stimulates digestive enzyme secretion. The mode of action of CCK has been extensively explored. CCK-stimulated enzyme secretion is believed to be initiated by the binding of CCK to CCK1 receptors localized on pancreatic acinar cells. Furthermore, it has been shown that the breakdown of phosphatidylinositol 4,5-biphosphate, which thereby produces both diacylglycerol and inositol trisphosphate (IP3), is activated by CCK1 receptor stimulation. Subsequent activation of Ca2+ phospholipid-dependent protein kinase by diacylglycerol and intracellular Ca2+ mobilization induced by IP3 have been considered to act synergistically to cause digestive enzyme secretion (Pandol et al., 1985). The insensitivity of CCK1 receptor inositol phosphate signaling to pertussis toxin suggests that its couples through the Gq family of G proteins (Pang and Sternweiss, 1990). Recently, a study using both phospholipase C (PLC) and G protein alpha -subunit-specific antibodies indicated that both Gq and G11alpha are present in pancreas and that the CCK1 receptor couples to Gq or G11 to activate PLC-beta 1 in pancreatic cell membranes (Piiper et al., 1997).

On the other hand, it has been demonstrated in rat pancreatic acini that the CCK1 receptors are coupled to the phospholipase A2 (PLA2)/arachidonic acid pathways to mediate Ca2+ oscillations and amylase secretion (Yule et al., 1993; Yoshida et al., 1997). Nevertheless, other studies have shown that there are at least two pathways responsible for the increased production of arachidonic acid in response to CCK1 receptor stimulation. One is the sequential effects of phospholipase C (PLC) and diglyceride lipase on phosphatidylinositol, and the other involves the action of the PLA2 effect on phosphatidylcholine. Both pathways cause stimulation of amylase release (Pandol et al., 1991). In addition to the activation of the PLC and PLA2 signal-transduction pathways, CCK1 receptor stimulation can lead to an increase in the adenylyl cyclase signal-transduction cascade (Marino et al., 1993).

Thus, CCK1 receptor is capable of coupling to both PLC and adenylyl cyclase at physiological concentrations in native cells. It is not clear whether this is a result of the independent coupling of CCK1 receptor to Gs and Gq or simply the result of G protein beta gamma -subunit activation of an isotope of adenylyl cyclase. A study using a chimeric CCK receptor in which the first intracellular loops between CCK1 and CCK2 receptors were exchanged showed that Arg68 and Asn69 belonging to the loop of CCK1 receptor are important for the stimulatory coupling of this receptor with adenylyl cyclase but are not involved in its coupling with Gq. These results support the idea that the CCK1 receptor is directly coupled with both Gs and Gq (Wu et al., 1997).

Recent studies (for reviews, see Müller and Lohse, 1995; Daaka et al., 1997) have shown that some GPCRs use the same effectors as those of the tyrosine kinase receptor pathway [e.g., Shc (adapter protein)/growth factor receptor-bound protein 2/product of son of sevenless (SOS)], resulting in Ras and mitogen-activated protein kinase (MAPK) activation and leading to expression of transcriptional factors, such as c-myc, c-jun, and c-fos. It was recently shown that MAPKs and c-Jun NH2-terminal kinases (JNKs, which phosphorylate serine residues of c-Jun) are rapidly activated by CCK-8 in rat pancreas both in vitro and in vivo (Dabrowski et al., 1996a,b; Tateishi et al., 1998). These results suggest that CCK might stimulate cell proliferation via its action at CCK1 receptors. Moreover, the activation of both MAPKs and JNKs may be of importance in the early pathogenesis of acute pancreatitis (Dabrowski et al., 1996a). The mechanism by which the Gq protein-coupled CCK receptor activates Ras is not well understood. Results obtained by Dabrowski et al. (1996b) suggest that formation of Shc/growth factor receptor-bound protein 2/SOS complex via a PKC-dependent mechanism may provide the link between Gq protein-coupled CCK receptor stimulation and Ras activation.

A case report of a woman with gallstones and obesity was ascribed to abnormal processing of transcripts from a normal CCK1 receptor gene that resulted in the predominance of mRNA with a 262-bp deletion corresponding to the third exon. Although this mutation could negatively affect expression or coupling to G proteins, neither in vivo nor in vitro data were obtained in support of such inferences. Unfortunately, other affected family members were not examined and expected splicing abnormalities in transcripts from other genes were not studied, so only an association could be established between the common phenotype of gallstones and obesity and the putative RNA processing abnormality in the affected patient (Miller et al., 1995).

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).

In contrast to CCK1 receptors, the signal-transduction cascade for CCK2 receptors has been rather poorly characterized, in large part because of the difficulty of working with isolated neurons or isolated gastric mucosal cells expressing CCK2 receptors. Thus, for a long time, central CCK2 receptors have not been proved to be linked to a well characterized second-messenger system in the brain, including the phosphoinositide system, although phosphoinositide metabolism was shown to be affected by CCK in neuroblastoma (Barrett et al., 1989) and in the embryonic pituitary cell line (Lo and Hughes, 1988). More recently, Zhang et al. (1992) showed that CCK-8 increased the turnover of phosphoinositides and IP3 labeling in dissociated neonatal rat brain cells, in which both CCK1 and CCK2 receptors were expressed. One study of CCK2 receptors, using synaptoneurosomes from guinea pig cortex, failed to provide support to their possible coupling with adenylyl cyclase or PLC, although Ca2+ release from intracellular stores, possibly via a G protein-independent mechanism, could be triggered by a CCK analog (Galas et al., 1992).

Expression of receptor cDNAs in a mammalian expression system allows for a readily available source of receptor for functional studies. In transfected cells (Cos, Chinese hamster ovary), it has been shown that like the CCK1 receptor, the CCK2 receptor couples to a pertussis toxin-insensitive G protein (Roche et al., 1990) that is probably related to the Gq/11 family, thereby causing activation of PLC (Tsunoda et al., 1988a,b, 1989; Delvalle et al., 1992). The region of the CCK2 receptor interacting with Gq was determined in CCK2 receptor with Lys333 Met, Lys334Thr, and Arg335Leu mutations transiently expressed in COS-7 cells and X. laevis oocytes. Indeed, these mutations resulted in the loss of Gq activation without affecting receptor affinity (Wang, 1997).

Site-directed mutagenic replacement of Asp