禁食和复投喂对大口黑鲈胆囊收缩素及其受体基因表达的影响

为研究大口黑鲈(Micropterus salmoides) 胆囊收缩素(Cholecystokinin, CCK)和其受体(Cholecystokinin receptor, CCKR)基因在摄食活动中的功能, 研究通过克隆得到CCK1、CCK2、CCK1R和CCK2R基因的编码区序列, 其长度分别为414、387、1368和1359 bp, 分别编码137、128、455和452个氨基酸。荧光定量结果表明CCK1和CCK2基因均在脑组织中高表达, 其次为肠道组织, 而CCK1R和CCK2R基因分别在胆囊和脑组织中高表达。在摄食后24h内, CCK1、CCK2、CCK1R和CCK2R基因的相对表达量呈先升高后下降趋势, 其中CCK1、CCK1R和CCK2R基因在摄食后3h相对表达量达到最高值, 而CCK2基因在摄食后12h相对表达量达到最高值(P<0.05)。禁食过程中CCK1、CCK1R和CCK2R基因相对表达量在禁食14d时显著升高(P<0.05)。复投喂后CCK1、CCK1R和CCK2R基因的相对表达趋势与餐后表达趋势相似, 呈先升高后降低趋势。但在禁食和复投喂过程中CCK2基因相对表达量并无显著变化。综上所述, 研究结果推测CCK1基因可能与CCK1R、CCK2R基因结合, 作为饱腹信号因子, 通过抑制食欲调控大口黑鲈摄食、消化等生理过程; 而CCK2基因可能作为短期食欲因子调节摄食活动。研究结果可为大口黑鲈摄食活动调节提供理论依据。     

Targeted disruption of the murine CCK1 receptor gene reduces intestinal lipid-induced feedback inhibition of gastric function

Cholecystokinin (CCK), acting at CCK1 receptors (CCK1Rs) on intestinal vagal afferent terminals, has been implicated in the control of gastrointestinal function and food intake. Using CCK1R(-/-) mice, we tested the hypothesis that lipid-induced activation of the vagal afferent pathway and intestinal feedback of gastric function is CCK1R dependent. In anesthetized CCK1R(+/+) ("wild type") mice, meal-stimulated gastric acid secretion was inhibited by intestinal lipid infusion; this was abolished in CCK1R(-/-) mice. Gastric emptying of whole egg, measured by nuclear scintigraphy in awake mice, was significantly faster in CCK1R(-/-) than CCK1R(+/+) mice. Gastric emptying of chow was significantly slowed in response to administration of CCK-8 (22 pmol) in CCK1R(+/+) but not CCK1R(-/-) mice. Activation of the vagal afferent pathway was measured by immunohistochemical localization of Fos protein in the nucleus of the solitary tract (NTS; a region where vagal afferents terminate). CCK-8 (22 pmol ip) increased neuronal Fos expression in the NTS of fasted CCK1R(+/+) mice; CCK-induced Fos expression was reduced by 97% in CCK1R(-/-) compared with CCK1R(+/+) mice. Intralipid (0.2 ml of 20% Intralipid and 0.04 g lipid), but not saline, gavage increased Fos expression in the NTS of fasted CCK1R(+/+) mice; lipid-induced Fos expression was decreased by 47% in CCK1R(-/-) compared with CCK1R(+/+)mice. We conclude that intestinal lipid activates the vagal afferent pathway, decreases gastric acid secretion, and delays gastric emptying via a CCK1R-dependent mechanism. Thus, despite a relatively normal phenotype, intestinal feedback in response to lipid is severely impaired in these mice.     

Evidence for a functional role of cholecystokinin receptors in the rat thyroid gland.

The aim of the present study was to examine the role of cholecystokinin (CCK) and/or cholecystokinin receptors subtypes (CCK1R and CCK2R) in the regulation of the thyroid gland structure and function. Animals were autopsied after 6 days of treatment with CCK or CCK receptor-specific antagonists (CCK1a--PD 140,548 or CCK2a--PD 135,158) solely or in combination with CCK. Results suggest that CCK exerts a stimulatory effect on follicular thyroid cells manifested by increased epithelium/colloid volume fraction ratio (E/C). Application of selective antagonists of CCK receptor subtypes has demonstrated that CCK acts through the CCK1 receptor subtype at the level of pituitary TSH. The model of endogenous hormone action reveals that thyroid CCK1 is responsible for the thyroid growth. It can be concluded that the physiological activity of CCK1 receptor plays a significant role in a complex interrelationship between TSH, vagal system and CCK1-dependent function of the thyroid gland.     

The biologically crucial C terminus of cholecystokinin and the non-peptide agonist SR-146,131 share a common binding site in the human CCK1 receptor. Evidence for a crucial role of Met-121 in the activation process.

The cholecystokinin (CCK) receptor-1 (CCK1R) is a G protein-coupled receptor, which mediates important central and peripheral cholecystokinin actions. Our aim was to progress in mapping of the CCK1R binding site by identifying residues that interact with the methionine and phenylalanine residues of the C-terminal moiety of CCK because these are crucial for its binding and biological activity, and to determine whether CCK and the selective non-peptide agonist, SR-146,131, share a common binding site. Identification of putative amino acids of the CCK1R binding site was achieved by dynamics-based docking of the ligand CCK in a refined three-dimensional model of the CCK1R using, as constraints, previous results that identified contact points between residues of CCK and CCK1R (Kennedy, K., Gigoux, V., Escrieut, C., Maigret, B., Martinez, J., Moroder, L., Frehel, D., Gully, D., Vaysse, N., and Fourmy, D. (1997) J. Biol. Chem. 272, 2920-2926 and Gigoux, V., Escrieut, C., Fehrentz, J. A., Poirot, S., Maigret, B., Moroder, L., Gully, D., Martinez, J., Vaysse, N., and Fourmy, D. (1999) J. Biol. Chem. 274, 20457-20464). By this approach, a series of residues forming connected hydrophobic clusters were identified. Pharmacological and functional analysis of mutated receptors indicated that a network of hydrophobic residues including Cys-94, Met-121, Val-125, Phe-218, Ile-329, Phe-330, Trp-326, Ile-352, Leu-356, and Tyr-360, is involved in the binding site for CCK and in the activation process of the CCK1R. Within this hydrophobic network, the physico-chemical nature of residue 121 seems to be essential for CCK1R functioning. Finally, the biological properties of mutants together with dynamic docking of SR-146,131 in the CCK1R binding site demonstrated that SR-146,131 occupies a region of CCK1R binding site which interacts with the C-terminal amidated tripeptide of CCK, i.e. Met-Asp-Phe-NH(2). These new and important insights will serve to better understand the activation process of CCK1R and to design or optimize ligands.     

Signaling through cholesterol esterification: a new pathway for the cholecystokinin 2 receptor involved in cell growth and invasion

Several studies indicate that cholesterol esterification is deregulated in cancers. The present study aimed to characterize the role of cholesterol esterification in proliferation and invasion of two tumor cells expressing an activated cholecystokinin 2 receptor (CCK2R). A significant increase in cholesterol esterification and activity of Acyl-CoA:cholesterol acyltransferase (ACAT) was measured in tumor cells expressing a constitutively activated oncogenic mutant of the CCK2R (CCK2R-E151A cells) compared with nontumor cells expressing the wild-type CCK2R (CCK2R-WT cells). Inhibition of cholesteryl ester formation and ACAT activity by Sah58-035, an inhibitor of ACAT, decreased by 34% and 73% CCK2R-E151A cell growth and invasion. Sustained activation of CCK2R-WT cells by gastrin increased cholesteryl ester production while addition of cholesteryl oleate to the culture medium of CCK2R-WT cells increased cell proliferation and invasion to a level close to that of CCK2R-E151A cells. In U87 glioma cells, a model of autocrine growth stimulation of the CCK2R, inhibition of cholesterol esterification and ACAT activity by Sah58-035 and two selective antagonists of the CCK2R significantly reduced cell proliferation and invasion. In both models, cholesteryl ester formation was found dependent on protein kinase zeta/ extracellular signal-related kinase 1/2 (PKCζ/ERK1/2) activation. These results show that signaling through ACAT/cholesterol esterification is a novel pathway for the CCK2R that contributes to tumor cell proliferation and invasion.     

Regulation of membrane cholecystokinin-2 receptor by agonists enables classification of partial agonists as biased agonists

Given the importance of G-protein-coupled receptors as pharmacological targets in medicine, efforts directed at understanding the molecular mechanism by which pharmacological compounds regulate their presence at the cell surface is of paramount importance. In this context, using confocal microscopy and bioluminescence resonance energy transfer, we have investigated internalization and intracellular trafficking of the cholecystokinin-2 receptor (CCK2R) in response to both natural and synthetic ligands with different pharmacological features. We found that CCK and gastrin, which are full agonists on CCK2R-induced inositol phosphate production, rapidly and abundantly stimulate internalization. Internalized CCK2R did not rapidly recycle to plasma membrane but instead was directed to late endosomes/lysosomes. CCK2R endocytosis involves clathrin-coated pits and dynamin and high affinity and prolonged binding of β-arrestin1 or -2. Partial agonists and antagonists on CCK2R-induced inositol phosphate formation and ERK1/2 phosphorylation did not stimulate CCK2R internalization or β-arrestin recruitment to the CCK2R but blocked full agonist-induced internalization and β-arrestin recruitment. The extreme C-terminal region of the CCK2R (and more precisely phosphorylatable residues Ser(437)-Xaa(438)-Thr(439)-Thr(440)-Xaa(441)-Ser(442)-Thr(443)) were critical for β-arrestin recruitment. However, this region and β-arrestins were dispensable for CCK2R internalization. In conclusion, this study allowed us to classify the human CCK2R as a member of class B G-protein-coupled receptors with regard to its endocytosis features and identified biased agonists of the CCK2R. These new important insights will allow us to investigate the role of internalized CCK2R·β-arrestin complexes in cancers expressing this receptor and to develop new diagnosis and therapeutic strategies targeting this receptor.     

Up-regulation of Cholesterol Absorption Is a Mechanism for Cholecystokinin-induced Hypercholesterolemia

Excessive absorption of intestinal cholesterol is a risk factor for atherosclerosis. This report examines the effect of cholecystokinin (CCK) on plasma cholesterol level and intestinal cholesterol absorption using the in vivo models of C57BL/6 wild-type and low density lipoprotein receptor knock-out (LDLR^−/−) mice. These data were supported by in vitro studies involving mouse primary intestinal epithelial cells and human Caco-2 cells; both express CCK receptor 1 and 2 (CCK1R and CCK2R). We found that intravenous injection of [Thr²⁸,Nle³¹]CCK increased plasma cholesterol levels and intestinal cholesterol absorption in both wild-type and LDLR^−/− mice. Treatment of mouse primary intestinal epithelial cells with [Thr²⁸,Nle³¹]CCK increased cholesterol absorption, whereas selective inhibition of CCK1R and CCK2R with antagonists attenuated CCK-induced cholesterol absorption. In Caco-2 cells, CCK enhanced CCK1R/CCK2R heterodimerization. Knockdown of both CCK1R and CCK2 or either one of them diminished CCK-induced cholesterol absorption to the same extent. CCK also increased cell surface-associated NPC1L1 (Niemann-Pick C1-like 1) transporters but did not alter their total protein expression. Inhibition or knockdown of NPC1L1 attenuated CCK-induced cholesterol absorption. CCK enhanced phosphatidylinositide 3-kinase (PI3K) and Akt phosphorylation and augmented the interaction between NPC1L1 and Rab11a (Rab-GTPase-11a), whereas knockdown of CCK receptors or inhibition of G protein βγ dimer (Gβγ) diminished CCK-induced PI3K and Akt phosphorylation. Inhibition of PI3K and Akt or knockdown of PI3K diminished CCK-induced NPC1L1-Rab11a interaction and cholesterol absorption. Knockdown of Rab11a suppressed CCK-induced NPC1L1 translocation and cholesterol absorption. These data imply that CCK enhances cholesterol absorption by activation of a pathway involving CCK1R/CCK2R, Gβγ, PI3K, Akt, Rab11a, and NPC1L.     

Over-expression of CCK1 Receptor Reverse Morphine Dependence

Studies demonstrated that cholecystokinin (CCK) system involved in morphine dependence and withdrawal. Our previous study showed that endogenous CCK system were up-regulated after chronic morphine exposure. Additionally, CCK1 receptor significantly blocked the inhibitory effect of exogenous CCK-8 on morphine dependence, but CCK2 receptor appears to be necessary for low concentrations of endogenous CCK to potentiate morphine dependence. Therefore, CCK1R and CCK2R function differently in chronic morphine dependence, but the mechanism is still unclear. In this study, HEK-293 cells co-transfected with µ-opioid receptors (HEK293-hMOR) and CCK1R or CCK2R were established. Cells were treated with 10 µM morphine for 6, 12, 16, 24 h and 100 µM naloxone precipitation for 15 min. cAMP overshoot was appeared at 12 h and was increased time dependently after morphine exposure in HEK293-hMOR cells. The cAMP overshoot did not appear in CCK1R-overexpressing HEK293-hMOR cells, while still appeared in CCK2R-overexpressing HEK293-hMOR cells. Over-expression of CCK1R reversed CREB and ERK1/2 activation in HEK293-hMOR cells exposed to morphine. Our study identifies over-expression of CCK1R significantly blocked morphine dependence, which was related with phosphorylation of CREB, and ERK1/2 signaling activation. While over-expression of CCK2R promoted morphine dependence, which was related with phosphorylation of CREB but not ERK1/2 signaling activation.     

A Type 1 Cholecystokinin Receptor Mutant That Mimics the Dysfunction Observed for Wild Type Receptor in a High Cholesterol Environment

Cholecystokinin (CCK) stimulates the type 1 CCK receptor (CCK1R) to elicit satiety after a meal. Agonists with this activity, although potentially useful for treatment of obesity, can also have side effects and toxicities of concern, making the development of an intrinsically inactive positive allosteric modulator quite attractive. Positive allosteric modulators also have the potential to correct the defective receptor-G protein coupling observed in the high membrane cholesterol environment described in metabolic syndrome. Current model systems to study CCK1R in such an environment are unstable and expensive to maintain. We now report that the Y140A mutation within a cholesterol-binding motif and the conserved, class A G protein-coupled receptor-specific (E/D)RY signature sequence results in ligand binding and activity characteristics similar to wild type CCK1R in a high cholesterol environment. This is true for natural CCK, as well as ligands with distinct chemistries and activity profiles. Additionally, the Y140A construct also behaved like CCK1R in high cholesterol in regard to its internalization, sensitivity to a nonhydrolyzable GTP analog, and anisotropy of a bound fluorescent CCK analog. Chimeric CCK1R/CCK2R constructs that systematically changed the residues in the allosteric ligand-binding pocket were studied in the presence of Y140A. This established increased importance of unique residues within TM3 and reduced the importance of TM2 for binding in the presence of this mutation, with the agonist trigger likely pulled away from its Leu³⁵⁶ target on TM7. The distinct conformation of this intramembranous pocket within Y140A CCK1R provides an opportunity to normalize this by using a small molecule allosteric ligand, thereby providing safe and effective correction of the coupling defect in metabolic syndrome.     

Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol

Recent studies indicate that membrane cholesterol can associate with G protein-coupled receptors (GPCRs) and affect their function. Previously, we reported that manipulation of membrane cholesterol affects ligand binding and signal transduction of the type 1 cholecystokinin receptor (CCK1R), a Class A GPCR. We now demonstrate that the closely related type 2 cholecystokinin receptor (CCK2R) does not share this cholesterol sensitivity. The sequences of both receptors reveal almost identical cholesterol interaction motifs in analogous locations in transmembrane segments two, three, four, and five. The disparity in cholesterol sensitivity between these receptors, despite their close structural relationship, provides a unique opportunity to define the possible structural basis of cholesterol sensitivity of CCK1R. To evaluate the relative contributions of different regions of CCK1R to cholesterol sensitivity, we performed ligand binding studies and biological activity assays of wild-type and CCK2R/CCK1R chimeric receptor-bearing Chinese hamster ovary cells after manipulation of membrane cholesterol. We also extended these studies to site-directed mutations within the cholesterol interaction motifs. The results contribute to a better understanding of the structural requirements for cholesterol sensitivity in CCK1R and provides insight into the function of other cholesterol-sensitive Class A GPCRs.     

Development of a CCK1R-membrane nanoparticle as a fish-out tool for bioactive peptides

The cholecystokinin receptor type 1 (CCK1R) is a G protein-coupled receptor (GPCR) that is involved in several biological processes including the regulation of the secretion of digestive enzymes. The peptide hormone cholecystokinin (CCK) binds to CCK1R, which is an important pharmacological target for several diseases, including obesity. Interestingly, nutritional dietary peptides also appear to activate CCK1R, and may play a role in CCK1R signaling in the gut. In this study, a novel technique to screen for CCK1R ligands based on affinity-selection is described. Functional expressed CCK1R is reconstituted into membrane nanoparticles called NABBs (nanoscale apo-lipoprotein bound bilayers). NABBs are native-like bilayer membrane systems for incorporation of GPCRs. CCK1R-NABBs were characterized using a fluorescently labeled CCK analog and can be used as a cutting-edge technology to screen for CCK1R ligands using affinity-selection mass spectrometry.     

Mechanism of activation of a G protein-coupled receptor, the human cholecystokinin-2 receptor

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.     

Permanent Photodynamic Cholecystokinin 1 Receptor Activation: Dimer-to-Monomer Conversion

The G protein-coupled cholecystokinin 1 receptor (CCK1R) is activated permanently by type II photodynamic action (i.e., by singlet oxygen) in the freshly isolated rat pancreatic acini, in contrast to reversible activation by CCK. But how CCK1R is photodynamically activated is not known. Therefore, in the present work, we subjected membrane proteins extracted from isolated rat pancreatic acini to photodynamic action with photosensitiser sulphonated aluminium phthalocyanine (SALPC), and used reducing gel electrophoresis and Western blot to detect possible changes in CCK1R oligomerization status. Photodynamic action (SALPC 1 µM, light 36.7 mW cm^??2 × 10 min) was found to convert dimeric CCK1R nearly quantitatively to monomers. Such conversion was dependent on both irradiance (8.51–36.7 mW cm^??2) and irradiation time (1–20 min). Minimum effective irradiance was found to be 11.1 mW cm^??2 (× 10 min, with SALPC 1 µM), and brief photodynamic action (SALPC 1 µM, 36.7 mW cm^??2 × 1 min) was effective. Whilst CCK stimulation of purified membrane proteins alone had no effect on CCK1R dimer/monomer balance, sub-threshold photodynamic action (SALPC 100 nM, 36.7 mW cm^??2 × 10 min) plus CCK revealed a bell-shaped CCK dose response curve for CCK1R monomerization, which was remarkably similar to the dose response curve for CCK-stimulated amylase secretion in isolated rat pancreatic acini. These two lines of evidence together suggest that during photodynamic CCK1R activation, CCK1R is permanently monomerized, thus providing a unique approach for permanent G protein-coupled receptor (GPCR) activation which has not been achieved before.     

SRC regulates constitutive internalization and rapid resensitization of a cholecystokinin 2 receptor splice variant

The third intracellular loop domain of G protein-coupled receptors regulates their desensitization, internalization, and resensitization. Colorectal and pancreatic cancers, but not the nonmalignant tissue, express a splice variant of the cholecystokinin 2 receptor (CCK2R) called CCK(2i4sv)R that, because of intron 4 retention, contains an additional 69 amino acids within its third intracellular loop domain. This structural alteration is associated with agonist-independent activation of Src kinase (Olszewska-Pazdrak, B., Townsend, C. M., Jr., and Hellmich, M. R. (2004) J. Biol. Chem. 279, 40400-40404). The purpose of the study was to determine the roles of intron 4 retention and Src kinase on CCK(2i4sv)R desensitization, internalization, and resensitization. Gastrin1-17 (G17) binds to both CCK2R and CCK(2i4sv)R and induces intracellular Ca2+ ([Ca2+]i) increases. Agonist-induced increases in [Ca2+]i were used to assess receptor activity. Src kinase activity was inhibited by transducing cells with a retrovirus containing a dominant-negative mutant Src (A430V). The subcellular location of enhanced green fluorescent protein-tagged receptors was monitored using laser scanning confocal microscopy. Both receptor variants desensitized at the same rate; however, CCK(2i4sv)R resensitized five times faster than CCK2R. Without agonist, 80% of CCK(2i4sv)R is located in an intracellular compartment. In contrast, 80% of CCK2R was located on the plasma membrane. Treatment with inverse agonist (YM022) or expression of dominant-negative Src blocked the constitutive internalization of CCK(2i4sv)R, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK(2i4sv)R resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK(2i4sv)R increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.     

Valine-286 residue in the third intracellular loop of the cholecystokinin 2 receptor exerts a pivotal role in cholecystokinin 2 receptor mediated intracellular signal transduction in human colon cancer cells

Although expression of the gastrin/cholecystokinin-2 receptor (CCK2R) is widely reported in human colorectal cancer, little is known on its role in mediating mature amidated gastrin (gastrin-17 amide, G-17) induced intracellular signal transduction in colon cancer cells. The purpose of this study was to explore the intracellular events of colorectal cancer cells after gastrin binding to CCK2R. Meanwhile, the influence of a natural point mutation 286V-->F in the third intracellular loop of CCK2R on gastrin-envoked intracellular signal transduction was also investigated. Firstly, Colo320 cells were stably transfected with wild type (Colo320 WT) and mutant CCK2R (Colo320 M), respectively. The intracellular signal transduction events in response to gastrin were investigated in both Colo320 WT and Colo320 M cells. In Colo320 WT cells, G-17 induced formation of intracellular cyclic AMP and inositol 1,4,5-trisphosphate, and stimulated intracellular calcium mobilization. G-17 also stimulated tyrosine phosphorylation of ERKl/2, p38, FAK, and paxillin, and up-regulated the mRNA expression of early response gene c-Jun and c-Fos. However, G-17 inhibited proliferation and induced apoptosis in Colo320 WT cells. Mutation 286V-->F in the third intracellular loop of CCK2R blocked G-17 induced biological without affecting binding affinity of CCK2R to G-17. Our results suggest that activation of CCK2R by gastrin stimulates heterotrimeric G-protein Gq and G(12/13) mediated intracellular signal transduction pathway in colon cancer cells. The valine-287 residue in third intracellular loop of CCK2R plays a pivotal role in CCK2R mediated intracellular signal transduction.     

An ITIM-like motif within the CCK2 receptor sequence required for interaction with SHP-2 and the activation of the AKT pathway

SHP-2 is a tyrosine phosphatase which functions as a positive regulator downstream of RTKs, activating growth-stimulatory signalling pathways. To date, very few G protein-coupled receptors (GPCRs) have been shown to be connected to SHP-2 and very little is known about the positive role of SHP-2 in GPCR signalling. The CCK2 receptor (CCK2R), a GPCR, is now recognized to mediate mitogenic effects of gastrin on gastrointestinal cells. In the present study, we demonstrate the role of SHP-2 in the activation of the AKT pathway by the CCK2R in COS-7 cells transfected with the CCK2R and in a pancreatic cancer cell line expressing the endogenous receptor. Using surface plasmon resonance analysis, we identified a highly conserved ITIM motif, containing the tyrosine residue 438, located in the C-terminal intracellular tail of the CCK2R which directly interacts with the SHP-2 SH2 domains. The interaction was confirmed by pull down assays and co-immunoprecipitation of the receptor with SHP-2. This interaction was transiently increased following gastrin stimulation of the CCK2R and correlated with the tyrosine phosphorylation of SHP-2. Mutational analysis of the key ITIM residue 438 confirmed that the CCK2R ITIM sequence is required for interaction with SHP-2 and the activation of the AKT pathway.     

Role of CCK1 and Y2 receptors in activation of hindbrain neurons induced by intragastric administration of bitter taste receptor ligands

G-protein-coupled receptors signaling bitter taste (T2Rs) in the oral gustatory system and the alpha-subunit of the taste-specific G-protein gustducin are expressed in the gastrointestinal (GI) tract. alpha-Subunit of the taste-specific G-protein gustducin colocalizes with markers of enteroendocrine cells in human and mouse GI mucosa, including peptide YY. Activation of T2Rs increases cholecystokinin (CCK) release from the enteroendocrine cell line, STC-1. The aim of this study was to determine whether T2R agonists in the GI tract activate neurons in the nucleus of the solitary tract (NTS) and whether this activation is mediated by CCK and peptide YY acting at CCK(1) and Y(2) receptors. Immunocytochemistry for the protooncogene c-Fos protein, a marker for neuronal activation, was used to determine activation of neurons in the midregion of the NTS, the region where vagal afferents from the GI tract terminate. Intragastric administration of the T2R agonist denatonium benzoate (DB), or phenylthiocarbamide (PTC), or a combination of T2R agonists significantly increased the number of Fos-positive neurons in the mid-NTS; subdiaphragmatic vagotomy abolished the NTS response to the mixture of T2R agonists. Deletion of CCK(1) receptor gene or blockade of CCK(1) receptors with devazepide abolishes the activation of NTS neurons in response to DB, but had no effect on the response to PTC. Administration of the Y(2) receptor antagonist BIIE0246 blocks the activation of NTS neurons to DB, but not PTC. These findings suggest that activation of neurons in the NTS following administration of T2R agonists to the GI tract involves CCK(1) and Y(2) receptors located on vagal afferent terminals in the gut wall. T2Rs may regulate GI function via release of regulatory peptides and activation of the vagal reflex pathway.     

Molecular mechanism underlying partial and full agonism mediated by the human cholecystokinin-1 receptor

The cholecystokinin-1 receptor (CCK1R) is a G protein-coupled receptor (GPCR) that regulates important physiological functions. As for other GPCRs, the molecular basis of full and partial agonism is still far from clearly understood. In the present report, using both laboratory experiments and molecular modeling approaches, we have investigated the partial agonism mechanism of JMV 180, on the human CCK1R. We first showed that efficacy of the CCK1R to activate phospholipase C is dependent on the correct orientation of the C-terminal end of peptidic ligands toward residue Phe(330) of helix VI. We have previously reported that a single mutation of Met(121) (helix III) markedly reduced the receptor-mediated inositol phosphate production upon stimulation by CCK. Computational simulations predicted that residue 121 affected orientation of the C-terminal end of CCK, thus suggesting that the molecular complex with a reduced inositol phosphate production observed with the mutated CCK1R resembles that resulting from binding of JMV 180 to the WT-CCK1R. Pharmacological, biochemical, and functional characterizations of the two receptor.ligand complexes with decreased abilities to signal were carried out in different cell types. We found that they presented the same features, such as total dependence of inositol phosphate production to Galpha(q) expression, single affinity of binding sites, insensitivity of binding to non-hydrolyzable GTP, absence of GTPgamma[S(35)] binding following agonist stimulation, similarity of dose-response curves for amylase secretion, and incapacity to induce acute pancreatitis in pancreatic acini. We concluded that helices VI and III of the CCK1R are functionally linked through the CCK1R agonist binding site and that positioning of the C-terminal ends of peptidic agonists toward Phe(330) of helix VI is responsible for extent of phospholipase C activation through Galpha(q) coupling. Given the potential therapeutic interest of partial agonists such as JMV 180, our structural data will serve for target structure-based design of new CCK1R ligands.     

Molecular basis for binding and subtype selectivity of 1,4-benzodiazepine antagonist ligands of the cholecystokinin receptor

Allosteric binding pockets in peptide-binding G protein-coupled receptors create opportunities for the development of small molecule drugs with substantial benefits over orthosteric ligands. To gain insights into molecular determinants for this pocket within type 1 and 2 cholecystokinin receptors (CCK1R and CCK2R), we prepared a series of receptor constructs in which six distinct residues in TM2, -3, -6, and -7 were reversed. Two novel iodinated CCK1R- and CCK2R-selective 1,4-benzodiazepine antagonists, differing only in stereochemistry at C3, were used. When all six residues within CCK1R were mutated to corresponding CCK2R residues, benzodiazepine selectivity was reversed, yet peptide binding selectivity was unaffected. Detailed analysis, including observations of gain of function, demonstrated that residues 6.51, 6.52, and 7.39 were most important for binding the CCK1R-selective ligand, whereas residues 2.61 and 7.39 were most important for binding CCK2R-selective ligand, although the effect of substitution of residue 2.61 was likely indirect. Ligand-guided homology modeling was applied to wild type receptors and those reversing benzodiazepine binding selectivity. The models had high predictive power in enriching known receptor-selective ligands from related decoys, indicating a high degree of precision in pocket definition. The benzodiazepines docked in similar poses in both receptors, with C3 urea substituents pointing upward, whereas different stereochemistry at C3 directed the C5 phenyl rings and N1 methyl groups into opposite orientations. The geometry of the binding pockets and specific interactions predicted for ligand docking in these models provide a molecular framework for understanding ligand selectivity at these receptor subtypes. Furthermore, the strong predictive power of these models suggests their usefulness in the discovery of lead compounds and in drug development programs.