The hormone cholecystokinin (CCK) was discovered in 1928 by Ivy and Oldberg, based on its ability to stimulate gallbladder contraction. Fifteen years later, Harper and Raper described a factor capable of stimulating pancreatic exocrine secretion (pancreozymin). When Jorpes and Mutt finally isolated the CCK peptide from porcine duodenum in 1966, it became clear that this single hormone was responsible for both of these classical physiological gastrointestinal regulatory activities. Many years later, in 1992, the receptor mediating these effects was finally identified and characterized as a family A guanine nucleotide-binding protein (G protein)-coupled receptor, the CCK-1 receptor (Liddle 1994). Of interest, the cDNA encoding this receptor was initially cloned and described in the same issue of the Proceedings of the National Academy of Sciences, USA, as the structurally closely related CCK-2 receptor (see the following chapter on this receptor). The CCK-1 receptor has subsequently been recognized as having multiple effects on tissues, including gallbladder, pancreas, gut, and neurons in the periphery and central nervous system, with most of these effects related to maintenance of nutritional homeostasis. This includes optimization of the digestive milieu, regulation of the rate of transit of intestinal chyme for optimal nutrient absorption, and the control of appetite. Indeed, this receptor has become the target of substantial efforts by pharmaceutical companies to develop an agonist drug that might be useful in the treatment of obesity as a noncaloric satiety agent.
Tissue Distribution and Physiological Actions of CCK-1 Receptors
The distribution and functions of CCK receptors differ among species, with much of the fundamental physiologic studies having been performed in rats, mice, guinea pigs, and dogs in the period of time before the CCK receptor genes had been cloned. Now that the molecular nature of the two types of CCK receptors have been established, and highly specific and sensitive assays for their presence and activity are now in place, the role of the CCK-1 receptor in human physiology can be better established. Human tissues with established physiological functions for the CCK-1 receptor include gallbladder smooth muscle, pancreatic neurons (and low levels of expression on acinar cells), gastric and intestinal smooth muscle, vagal afferent neurons, and distinct central nervous system nuclei (Noble et al. 1999). These targets are involved in the normal physiologic stimulation of gallbladder emptying, stimulation of pancreatic exocrine secretion, regulation of gastric emptying and bowel transit, and induction of postcibal satiety. These functions naturally follow from the distribution and secretory pattern of the hormone that activates them. CCK is produced in I cells scattered within the mucosa of the proximal small intestine and is secreted into the bloodstream in response to luminal nutrients, such as protein and fat. These complex nutrients require micelle formation (fat), digestion (protein and fat), and regulated enteric transit of chyme for optimal absorption. CCK is also produced in some neurons where it can have neurocrine effects. Other possible effects of this hormone on CCK-1 receptors expressed on healthy cells, with less clearly established physiological roles, include stimulation of the secretion of somatostatin from gastric D cells and secretion of pepsinogen and leptin from chief cells, trophic effects on the exocrine pancreas, and the regulation of secretion of insulin, pancreatic polypeptide, and somatostatin from the pancreatic islets.
Structure of CCK-1 Receptors and Molecular Basis of Ligand Binding
The natural peptide agonist ligand for the CCK-1 receptor binds at the extracellular surface of this receptor, with key determinants within loop and amino-terminal tail epitopes (Miller and Gao 2008). This has been well established in receptor truncation and mutagenesis studies, chimeric receptor studies, ligand structure-activity studies, fluorescence probing, and photoaffinity labeling studies. The latter represent the most direct evidence for mode of docking and have been performed to establish spatial approximation constraints involving six of the seven residues within the focused pharmacophoric domain of this hormone, as well as the amino-terminal region of the hormone just outside this domain. Of note, analogous studies with the CCK-2 receptor have suggested a distinct mechanism for the binding of the same peptide to that receptor, with the possibility that the carboxyl terminus of CCK dips down within the helical bundle of the CCK-2 receptor. It is noteworthy that the structure-activity characteristics for peptide binding and activation of the two types of CCK receptors are quite distinct, with the CCK-1 receptor requiring the carboxyl-terminal heptapeptide-amide of CCK for high-affinity binding and activation, while only the carboxyl-terminal tetrapeptide-amide is required for the CCK-2 receptor. With gastrin sharing the carboxyl-terminal pentapeptide-amide with all CCK peptide species, it is clear that both gastrin and CCK bind and activate the CCK-2 receptor with high affinity and potency, while only CCK peptides have these actions at the CCK-1 receptor.
For the CCK-1 receptor, in addition to the orthosteric natural peptide hormone-binding site, there is an allosteric ligand-binding pocket within the intramembranous helical bundle that is the site of docking a series of non-peptidyl small-molecule ligands. This pocket has been shown to be totally distinct from the CCK-binding site using classical manipulations, such as the ability of a benzodiazepine to slow the dissociation of prebound orthosteric ligand, CCK (Gao et al. 2008). Molecular models also support the separate and distinct nature of the peptide and nonpeptidyl small molecule–binding sites within the CCK-1 receptor. Additionally, the conformation of the allosteric, small molecule–binding pocket has been shown to have distinctive features in the CCK-1 receptor and the closely related CCK-2 receptor (Cawston and Miller, 2010), and to assume different conformations in the activated and inactive states of the CCK-1 receptor (Harikumar et al. 2013). These conformations have been shown to possess predictive power in identifying ligands for these receptors.
Signaling at the CCK-1 Receptor
The classical pathway of signaling stimulated by natural agonist action at the CCK-1 receptor is mediated via coupling with heterotrimeric G proteins in the Gq family (Gq, G11, and G14), involving the rapid hydrolysis of phosphatidylinositol bisphosphate by phospholipase C enzymes (principally β isoforms) to generate inositol trisphosphate and diacylglycerol. This results in calcium mobilization from intracellular stores and the subsequent activation of various isoforms of protein kinase C (Williams 2001). High concentrations of CCK can also stimulate coupling of this receptor with Gs, with subsequent activation of adenylate cyclase, increase in cAMP, and activation of protein kinase A, although the physiologic nature of this signaling pathway is not clear. There have also been reports of CCK acting through this receptor to stimulate mitogen-activated protein kinases (MAPKs). These include extracellular signal-regulated kinases 1 and 2 (ERK 1 and ERK 2), c-jun kinases (JNKs), and ERK 5 and p38 MAPK. These signaling events are downstream of the G protein coupling events. Phosphatidylinositol 3-kinases (PI 3-kinases), p125-focal adhesion kinase (FAK), and Janus kinases (JAKs) are also activated by CCK action on this receptor. The CCK-1 receptor has also been reported to couple with another distinct G protein, G13, to result in the activation of RhoA (Sabbatini et al. 2010). This is believed to affect the cytoskeleton. Current understanding of the linkage and interrelationship of these signaling molecules have been reviewed elsewhere (Williams 2001; Cawston and Miller 2010).
In addition to association with the classical proximal mediator of signaling, representing coupling of the heterotrimeric G proteins with the CCK-1 receptor, this receptor is also known to associate with arrestin molecules. Arrestin binding to a G protein-coupled receptor can occur as a result of agonist binding with signaling kinase action leading to phosphorylation of the receptor, as well as direct interaction of arrestin with nonphosphorylated portions of the receptor. Arrestin is a known multifunctional adapter protein that can contribute to receptor endocytosis and to signaling events in the cell, including some of those described above (Whalen et al. 2011).
Biochemical and Cellular Regulation of the CCK-1 Receptor
Like most G protein-coupled receptors, the CCK-1 receptor is regulated by both biochemical and cellular mechanisms to protect the cell from overstimulation (Miller and Gao 2008). Indeed, hyperstimulation through this receptor is the basis for one of the most frequently used animal models of pancreatitis, in which very high doses of the CCK analogue caerulein is administered and results in fusion of the zymogen granules with the lateral (rather than the apical) plasmalemma and release of these enzymes into the pancreatic parenchyma where they become activated and initiate autodigestion of this organ and surrounding tissues. The most common biochemical mechanism of regulation of this receptor involves phosphorylation. This occurs exclusively on serine and threonine residues within the intracellular loops and carboxyl-terminal tail domains. The earliest and most sensitive phosphorylation events result from activation of protein kinase C. Of interest, the phosphorylation of key protein kinase C substrates within the third intracellular loop has been shown to produce a conformational change that makes the second loop accessible for its phosphorylation and for its interaction with cellular regulatory molecules. Higher concentrations of CCK also stimulate the slower action of a G protein-coupled receptor kinase that adds to the phosphorylation of this receptor. These phosphorylation events have been shown to interfere with the normal coupling of this receptor with its G proteins. Presumably, this also contributes to arrestin association, known to effect such disruption of coupling.
Because the natural agonists for the CCK-1 receptor are hydrophilic peptides that are unable to cross the lipid bilayer, internalization of the receptor is another very effective mechanism for desensitization in which the hormone is unable to bind to and stimulate the receptor. Indeed, the agonist occupation of this receptor has been shown to result in the internalization of this receptor, with clathrin-dependent endocytosis representing the major pathway (Roettger et al. 1993). In CHO cell lines, potocytosis into caveolae has also been described. Another interesting cellular mechanism for desensitization that has been described was identified as insulation in which the receptor is moved into a highly specialized plasmalemmal domain that is devoid of G proteins for coupling with the receptor. These cellular regulatory mechanisms are typically slower of onset and slower to reverse than the biochemical mechanisms described above.
Clinical Relevance of the CCK-1 Receptor
The CCK-1 receptor has been implicated in mediating some of the intestinal dysmotility responsible for abdominal crampy pain and abnormal bowel habits in irritable bowel syndrome, as well as the gallbladder inertia and incomplete gallbladder emptying found in cholesterol gallstone disease. It has also been proposed as playing a role in the reduced satiety and increased food consumption present in some patients with obesity and obesity-related medical disorders. Indeed, it is the latter that has stimulated most of the work by pharmaceutical companies to develop noncaloric agonists of this receptor as a means for inducing satiety and reducing food intake. There are data suggesting the possibility that different forms of CCK may affect appetite differently, with CCK-8 reducing meal size, but sometimes having more frequent meals offsetting this, while CCK-58 is reported to both reduce meal size and to prolong inter-meal interval (Sayegh et al. 2014).
There has also been substantial work focused on CCK receptors in neoplastic diseases (Reubi et al. 1997). This follows the established trophic effects of this hormone on target tissues that are mediated by these receptors. Indeed, many epithelial cancer cell lines have been described to express CCK-1 and/or CCK-2 receptors, or misspliced variants of these receptors (Korner and Miller 2009). The major cancers that can express the CCK-1 receptor include pancreatic ductal adenocarcinoma, colorectal carcinoma, meningiomas, and some neuroblastomas. Antagonists of this receptor are being studied to examine their potential therapeutic role in pancreatic cancer. Additionally, reagents directed to this receptor may have an imaging, staging, or stratification role for these tumors.
The CCK-1 receptor is physiologically quite important for normal digestion, bowel transit, and appetite control, all roles key for normal nutritional homeostasis. Additionally, this receptor plays various roles in common gastrointestinal disorders, including irritable bowel syndrome and gallstone disease, as well as likely contributing to the development of obesity and potentially playing a therapeutic role in this disorder and in obesity-related medical diseases. It also can be expressed on various cancers, where agents targeting this receptor can be used in diagnosis, staging, stratification, and possibly also treatment.
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