CCK-B receptor: chemistry, molecular biology, biochemistry and pharmacology

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Abstract

Cholecystokinin (CCK) is a peptide originally discovered in the gastrointestinal tract but also found in high density in the mammalian brain. The C-terminal sulphated octapeptide fragment of cholecystokinin (CCK8) constitutes one of the major neuropeptides in the brain; CCK8 has been shown to be involved in numerous physiological functions such as feeding behavior, central respiratory control and cardiovascular tonus, vigilance states, memory processes, nociception, emotional and motivational responses.

CCK8 interacts with nanomolar affinities with two different receptors designated CCK-A and CCK-B. The functional role of CCK and its binding sites in the brain and periphery has been investigated thanks to the development of potent and selective CCK receptor antagonists and agonists.

In this review, the strategies followed to design these probes, and their use to study the anatomy of CCK pathways, the neurochemical and pharmacological properties of this peptide and the clinical perspectives offered by manipulation of the CCK system will be reported. The physiological and pathological implication of CCK-B receptor will be confirmed in CCK-B receptor deficient mice obtained by gene targeting (Nagata et al., 1996. Proc. Natl. Acad. Sci. USA 93, 11825–11830). Moreover, CCK receptor gene structure, deletion and mutagenesis experiments, and signal transduction mechanisms will be discussed.

Introduction

Cholecystokinin (CCK) is a gut–brain peptide that exerts a variety of physiological actions in the gastrointestinal tract and central nervous system through cell surface CCK receptors. CCK was initially isolated from the porcine duodenum as a 33 amino acid peptide (Mutt and Jorpes, 1968). A number of biologically-active molecular variants were subsequently described (Rehfeld et al., 1982) and the most abundant peptide present in the brain was shown to be CCK8: Asp–Tyr(SO3H)–Met–Gly–Trp–Met–Asp–Phe–NH2. On the basis of their pharmacological properties and specificities for ligand binding, CCK receptors have been divided into two subtypes, namely, the CCK-A and CCK-B receptors both belonging to the class of G protein-coupled receptors characterized by seven transmembrane (TM) domains. CCK-A receptors are located mainly in the periphery but are also found in some regions of the brain (Hill et al., 1987a, Hill et al., 1987b). The major population of central CCK receptors are of CCK-B subtype (Hill et al., 1987a), which is also found in the stomach and vagus nerve. The gastrin receptor was found to be identical to the CCK-B receptor.

Considerable interest is devoted to the pharmacology of CCK-B receptors, since administration of selective agonists produces behavioral changes such as anxiety, perturbation of memory and hyperalgesia, and dysfunctioning of CCK-B related neural pathways could be involved in neuropsychiatric disorders. Accordingly, CCK-B antagonists have been shown to block panic attacks induced in humans by systemic administration of low doses of CCK4 (Bradwejn et al., 1991), Trp–Met–Asp–Phe–NH2, which has a 300-fold higher affinity for the CCK-B receptor than for the peripheral CCK-A receptor (Daugé et al., 1990).

Section snippets

Design of selective agonists for CCK-B receptors

At CCK-A receptors, sulphated CCK8 [Asp–Tyr(SO3H)–Met–Gly–Trp–Met–Asp–Phe–NH2]was the minimal sequence for high affinity binding, whereas at central binding sites, CCK4, gastrin and unsulphated CCK8 can be bound, albeit they have somewhat lower potency compared to sulphated CCK8.

Different strategies have been followed to design potent and selective agonists and antagonists of CCK-B receptors. In spite of its intrinsic flexibility, CCK8 was found by NMR to exist preferentially under folded form

Cloning and characterization of the CCK-B receptor

Although there is general agreement that the CCK-A receptor has a distinct agonist and antagonist binding profile from the gastrin and CCK-B receptors, controversy remains regarding the existence of distinct CCK-B and gastrin receptor subtypes. Gastrin, CCK and CCK-related peptides comprise a hormone family, characterized by the identical carboxyl-terminal pentapeptide amide structure, a domain critical for receptor binding. Agonist binding studies on brain membranes and parietal cells show a

CCK-B receptor gene structure

The gene for the CCK-B receptor has been cloned in humans (Song et al., 1993). This gene exceeded 8 kb in length and contained a 1356-bp open reading frame which was interrupted by four introns of 164–1177 bp. Exon 1 encodes the putative extracellular amino terminus of the receptor. Exons 2 and 3 encode TM regions I–IV, and exon 4 encodes the fifth TM region and an initial portion of the third intracellular loop. Exon 5 encodes the remainder of this intracellular loop, the remaining TM regions VI

CCK-B receptor localization

CCK-A receptors are found principally in the gastrointestinal tract and select areas of the CNS, while CCK-B/gastrin receptors are found principally in the CNS and select areas of the gastrointestinal tract, on pancreatic acinar cells and parietal cells.

Autoradiographic studies using CCK-related peptide-binding sites in the rat brain, such as [125I]CCK8 (Niehoff, 1989; Pélaprat et al., 1987), [3H]BDNL (Pélaprat et al., 1987), [3H]CCK8 (Dijk et al., 1984), [3H]pentagastrin (Gaudreau et al., 1983

Signal-transduction cascade for CCK-B receptors

The signal transduction mechanism of CCK-A receptors has been best characterized in pancreatic acinar cells, where CCK stimulates digestive enzyme release, usually assayed as amylase activity. One physiologically important signaling pathway is the hydrolysis of polyphosphoinositides (PPI) by phospholipase C and the subsequent formation of the second messengers, inositol 1,4,5-triphosphate (1,4,5-IP3) and 1,2-diacylglycerol (DAG), leading to the release of intracellular Ca2+ and the activation

Site directed mutagenesis of the CCK-B receptor: characterization of residues involved in binding of ligands and functional coupling

In order to develop more specific and selective compounds, it is important to elucidate the molecular interactions involved in CCK receptor ligand binding. In contrast to the endogenous peptide ligands, nonpeptide antagonists often show substantial differences in affinity among species. These species-related differences in binding affinity often reflect differences in the primary structure of the receptors. Thus, although canine and human CCK-B receptors share ∼90% amino acid identity and have

CCK-B receptor heterogeneity

On the basis of the pharmacological data obtained receptor subtypes has been proposed to exist in the CCK family. However, despite extensive searching by hybridization screening of cDNA libraries from different tissues [reviewed in Wank (1995)], only two CCK receptors have been identified. Southern blot hybridization using human, guinea pig and rat DNA with either CCK-A or CCK-B receptor species-specific, full-length coding sequence probes under both high- and low-stringency conditions has also

Gastric acid secretion

Gastrin and CCK, which are two related peptides that share homology at their biologically active pentapeptidic C-terminal sequence, have been shown to stimulate gastric acid secretion in vitro. Thus, both peptides bind to receptor sites located on parietal (Magous et al., 1989) and induce an increase in phosphoinositide turnover (Roche and Magous, 1989; Chew and Brown, 1986) and an uptake in [14C]aminopyrine ([14C]AP) (an index of acid secretion in vitro) with the same efficacy and potency (

Conclusion

The peptide CCK exists in numerous brain and peripheral regions where it serves as a neurotransmitter and neuromodulator or hormone. The development of selective and highly potent agonists and antagonists has constituted a major breakthrough in the field of CCK research. Numerous data support the existence of physiological interactions between endogenous CCK system and other systems, as opioid or dopaminergic systems.

The obvious neuroanatomical association between dopamine and CCK continues to

Acknowledgements

The authors would like to thank C. Dupuis for typing the tables. All members of the laboratory and colleagues whose names appear in the references cited in this review are acknowledged. They thank Rhone-Poulenc-Rorer for their financial supports.

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