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Institut National de la Santé et de la Recherche Médicale U. 531, Institut Louis Bugnard, Centre Hospitalier Universitaire Rangueil, Toulouse, France
ABSTRACT I. INTRODUCTION II. THE CHOLECYSTOKININ RECEPTORS A. Nomenclature B. cDNAs Cloning and Deduced Protein Structures C. Genes D. Binding Properties and Ligands III. STRUCTURE-FUNCTION RELATIONSHIP OF CHOLECYSTOKININ RECEPTORS A. Localization of Ligand Binding Sites B. Activation Mechanism and Regulation IV. SIGNALING TRANSDUCTION PATHWAYS ACTIVATED BY CHOLECYSTOKININ RECEPTORS A. Phospholipases/Calcium Mobilization and Protein Kinase C Activation B. Adenyl Cyclase and cAMP Production C. Nitric Oxide and cGMP Pathway D. Mitogen-Activated Kinase Cascades E. Phosphatidylinositol 3-Kinase F. Focal Adhesion Kinase and Associated Proteins G. The JAK/STAT Pathway H. Other Signaling Molecules 1. Small GTPases 2. NFkappaB/IkappaB V. TARGET GENES OF CHOLECYSTOKININ RECEPTORS A. Gastrin-Dependent Gene Regulation B. CCK-Dependent Gene Regulation VI. TISSUE DISTRIBUTION AND PHYSIOLOGICAL ACTIONS OF CHOLECYSTOKININ RECEPTORS IN THE GASTROINTESTINAL TRACT AND OTHER PERIPHERAL ORGANS A. Gastric Mucosae, Exocrine and Endocrine Pancreas 1. Stomach 2. Exocrine pancreas 3. 3. Endocrine pancreas 4. 4. Liver and intestine B. Gastrointestinal Smooth Muscles 1. Gallbladder 2. Stomach 3. Bowel C. Other Peripheral Organs and Tissues 1. Adipocytes 2. Adrenal gland 3. Blood mononuclear cells 4. Kidney 5. Vagal afferent fibers VII. PATHOLOGICAL ACTIONS OF PERIPHERAL CHOLECYSTOKININ RECEPTORS AND THEIR RELEVANCE TO CLINICAL DISORDERS IN HUMANS A. Digestive and Metabolic Diseases 1. Peptid ulcer 2. Irritable bowel syndrome 3. Gallbladder diseases 4. Pancreatitis 5. Obesity B. Cancers 1. 1. Gastric cancer 2. Pancreatic adenocarcinoma 3. Barrett's esophagus and esophageal adenocarcinoma 4. Other tumors expressing CCK receptors 5. Colon cancer and gastrin precursors C. Peptide and Receptor Targeting 1. Gastrimmune 2. CCK receptors VIII. CONCLUSION/PROSPECTS ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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II. THE CHOLECYSTOKININ RECEPTORS |
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Two types of CCK receptors (type A, "alimentary," and type B, "brain") have been identified on a pharmacological basis. The CCK-A receptor was first characterized in pancreatic acini from rodents (95, 120, 224, 415, 432), whereas the CCK-B receptor was first found in the brain (217, 427). Based on recommendations of the International Union of Pharmacology (IUPHAR) committee regarding receptor nomenclature and drug classification, the CCK-A receptor has been renamed CCK1 receptor (CCK1R), and the CCK-B receptor has been renamed CCK2 receptor (CCK2R). CCK1R binds and responds to sulfated CCK with a 500- to 1,000-fold higher affinity or potency than sulfated gastrin or nonsulfated CCK. The CCK2R binds and responds to gastrin or CCK with almost the same affinity or potency and discriminates poorly between sulfated and nonsulfated peptides. In the periphery, the CCK2R can be considered as the "gastrin receptor" (see sect. IID).
B. cDNAs Cloning and Deduced Protein Structures
Historically, a cDNA encoding gastric gastrin receptor was originally cloned by A. S. Kopin and co-workers (253) through expression of a canine parietal cell cDNA library in COS-7 cells and isolation of a cDNA clone encoding a 453-amino acid protein. Binding of 125I-Bolton-Hunter-CCK-8 to COS-7 cells expressing this protein was inhibited by CCK- and gastrin-related peptides with potencies in agreement with CCK2R pharmacology. However, the nonpeptide antagonist for the CCK1R (L-364,718) competed with labeled CCK-8 to the recombinant receptor with higher potency (19 nM) than the nonpeptide antagonist of the CCK2R (L-365,260, 130 nM), but this atypical pharmacological feature was further shown to be species specific (38). In addition to binding parameters, intracellular Ca2+ mobilization, inositol 1,4,5-trisphosphate elevation in response to gastrin, and affinity labeling of the 76,000 component confirmed that the cloned receptor was indeed the gastric gastrin receptor. The human brain CCK2R cDNA was then isolated and sequenced, providing evidence that the brain and gastric CCK2R represent a unique molecular entity encoded by a single gene (219, 273, 380).
Report of the cloning of the pancreatic rat CCK1R cDNA by S. A. Wank appeared in the same issue of the Proceeding of the National Academy of Sciences (USA) as that of the canine CCK2R (559). Indeed, Wank and colleagues purified to homogeneity a sufficient amount of receptor protein to obtain partial sequences of five fragments. From these peptides, degenerate oligonucleotides were designed and used as primers to amplify, by polymerase-chain reaction, a cDNA fragment, which was then utilized as a probe to screen a cDNA library obtained from rat pancreas. A complete cDNA, with an open reading frame encoding a protein of 444 amino acids, was thus cloned. In vitro transcripts of the cloned cDNA were injected into Xenopus oocytes and shown to display CCK-induced chloride currents that were inhibited by a specific antagonist of the CCK1R (559).
The CCK1R and CCK2R exhibit a relatively low degree of sequence homology (50%) but present seven hydrophobic segments, likely corresponding to transmembrane domains, with extracellular NH2-terminal and intracellular COOH-terminal ends. Such structures are characteristic of G protein-coupled receptors (GPCRs) in agreement with high-resolution three-dimensional structure of rhodopsin (366). Other sequence signatures of members of the family I of GPCRs that are essential for receptor activation are also present in the CCK1R and CCK2R, such as an E/DRY motif at the bottom of transmembrane domain III, and a NPXXY motif within transmembrane domain VII. Cloning of cDNAs encoding CCK1R in guinea pig gallbladder and pancreas, mouse pancreas, rabbit stomach, and human gallbladder as well as those encoding CCK2R in the rat pancreatic cancer cell line AR4–2J, an enterochromaffin-like carcinoid tumor of Mastomys natalensis, rat stomach, and bovine pancreas, demonstrated a high degree of sequence homology well within the range expected for interspecies variations of the same receptor type (122, 140, 165, 345, 403, 534, 560). CCK1R and CCK2R contain three to four potential N-linked glycosylation sites in their amino termini, which is consistent with a high and heterogeneous degree of glycosylation, experimentally noticed in both affinity and photoaffinity labeling experiments (152, 372). For instance, pancreatic native CCK1R, which migrates as a 85,000- to 100,000-Da component in SDS-PAGE, is shifted to a 38,000- to 42,000-Da protein after endoglycosidase-F treatment (152, 372). SDS-PAGE analysis suggested that the degree of glycosylation of affinity labeled CCK2R differs according to the cell type in which the receptor is expressed. Indeed, photoaffinity labeling of canine CCK2R in isolated parietal cells identified a component of 76,000 Da, while photoaffinity labeling of canine CCK2R in pancreatic membranes identified a component of 47,000 Da (153, 305). The bovine CCK2R, which appeared as a 42,000- to 47,000-Da protein in pancreatic membranes, was further identified as a 85,000-Da recombinant protein in COS-7 cells, with both components yielding a 37,000-Da protein by endoglycosidase-F treatment (140, 275). The precise role of the carbohydrate moieties of these receptors remains largely unknown.
The CCK1R gene is on human chromosome 4p15.1-p15.2 and mouse chromosome 5 (216, 430). CCK2R has been assigned to human chromosome 11p15.4 and to distal chromosome 7 in mice (216, 430). The transcriptional start site of human CCK1R was identified 206 bp upstream of the translation start site. In the mouse CCK2R gene, the transcriptional start site was identified at 199 bp upstream of the translation start site in a region devoid of any TATA-like sequences (157, 262). So far, no precise data have been reported on CCKR gene regulation. However, several studies have documented polymorphism in CCKR gene promoters associated with diseases such as panic disorder, alcohol dependence, and obesity (245, 325, 326, 555).
The two receptor genes are each organized into five exons (Fig. 2). In the CCK2R gene, the presence of an exon I variant within intron I was described (317). According to the authors, in some cells, especially cancer cells, exon I would be used alternatively to exon I leading to synthesis of transcripts encoding an NH2-terminally truncated CCK2R (317). Gene organization in exon/introns may theoretically lead to protein diversity. The existence of two mRNA isoforms for the CCK2R, produced by alternate splicing of exon 4 in human stomach, was also reported (491). Although this possibility exists and affects the sequence of the third intracellular loop by introducing a five-amino acid cassette, its functional significance remains controversial. In fact, recombinant human receptors encoded by these two isoforms are undistinguishable in terms of pharmacology and signal transduction features (218). Moreover, the ratio between the expression levels of the two isoforms was found to be 
1:99, supporting the view that the splice site in exon 4 of the human CCK2R gene is dominant (218). However, the nature of the predominant variant differs according to species: small variant in human; long variant in mouse, rat, dog, and calf (140, 219, 227, 253, 273, 380, 384, 559, 560). Additionally, a misspliced cDNA clone that encodes a receptor with retention of intron IV in the third intracellular loop (CCK2Ri4sv) was identified from colorectal and pancreatic tumors (130, 199). This misspliced variant seems to exhibit spontaneous ligand-independent oscillatory increases in intracellular Ca2+ and increases cell growth rate (199). Concerning CCK1R, the presence of a seven-amino acid glycine-rich cassette in the third intracellular loop of the murine receptor has been reported to contribute to species-specific aspects of signaling (384).
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Many studies have been devoted to the pharmacological characterization of naturally expressed, and more recently, of recombinant CCK receptors. Radiolabeled analogs of CCK and gastrin as well as tritiated antagonists have been used in such works. Binding experiments using labeled agonists as radioligands yielded the binding parameters of the G protein-coupled state of the receptor, whereas binding studies with labeled antagonists provided binding parameters of the resting and inactive state of the receptor. This explains, at least in part, why binding experiments with antagonists identified a greater number of binding sites compared with experiments using agonists. The binding affinities of CCK and gastrin agonists for the different affinity states of the CCK receptors are different. In general, Kd values for binding of CCK to high- and low-affinity sites of the CCK1R are in the range of 50–300 pM and 50–200 nM, respectively (224, 225, 432). While high-affinity sites are much less numerous than low-affinity sites, both components are functionally important, at least in pancreatic acinar cells (see sect. IVA). On the other hand, Kd values for high- and low-affinity binding of CCK to CCK2R are 100–300 pM and 2–5 nM, respectively (264). In addition to these two sites, there exist very-low-affinity sites as well with Kd values of 10 µM in both the CCK1R and CCK2R (213).
The natural ligand with the highest affinity for CCK1R is the sulfated octapeptide of CCK (CCK-8) (Fig. 1). Other natural molecular variants of CCK such as CCK-33, CCK-39, and CCK-58 bind to CCK1R with similar affinity to CCK-8 (396, 490). Gastrin, at physiological concentrations, is likely a poor activator of CCK1R, its affinity being 100- to 500-fold lower than that of sulfated CCK-8. Structure-activity relationship studies with synthetic CCK analogs have indicated that sulfation of the position 2 tyrosine in CCK-8 (Fig. 1) is critical for binding to CCK1R, since its removal causes a 500-fold drop in affinity. The two natural ligands with the highest affinities for CCK2R are sulfated gastrin-17 (often abbreviated G-17II) and sulfated CCK-8 (140, 214). Nonsulfated gastrin-17 (G-17I) exhibits a 3- to 10-fold lower affinity than sulfated gastrin-17. This affinity order and the fact that postprandial blood levels of gastrins are 5- to 10-fold more elevated than those of CCK lead one to consider that sulfated gastrin-17 is the preferred ligand and probably the physiological ligand of most of the peripheral CCK2R. On the other hand, due to the abundance of sulfated CCK-8 in the central nervous system, it is likely the ligand which naturally activates brain CCK2R. Compared with sulfated gastrin-17, the COOH-terminal tetrapeptide common to gastrin and CCK and nonsulfated CCK-8 interacts with the CCK2R with only a 10- to 50-fold decreased affinity.
The variety of physiological functions that can be regulated through the CCK receptors and their potential use as targets for the treatment of several human diseases have stimulated searches for specific, potent agonists and antagonists of these receptors. For most of these molecules, their chemistry and pharmacological properties are extensively described elsewhere (Table 1) (201, 351). Historically, amino acid derivatives such as benzotript and proglumide were the first CCK receptor antagonists reported. They were followed by peptidic analogs of CCK and gastrin and, more recently, by nonpeptide ligands with distinct chemical structures.
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Although the COOH-terminal tetrapeptide of CCK presents a low affinity for the CCK1R, chemical manipulation of this peptidic fragment successfully generated selective high-affinity agonists of this receptor which surprised scientists in the field who believed that sulfated tyrosine was required for optimal binding and activity of CCK at CCK1R. Substitution of Met by an o-tolylaminoacarbonyl-Lys residue and of Phe by (N-Me)Phe in CCK-4 were key modifications that gave these peptides a CCK1R personality (288). Interestingly, high-affinity agonists of CCK2R were obtained by replacement of Met by (NMet)Nle (351). Such work, which yielded a large variety of CCK-4 analogs, also indicated that minor structural differences in CCK-4-based peptides dictate affinity and selectivity for CCK1R and CCK2R.
In the benzodiazepine derivative family, L364,718 and L365,260 appeared as the first potent nonpeptide antagonists of CCK1 and CCK2R, respectively (82, 291). They were followed by a large series of potent compounds (201). Interestingly, from a drug design point of view, CCK1R antagonist L364,718 (MK-329) was generated from asperlicin, a fermentation product isolated from the fungus Aspergillus alliaceus, which presents micromolar affinity for CCK1R (83).
Several of the nonpeptide agonists and antagonists have reached phase I and II clinical trials. Indications for these compounds can be deduced from the pathophysiological role of CCK1R and CCK2R in humans (see sect. VII). In this context, one major question that recently arose with some of the nonpeptide antagonists was whether these were pure antagonists rather than partial agonists. Indeed, some so-called antagonists turned out to present some agonist activity in the stomach and pancreas and on cells transfected with the cDNAs encoding CCK1R or CCK2R (48, 257, 447). Presumably, newly generated molecules are more capable of efficiently blocking CCK1R- or CCK2R-related physiological effects (201).
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III. STRUCTURE-FUNCTION RELATIONSHIP OF CHOLECYSTOKININ RECEPTORS |
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A large set of converging data related to binding sites of cholecystokinin receptors is currently available, giving a good picture of the binding mode of natural and synthetic ligands to their cognate receptors. The data were provided using essentially four complementary approaches, site-directed mutagenesis, photoaffinity labeling, NMR-NOE transfer, and three-dimensional modeling. The binding site for CCK on the CCK1R is of particular interest due to the high selectivity of this receptor for sulfated versus nonsulfated CCK and for sulfated CCK versus gastrin. The two key interactions, which account for the 500- to 1,000-fold selectivity of CCK1R for sulfated versus nonsulfated CCK, involve a Met and an Arg in the second extracellular loop (169, 170). Proximity of the sulfated moiety of CCK with Arg was recently confirmed by photoaffinity labeling (17). The NH2-terminal moiety of CCK is tightly linked to extracellular residues of CCK1R, including residues in the second extracellular loop at the top of transmembrane (TM) helix I and within the third extracellular loop (16, 237). The COOH-terminal tetrapeptide of CCK appears to be embedded between TM helices III, V, VI, and VII through a network of both ionic and hydrophobic interactions (16, 145, 168). This binding mode of the COOH terminus of CCK into CCK1R is in agreement with an NMR study of the interactions between CCK and a fragment of CCK1R comprising the top portion of helix VI and the third extracellular loop as well as a fragment including amino acids at the top of transmembrane segment I (173, 373). With the use of photoaffinity labeling, two hits in the CCK1R were identified. The first was a Trp at the top of TM I using a photoprobe with the reactive moiety within the COOH-terminal Phe of CCK and the second was an His within the third extracellular loop using a probe with a benzophenone in the place of the Gly of CCK (Fig. 3) (193, 228). Accordingly, a model of binding of CCK to the CCK1R was proposed in which the COOH terminus of CCK and the tyrosine sulfate were in interaction with Trp39 and Arg197, respectively, and the NH2-terminal moiety was in contact with the third extracellular loop of the receptor (132). This second model of CCK positioning into the CCK1R binding site is divergent from that obtained on the basis of site-directed mutagenesis results (16, 237).
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An exciting issue that emerged in the course of binding site studies with CCK receptors was whether synthetic molecules that are structurally divergent with the natural ligands of the receptors share the same binding site or have distinct binding pockets. This issue has been addressed by several authors using site-directed mutagenesis. Regarding CCK1R, it was shown that certain residues of CCK1R were critical for binding and response to CCK but were without any importance for binding and activity of several nonpeptide antagonists. The best illustration was achieved through mutation of the Arg in the second extracellular loop of CCK1R, which caused dramatic decreases in affinity and potency of CCK but did not change affinity and antagonistic potency of L364,718 and SR27,897. In contrast, mutation of Asn333 at the top of transmembrane helix 6 affected affinity and potency of both CCK, L364,718, and SR27,897 (168, 170).
A large set of data exists concerning the binding sites for nonpeptide ligands of CCK2R that were essentially obtained in the course of studies related to the analysis of the impact of intra- and interspecies polymorphisms of this receptor on affinity and partial agonism of a series of CCK2R antagonists. The first striking interspecies difference to be studied was that between dog and human CCK2R. Indeed, the CCK1R antagonist L364,718 binds to the canine CCK2R with an affinity similar to that with which the CCK2R antagonist L365,260 binds to the human CCK2R, and vice versa. Mutation of nonconserved amino acids from transmembrane helices led to the identification of a single amino acid in the canine sequence (Leu355, TM VI) that is responsible for this reversal of specificity (38). In general, studies with so-called nonpeptide antagonists of CCK2R revealed that although the amino acid sequence homology of CCK2R in the different species is near 90%, the efficacy of the nonpeptide molecules to stimulate phospholipase C varied from 0 to 60% of the CCK-induced maximal response according to the species and the compound tested (L365,260, L740,093, YM022, PD135158, PD136,450, PD134,308) (39, 47, 48, 227, 232, 255, 256). Incorporation of nonconservative amino acids in the human CCK2R sequence enabled identification of amino acids in transmembrane helices that account for these variations (47, 48). These studies contributed to our understanding of the structural basis by which CCK2R ligands possess agonist activity. Furthermore, they point out that polymorphisms among receptors from different species can cause alterations in the apparent pharmacological profile of a drug.
Another interesting issue, raised by the generation of nonpeptide ligands for peptide-binding GPCRs, was whether or not compounds with similar structures but opposite biological activities share the same binding site and, so, which intrinsic mechanism(s) govern(s) receptor function at the binding site level. A nonpeptide agonist of the CCK1R, SR146,131, was generated starting from the structure of the antagonist SR27,897 (44). Pharmacological analysis of CCK1R mutants using these compounds, together with molecular modeling, agreed with the view that the binding sites of the two nonpeptidic ligands and of CCK are largely overlapping. Within these binding sites, a Leu in TM VII interacts with SR146,131 but not SR27897 or L364,718, suggesting that one underlying mechanism of activation by the nonpeptide ligand resides in this additional site of interaction (145, 182).
B. Activation Mechanism and Regulation
Residues located inside the ligand binding site in GPCRs play a key role in the GPCR activation process. Conserved motifs such as the E/DRY (TM III) and NPXXY (TM VII) motifs found in members of family I of GPCRs are critical as mutation in either of these two respective regions in CCK2R yielded a constitutively active or inactive receptor (160, 162). The constitutively active CCK2R mutant bearing a mutation in E/DRY motif (Asp mutated to Ala) was associated with dramatic alterations in cell morphology in which it was expressed and enhanced cell proliferation and invasion. On the other hand, while CCK2R bearing a mutation in the NPXXY motif (Asn mutated to Ala) was unable to activate phospholipase C, it still physically coupled to G
q protein, thereby demonstrating that the Asn of this motif is essential for G protein activation (160). Other residues, which are conserved in GPCRs, were also shown to be important for activation of phospholipase C and adenylyl cyclase by CCK2R and CCK1R. Transmembrane residues of the CCK2R such as Asp of TM II, triple basic motif (KKR) at the COOH terminus of the third intracellular loop, and a Phe residue of TM VI were shown to play a key role in the coupling of CCK2R to phospholipase C (222, 383, 554). Interestingly, mutation of two of these amino acids or motifs, namely, the Phe of TM VI and the KKR motif of the third intracellular loops, were also demonstrated to be without importance for CCK2R-induced stimulation of arachidonic acid release, supporting distinct mechanisms of CCK2R coupling to phospholipase C and phospholipase A2 (383). The chemical nature of residue 121 within TM III of the CCK1R seems to be very important for activation of the receptor. In fact, biological experiments with mutants as well as dynamic simulations of modeled liganded CCK1R support the conclusion that introduction of hydrophobic residues near position 121 determines positioning of the aromatic ring of the Phe of CCK within the binding pocket (145). This was further documented in a study with JMV 180, a partial agonist of the CCK1R, which partly shares its binding site with that of CCK (15). In this study, it was shown that helices III and VI of the CCK1R are functionally linked through the CCK1R agonist binding site and that positioning of the COOH-terminal ends of peptidic agonists towards Phe330 of helix VI is responsible for extent of phospholipase C activation through Gq coupling (15). The molecular basis for CCK1R coupling to adenylyl cyclase was investigated based on the data showing that CCK1R, but not CCK2R, stimulates cAMP formation. Chimeric receptors constructed and expressed in HEK cells provided evidence that a small peptidic sequence of the first intracellular loop of CCK1R is essential for cAMP signaling (577). The importance of the first intracellular loop for CCK1R coupling to adenylyl cyclase was further supported by substitution of a single residue (Ser) in the CCK2R by an Asn, which confers a full cAMP response to CCK and gastrin (575).
CCK receptors, like other GPCRs, were initially thought to trigger intracellular signals as monomeric membrane proteins. Biochemical and biophysical data have recently challenged this concept and provided evidence that many GPCRs, if not all, can form homo-oligomeric and hetero-oligomeric assemblies within the plasma membrane (65, 368). CCK1R may exist as dimers that dissociate under CCK activation (90). A functional impact of CCK1R+CCK2R heterodimerization was suggested on the basis of the observations that coexpression of CCK1R and CCK2R in CHO cells yields enhanced signaling and cell growth compared with the expression of a single receptor type (89). This finding could explain why transgenic mice expressing CCK2R in the pancreas together with endogenous CCK1R develop preneoplastic lesions and pancreatic cancers. These intriguing data deserve further investigation to establish the pathophysiological relevance of CCKR heterodimerization.
GPCR-mediated internalization is a biological process that contributes both to the agonist response and to its desensitization. Both CCK1R and CCK2R have been shown to undergo internalization in pancreatic acini and transfected NIH 3T3 or CHO cells (177, 418, 522). Interestingly, in the case of CCK1R, agonist and antagonist-induced desensitization of CCK1R were described (417). Under CCK stimulation, the CCK1R is rapidly phosphorylated, mostly in the third intracellular loop, both by protein kinase C and a G protein receptor kinase. There are at least five distinct phosphorylatable amino acids in CCK1R. While exchange of two phosphorylatable amino acids within the third intracellular loop for alanine completely abolishes CCK1R phosphorylation, they do not alter its signaling and internalization (363). A study supports that an internal region of the COOH-terminal tail of the CCK1R which is devoid of phosphorylation sites is important for normal CCK1R trafficking in CHO cells (177). Analysis of the molecular basis for CCK2R internalization using COOH-terminal truncated receptors indicated that phosphorylation sites involved in CCK2R endocytosis are mainly located on the COOH terminus of the receptor (381).
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IV. SIGNALING TRANSDUCTION PATHWAYS ACTIVATED BY CHOLECYSTOKININ RECEPTORS |
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Typically, the superfamily of GPCRs is capable of inducing a rapid hydrolysis of phosphatidylinositol bisphosphate by phospholipase C (PLC) family members to generate inositol trisphosphate (IP3) and diacylglycerol (DAG), which respectively induce calcium mobilization and stimulate several protein kinase C (PKC) isoforms.
In different cell types CCK1 or CCK2 receptors (CCK1R, CCK2R) activate principally PLC-
isoforms, likely through heterotrimeric G proteins of the Gq family as demonstrated by immunoblocking experiments with anti-PLC- or anti-Gq/ antisera (341, 371, 379, 588). However, two publications suggested that 
-subunits of G proteins might be also involved in PLC activation by CCK1R (587, 590).
PLC-1 isozyme has also been implicated in IP3 formation through CCK2R (582, 583). Recently, a direct association between CCK2R and PLC-1 has been demonstrated implicating the COOH-terminal phospho-Tyr438 of the receptor and the SH2 domains of the PLC-1 (19).
IP3 produced by PLC isozymes leads to the subsequent release of calcium from intracellular stores. In numerous cell types naturally expressing endogenous CCK receptors or stably transfected with CCK1R or CCK2R, CCK and gastrin induce calcium mobilization. In particular, calcium signaling in response to CCK has been extensively studied in rat pancreatic acinar cells, which naturally express CCK1R. In this model, the two affinity states of CCK1R generate different patterns of cytosolic calcium elevations. Activation of the high-affinity binding sites with physiological concentrations of CCK generates calcium oscillations resulting from the complex regulation of different intracellular receptors that control calcium mobilization (374). These oscillations are mediated through the activation of IP3 receptors, although weak IP3 production has been observed in response to physiological concentrations of CCK (158, 526). This primary release of calcium from IP3-sensitive stores may be relayed through calcium mobilization from IP3-insensitive pools by a mechanism that involves calcium and other types of receptors (551). Among them, two receptors types, the ryanodine receptors and the nicotinic acid adenine dinucleotide phosphate (NAADP) receptors, which are activated by two different calcium mobilizing messengers, cADP-ribose (cADPr) and NAADP, have also been implicated in calcium oscillations evoked by CCK1R (71–75, 525). The cellular distribution of these receptors, mainly apical for the IP3 receptor (346), basolateral for the ryanodine receptor (274), and likely over the whole cell for the NAADP receptor (70) as well as the cooperation that exists between these receptors may explain the spreading of the calcium wave from the apical region throughout the whole cell in response to CCK. Recently, CCK1R has been shown to activate a cytosolic ADP-ribosyl cyclase that may be responsible for the production of the calcium mobilizing messenger cADPr (260, 500).
In rat pancreatic acini, calcium oscillations play a crucial role in enzyme secretion regulated by low doses of CCK (570).
Several signaling molecules modulate calcium oscillations induced by CCK1R. Gq, G11, G14, as well as the and subunits likely released from Gq family members, play an important role in mediating the oscillatory calcium response (588, 590). The pattern of calcium oscillations is also regulated by the phosphorylation of IP3 receptors in response to physiological doses of CCK through a mechanism dependent on the protein kinase A (PKA) pathway (271, 502). In addition, the high-affinity binding site of CCK1R is coupled to the phospholipase A2/arachidonic acid cascade that inhibits IP3 and ryanodine receptors (180, 469). Activation of the low-affinity binding sites by high concentrations of CCK generates a very different pattern of calcium mobilization, which consists of a rapid global elevation of intracellular calcium that decreases to a sustained plateau (394). The initial peak of calcium corresponds to IP3 production and the subsequent release of calcium from IP3-sensitive intracellular stores, whereas the plateau is dependent on extracellular calcium influx. The mechanisms regulating calcium signals in response to CCK2R activation remain poorly understood. Both a rapid mobilization of intracellular calcium that decreases to a sustained plateau and calcium oscillations have been described in numerous cell types naturally expressing endogenous CCK2R as well as stably transfected cell lines (6, 462, 463, 517).
In addition to - and -PLC isoforms, two other phospholipases are activated by the CCK receptors: cytosolic phospholipase A2(cPLA2), an enzyme that produces arachidonic acid from membrane phospholipids (166, 180, 266, 469, 474, 531), and phospholipase D (PLD), which induces a sustained diacylglycerol (DAG) production and activates PKCs (7, 54, 180, 414). Whereas the high-affinity binding site of CCK1R activates cPLA2, the low-affinity binding site is coupled to PLD (180, 454).
The PKC family includes three subgroups: conventional PKCs, which are dependent on calcium and DAG (, , ); novel PKCs, which show sensitivity to DAG but are calcium independent (
, 
, 
, 
); and atypical isoforms that are unresponsive to calcium and DAG (
, 
, 
).
Numerous studies have shown the involvement of PKCs in both CCK1R and CCK2R signaling using broad-spectrum PKC inhibitors. In particular, mitogen-activated protein kinase pathways are activated by CCK1R or CCK2R through PKC-dependent mechanisms, although the specific isoforms of PKC involved were not identified in these studies (107, 111, 116, 528). More recently, the activation of several PKC isoforms by gastrin and CCK has been reported. In rodent pancreatic acinar cells, low-affinity CCK1R occupancy leads to the stimulation of PKC-, -, -, and - (33, 280, 377, 435, 519). In different human gastric tumor cell lines, CCK2R activates PKC-, -, -, and - (355, 503).
Like conventional and novel PKCs, protein kinase D (PKD, also called PKC-µ) and PKD2, which shows a high homology to PKD, are serine/threonine kinases targets of both DAG and phorbol esters. However, their structures and the regulation of their enzymatic activities can be distinguished from the members of the PKC family. Only CCK2R, stably transfected in fibroblasts or human gastric cancer cells, induces phosphorylation and activation of PKD and PKD2 through a PKC-dependent mechanism (93, 503, 504).
B. Adenyl Cyclase and cAMP Production
Although both CCK1R and CCK2R activate the PLC pathway via a Gq/11 protein, only CCK1R is also coupled to Gs. In pancreatic acinar cells, CCK induces adenylate cyclase activity and in CHO cells stably transfected with CCK1R, high doses of CCK increase intracellular cAMP by stimulating this enzyme (480, 589). The structural basis for this activation is described in section IIIB. Although some studies have reported the effect of pertussis toxin on signaling pathways activated by CCK receptors (210, 338, 382), coupling to Gi proteins and adenyl cyclase inhibition remain controversial (378). Numerous publications have shown that CCK1R is not coupled to Gi in pancreatic acinar cells (303, 543, 576), and only one publication has reported that CCK2R inhibits adenyl cyclase activity via a mechanism likely to involve Gi (440).
C. Nitric Oxide and cGMP Pathway
Nitric oxide (NO), a molecule produced from L-arginine by NO synthase (NOS), can initiate cGMP-dependent or cGMP-independent signaling pathways. Production of cGMP, through the stimulation of soluble guanylate cyclase by NO, leads principally to the activation of cGMP-dependent protein kinases but can also indirectly activate cAMP-dependent protein kinases by blocking enzymes involved in the degradation of cAMP (147).
The NO/cGMP pathway has been shown to be activated by CCK1R in different cellular models. In CHO cells expressing CCK1R, CCK increases NOS activity, NO production, and the intracellular concentration of cGMP. In this cell model, activation of this signaling pathway by CCK1R is linked to cell proliferation (101, 102). Stimulation of the NO/cGMP signaling cascade in response to CCK has also been observed in rodent pancreatic acini and could be involved in pancreatic secretion in vivo. Indeed, nonselective NOS inhibitors as well as deletion of NOS in transgenic mice decrease pancreatic secretion induced by CCK (2, 125). In addition, the gastroprotective role of CCK on gastric mucosal is dependent on this pathway (63, 64, 568).
D. Mitogen-Activated Kinase Cascades
Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases known to be activated by several families of receptors including tyrosine kinase receptors, GPCRs, or cytokines receptors. They include four subgroups: extracellular signal-regulated kinase 1/2 (ERK1/2 also known as p44MAPK and p42MAPK), c-jun NH2-terminal kinases (JNKs), ERK5, and p38 MAPKs. These kinases are implicated in numerous cellular functions such as cell growth, differentiation, survival, and apoptosis.
Several studies using ERK kinases (MEKs) inhibitors have shown the involvement of the ERK1/2 pathway in different cellular processes regulated by CCK receptors. In particular, this signaling cascade controls cell proliferation and migration induced by CCK2R and the transcriptional regulation of gastrin-sensitive genes (208, 353, 496). In pancreatic acinar cells, the ERK pathway also regulates protein translation induced by CCK1R, a cellular event involved in digestive enzyme synthesis (433).
Activation of ERK1 or ERK2 by growth factor receptors with intrinsic tyrosine kinase activity has been well documented. The best understood mechanism involves the recruitment of Grb2/Sos or Shc/Grb2/Sos complexes to tyrosine-phosphorylated receptors. These complexes subsequently activate the following cascade: Ras/Raf/ERK-kinases/ERK1/2.
In pancreas-derived AR42J cells, which are known to express endogenous CCK2R or in stably transfected CHO cells, gastrin stimulates the Ras-dependent ERK1/2 pathway via tyrosine phosphorylation of Shc, which enables interaction with the Grb2/Sos complex. This mechanism is PKC dependent, and Src family kinases, also activated by CCK2R, have been identified as the tyrosine kinases mediating gastrin-induced Shc phosphorylation (111, 113, 465). The Ras-dependent MEK/ERK1/2 cascade is also activated by CCK1R in pancreatic acinar cells (138, 139). In this cell model, CCK1R stimulates the Shc/Grb2/Sos complex through a PKC-dependent mechanism. Although the tyrosine kinase upstream of this cascade has not been clearly identified, PYK2, a tyrosine kinase related to p125-FAK, which is activated by both high- and low-affinity CCK1R, might be involved (107, 520). In addition, it is possible that Src family kinases, also known to be stimulated by the CCK1R in pancreatic acini, could play a role in Shc phosphorylation (533). CCK1R also activates a downstream effector of the ERKs, the 90-kDa ribosomal S6 kinase (p90rsk), known to play an important role in protein synthesis (56). Alternately, ERK1/2 activation by CCK receptors might be mediated by a Ras-independent mechanism involving the direct activation of Raf by PKCs (348, 463).
ERK1/2 activation by CCK2R can occur through a very different mechanism in gastrointestinal epithelial cells. This mechanism involves transactivation of the epidermal growth factor (EGF) receptor. In gastric epithelial cells, gastrin induces the expression and processing of proHB-EGF leading to the release of HB-EGF, tyrosine phosphorylation of EGF receptors and the subsequent activation of downstream signaling pathways (327, 475). In contrast, gastrin induces EGF receptor transactivation in intestinal cells via an intracellular signaling pathway mediated by Src family kinases (191). The transactivation of a receptor tyrosine kinase has never been reported for CCK1R.
Phosphorylation of GPCRs by specific GPCR kinases (GRKs) is a mechanism that contributes to receptor desensitization. It also plays an important role in regulation of MAPK cascades. Particularly, for the 2-adrenergic receptor, serine and threonine phosphorylated residues bind -arrestins, which serve as adapter proteins in the recruitment of signaling molecules. The resulting complex is internalized with the 2-adrenergic receptor and leads to activation of MAPKs (315). Although CCK receptors are known to be phosphorylated by serine/threonine kinases such as PKCs or GRKs and internalized (164, 177, 363, 381, 393), MAPK stimulation by a -arrestins-dependent mechanism has never been described for the CCK receptors.
JNK and p38-MAPK were initially identified as two signaling cascades mediating cellular stress induced by exposure to ultraviolet radiation, proinflammatory cytokines, and osmotic shocks. Afterwards, several studies have shown that they are also activated by receptor tyrosine kinases and GPCRs (192). JNK and p38-MAPK cascades are both stimulated by CCK receptors. Activation of these pathways by CCK2R can be blocked by PKCs or Src kinases inhibitors and is involved in the regulation of cell proliferation and survival by gastrin (115, 116).
In rat pancreatic acini, which naturally express CCK1R, supraphysiological concentrations of CCK activate JNK. In this model, CCK-induced JNK activation is mediated through a Ras-dependent mechanism that does not involve PKCs or calcium mobilization (106). Little is known about the role of the JNK pathway in CCK1R-mediated effects. However, it has been suggested that JNK activation by high doses of CCK might be a stress response, since these doses also induce pancreatitis (443, 549). In addition, the JNK pathway may be also involved in DNA synthesis stimulated by CCK1R in pancreatic cells (348).
In contrast, in the same cellular model, the p38-MAPK pathway is induced by physiological doses of CCK and mediates actin cytoskeleton reorganization, likely through the phosphorylation of the small heat shock protein Hsp27 (442, 550).
In addition to ERK1/2, JNK, and p38-MAPK, the ERK5 pathway is activated by gastrin in the intestinal cell line RIE-1 transfected with CCK2R. In this cell line, ERK5 may participate in the activation of the transcription factor MEF2 and the regulation of COX-2 downstream of CCK2R (191).
E. Phosphatidylinositol 3-Kinase
Class I phosphatidylinositol (PI) 3-kinases are a family of lipid kinases that play a central role in numerous cellular processes including cell proliferation and survival, protein synthesis, motility, and adhesion. These lipid kinases phosphorylate the D3 position of the inositol ring on phosphatidylinositols which serve as intracellular second messengers and recruits pleckstrin homology (PH) domain-containing proteins, such as AKT, to the plasma membrane. These kinases are heterodimers composed of the p110 catalytic subunit constitutively associated with the p85 adaptor/regulatory subunit.
Class I PI 3-kinases are subdivided into class IA and class IB. The PI 3-kinase , belonging to class IB, is mainly activated by GPCRs. Recently, using pancreatic acini isolated from mice deficient for the catalytic subunit of PI 3-kinase , Gukovsky et al. (188) have shown the role of this PI 3-kinase isoform in several intracellular events induced by supramaximal concentrations of CCK including calcium mobilization, calcium influx, and activation of NF
B and trypsinogen (188). All these events may play an important role in acute pancreatitis induced by supraphysiological doses of CCK. However, other PI 3-kinase isoforms may be activated by CCK1R.
In pancreatic acinar cells, the PI 3-kinases also play an important role in protein synthesis activated by low doses of CCK. In this model, Bragado et al. (57, 58) demonstrated that activation by CCK1R of two components of the translational machinery, p70S6kinase, which phosphorylates the ribosomal protein S6, and the initiation factor eIF4E, which is involved in cap-dependent translation, can be blocked by PI 3-kinase inhibitors (57, 58). However, the isoform involved in this process remains to be identified. Mechanisms leading to PI 3-kinase activation by CCK1R are poorly understood. They might involve Src family kinases. Indeed, immunoprecipitation studies in pancreatic acini have shown that CCK induces an association between Src and PI 3-kinase (240).
Class IA PI 3-kinases are known to be activated by receptor tyrosine kinases (RTKs) through at least two different mechanisms. First, the SH2 domains of p85 can bind directly to specific phosphotyrosine-containing sequences on tyrosine kinase receptors. Conformational changes in the p85/p110 complex following recruitment by the receptor and the proximity to lipid substrates lead to kinase activation. Another mechanism has been described for insulin and insulin-like growth factor (IGF)-I receptors, in which receptor autophosphorylation following ligand stimulation permits the binding of scaffold proteins called insulin receptor substrates (IRS). Subsequent phosphorylation of IRS proteins by the receptor recruits the p85/p110 PI 3-kinase and leads to its activation.
This class of PI 3-kinase has been reported to be activated in response to gastrin in different cell lines expressing endogenous CCK2R as well as stable transfectant (43, 113, 149, 258). The molecular mechanism involves Src phosphorylation of the adaptor protein IRS-1 on tyrosine residues, which serve as binding sites that recruit and activate p85/p110 PI 3-kinase complex (113, 259). The downstream effector of p85/p110 PI 3-kinase, AKT (also known as PKB), has also been identified to play a role in CCK2R signaling and is rapidly activated by phosphorylation in response to gastrin. The PI 3-kinase/AKT pathway is involved in the proliferative and antiapoptotic action of CCK2R as well as the regulation of cell adhesion and migration mediated by this receptor (43, 149, 198, 527). Another role of this pathway in CCK2R signaling is to control protein synthesis by regulating components of the translational machinery. In particular, CCK2R activates p70S6K and the initiation factor eIF4E (119, 392, 466).
F. Focal Adhesion Kinase and Associated Proteins
p125-focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase, localized to focal adhesion sites, that controls multiple intracellular signaling pathways involved in cell morphology, cell motility, and invasion. This protein is activated by numerous membrane receptors including integrins, RTKs, and GPCRs. In many cell types, p125-FAK phosphorylation leads to the recruitment of Src kinases and the formation of an activated p125-FAK/Src complex. This complex associates and phosphorylates integrin-associated proteins such as paxillin and talin, as well as adaptor proteins such as Shc and p130Cas (446). Phosphorylation of p125-FAK, p130Cas, and paxillin have been reported for both CCK1R and CCK2R in numerous cell models. These phosphorylations are regulated by the small GTPase Rho that also plays an important role in stress fiber formation and modulation of cell morphology observed in response to activation of CCK receptors (163, 239, 365, 463, 517, 518, 586).
Formation of an activated p125-FAK/Src complex has also been observed following CCK2R activation, and phosphorylation of p130Cas by gastrin has been shown to be Src dependent (112, 115).
PYK2 is also a nonreceptor focal adhesion tyrosine kinase closely related to p125-FAK. In pancreatic acini, CCK, through CCK1R, induces the phosphorylation and the activation of PYK2 by a calcium- and PKC-dependent mechanism. Once phosphorylated, PYK2 can associate with Grb2 or a p130Cas-associated protein, CrkII, leading to the activation of downstream signals (520).
Janus kinases (JAKs) are a family of nonreceptor tyrosine kinases which includes four members: the ubiquitously expressed JAK1, JAK2, TYK2, and JAK3 which is found in hematopoietic cells. They are known to phosphorylate and activate the STAT family of transcription factors (signal transducers and activators of transcription). Once phosphorylated, STAT proteins dimerize and translocate to the nucleus where they bind to target genes. The JAK/STAT signaling pathway that is well known to be activated by cytokines and growth factor receptors is involved in a wide variety of cellular processes including immune response, differentiation, cell survival, proliferation, and oncogenesis. The mechanism of JAK activation by cytokine receptors has been elucidated. Ligand binding induces dimerization of the receptors and a transphosphorylation of the associated JAK tyrosine kinases. Activated JAKs in turn phosphorylate the receptor that recruits the STAT proteins. To date, very few GPCRs have been shown to be connected to the JAK/STAT pathway. Recently, CCK2R has been shown to activate the JAK2/STAT3 pathway in different cell lines in vitro and in vivo, such as in transgenic mice expressing the receptor in pancreatic acini (148, 149). The mechanism of JAK2 activation involves Gq proteins and requires the NPXXY motif, located at the end of the seventh TM domain of CCK2R This motif, which is critical for Gq-dependent signaling pathways, is also required for STAT3 activation by CCK2R. This signaling pathway participates in CCK2R-mediated growth effects. JAK2 could also be involved in gastrin-induced modulation of cell-cell adhesion.
The small G protein superfamily, which includes at least five subfamilies (Ras, Rho/Rac/Cdc42, Rab, Arf/Sar1, and Ran), plays a key role upstream of numerous signaling cascades and regulates a large number of cellular processes.
CCK2R stimulates the small GTPase Ras upstream of the ERK1/2 and PI 3-kinase/AKT pathways, mediating the proliferative and antiapoptotic action of gastrin (495).
Ras is also activated by CCK1R, in pancreatic acini. This activation does not appear to be involved in pancreatic enzyme secretion stimulated by CCK but could lead to a stimulation of DNA synthesis through a mechanism independent of the ERK1/2 pathway (139, 348).
Among the Rho family members, Rho, Rac, and Cdc42 have been reported to be activated by CCK2R. Rho and Cdc42 appear to regulate the PI 3-kinase pathway and the proliferative effects mediated by this receptor (495). In addition to their role in regulating cell proliferation, Rho and Rac are able to switch on signaling pathways involved in amylase secretion evoked by CCK1R (41, 354) and stress fiber formation and morphological changes induced by both CCK receptors (see sect. IVF).
In numerous cell systems, Rho can be activated through the -subunits of the heterotrimeric G proteins, G12 and G13. In intestinal smooth muscle cells, high concentrations of CCK activate G12, G13, and RhoA (342). More recently, it has been reported in NIH3T3 cells transfected with CCK1R that occupation of the low-affinity binding site of the receptor leads to Rho activation and cytoskeletal remodeling mainly through a G13-dependent mechanism (276). In addition to the Ras and Rho families, several members of the Rab family have been implicated in CCK1R signaling. Although the precise mechanisms are not known, a role for Rab3D, Rab4, Rab11, and Rab27b in regulating CCK-induced acinar exocytosis has been reported by several groups (87, 88, 212).
Supramaximal concentrations of CCK, known to induce pancreatitis in rats via CCK1R, activate NFB, a transcription factor that regulates inflammatory processes. In most cells, NFB is sequestered in the cytoplasm through interactions with the IB (inhibitor of NFB) family of inhibitory proteins. The mechanism leading to CCK induction of NFB involves the activation of an IB kinase. Phosphorylation of IB and its subsequent ubiquitination and degradation by proteasome activity dissociates NFB from IB. Nuclear translocation of NFB leads to transcription of target genes such as the mob-1 chemokine. Several signaling pathways triggered by CCK1R are upstream NFB activation, including calcium mobilization, the novel PKCs and , and PI 3-kinase (197, 435, 514).
In gastric epithelial cells, CCK2R also activates NFB, leading to expression of proinflammatory genes such as interleukin (IL)-8 (206).
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V. TARGET GENES OF CHOLECYSTOKININ RECEPTORS |
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Gastrin is known to regulate gastric acid secretion by acting on enterochromaffin-like (ECL) cells that control synthesis and secretion of histamine. This process requires expression of three target genes regulated by gastrin and CCK2R in ECL cells: 1) histidine decarboxylase (HDC), the rate-limiting enzyme for histamine biosynthesis; 2) vesicular monoamine transporter 2 (VMAT2), involved in histamine accumulation into secretory vesicles; and 3) chromogranin A (CgA), which plays a role in vesicle stability and propeptide processing (208). In gastric epithelial cells, CgA expression is regulated by gastrin via the binding of three transcription factors, SP1, CREB and Egr-1, to a GC-rich element of the promoter. In the VMAT2 gene promoter, two sequences are involved in gastrin-mediated effects: a cAMP responsive element (CRE) that binds the transcription factor CREB and an overlapping AP2/SP1 site regulated by an uncharacterized nuclear protein. A gastrin-responsive element has also been identified in the HDC gene promoter that allows the association of two uncharacterized nuclear factors, gastrin responsive element binding proteins 1 and 2. In gastric epithelial cells, gastrin likely stimulates the expression of these three target genes through the activation of a Raf/MEK/ERK1/2 cascade in a ras-independent mechanism involving the direct activation of Raf by PKCs (208).
In glucagon-producing pancreatic cells, CCK2R also regulates glucagon gene expression via activation of the ERK1/2 pathway and binding of the transcription factor Egr-1 to the islet-specific G4 element present in the proximal glucagon promoter (277).
In addition to secretion, CCK2R is known to regulate cell proliferation by stimulating the expression of early response genes and other growth-related genes. In the tumor pancreatic cell line AR4–2J, in ECL cells, and in fibroblasts, c-Fos expression has been shown to be regulated by CCK2R. In particular, the CA-rich G box of the serum response element (SRE) has been reported to play a crucial role in gastrin-induced c-Fos promoter activation. However, maximal activation of the SRE by gastrin also requires the binding of ternary complex factors (TCFs) such as Elk1 and SAP1a to an E26 transformation specific (Ets) motif. This activation is PKC dependent and mediated by the ERK pathway. In addition, specific activation of the CArG box by CCK2R involves the small G protein RhoA (496, 499). One publication also mentions the importance of the CRE promoter element in gastrin-induced c-Fos expression that might cooperate with the two other binding sites (523). In gastrointestinal cells, CCK2R was also described to induce the expression of the protooncogenes c-Jun and c-Myc, although the mechanisms involved are unknown (517, 556).
Cyclins, that control the G1/S transition during the cell cycle, play a crucial role in cell proliferation. Several studies have shown an increase in the transcription of cyclin D1, D3, and E in response to gastrin (388, 594). The mechanism by which gastrin induces cyclin D1 transcription has been studied in a model of gastric tumor cells expressing CCK2R. The CRE site of the cyclin D1 promoter was shown to predominantly mediate cyclin D1 induction by gastrin via the binding of two transcription factors CREB and -catenin (388).
Proteins of the Reg family have been recognized as novel growth factors whose expression is increased under cellular stress, during inflammatory processes and tumor development. Reg-1 gene expression in response to gastrin has been reported in ECL cells and may be involved in gastric mucosal cell growth (20, 156). A C-rich region has been identified in the Reg-1 promoter sequence that binds transcription factors of the Sp-family, SP1 and SP3, in response to gastrin and mediates the effects of CCK2R activation. This transcriptional regulation of Reg-1 involves, in gastric epithelial cells, the activation of PKCs and the small G protein RhoA (20). In the same cells, CCK2R also increases the expression of plasminogen activator inhibitor-2 (PAI-2), known to be upregulated in gastric cancers. The binding of CREB to a CRE and of c-jun to an AP-1 site have been shown to be responsible for PAI-2 induction by gastrin. As for c-Fos gene expression, the transcriptional regulation of PAI-2 by CCK2R is mediated by Rho, PKCs, and the ERK1/2 pathway (539).
In gastric epithelial cells, the growth stimulatory effect of CCK2R could be partially mediated through the expression of HB-EGF, release of this factor, and stimulation of the EGF receptor. In these cells, the mechanism by which CCK2R induces HB-EGF gene transcription
