Evidence That Interspecies Polymorphism in the Human and Rat Cholecystokinin Receptor-2 Affects Structure of the Binding Site for the Endogenous Agonist Cholecystokinin

Abstract

The cholecystokinin (CCK) receptor-2 exerts very important central and peripheral functions by binding the neuropeptides cholecystokinin or gastrin. Because this receptor is a potential therapeutic target, great interest has been devoted to the identification of efficient antagonists. However, interspecies genetic polymorphism that does not alter cholecystokinin-induced signaling was shown to markedly affect activity of synthetic ligands. In this context, precise structural study of the agonist binding site on the human cholecystokinin receptor-2 is a prerequisite to elucidating the molecular basis for its activation and to optimizing properties of synthetic ligands. In this study, using site-directed mutagenesis and molecular modeling, we delineated the binding site for CCK on the human cholecystokinin receptor-2 by mutating amino acids corresponding to that of the rat homolog. By doing so, we demonstrated that, although resembling that of rat homolog, the human cholecystokinin receptor-2 binding site also displays important distinct structural features that were demonstrated by susceptibility to several point mutations (F120A, Y189A, H207A). Furthermore, docking of CCK in the human and rat cholecystokinin receptor-2, followed by dynamic simulations, allowed us to propose a plausible structural explanation of the experimentally observed difference between rat and human cholecystokinin-2 receptors. Cholecystokinin (CCK)1 and gastrin are two regulatory peptides having high affinity for CCK receptors. Both peptides share an identical carboxyl-terminal pentapeptide sequence but differ in their selectivity for the two CCK receptor subtypes, the CCK1 (CCK1R) and the CCK2 (CCK2R) receptors, on the basis of tyrosine sulfation at the seventh position (CCK) or at the sixth position (gastrin) from the carboxyl terminus.Although both receptors recognize sulfated CCK with comparable high affinity, the CCK2R has high affinity for both sulfated and non-sulfated gastrin (1, 2). CCK1R and CCK2R are seven-transmembrane spanning receptors that belong to the superfamily of G protein-coupled receptors and have ϳ50% homology (3-5). CCK1Rs are mainly found in the peripheral organs, where they regulate pancreatic secretion and gallbladder and gastrointestinal motility, but are also found in some areas of the central nervous system, where they regulate satiety and analgesia. CCK2Rs are predominantly present throughout the central nervous system, where they regulate anxiety/panic attacks and dopamine release, implicated in the pathogenesis of dopaminergic related movement and behavioral disorders in humans. In the gut, CCK2Rs regulate acid and histamine secretions, gastrointestinal motility, as well as growth in the gastric mucosa (1, 2). The important physiological functions mediated by CCK receptors, and therefore their possible implication in associated disorders, have generated considerable interest in the identification of ligands that selectively activate or block CCK1R and CCK2R (6). To date, a large panel of such molecules has b...

Cholecystokinin and Gastrin Receptors

“…IVA). On the other hand, K d values for high-and low-affinity binding of CCK to CCK2R are ϳ100 -300 pM and 2-5 nM, respectively (264). In addition to these two sites, there exist very-low-affinity sites as well with K d values of ϳ10 M in both the CCK1R and CCK2R (213).…”

Partial Agonism, Neutral Antagonism, and Inverse Agonism at the Human Wild-Type and Constitutively Active Cholecystokinin-2 Receptors

“…The presence of the desired and the absence of undesired mutations were confirmed by automated sequencing of the complete CCK2R coding sequence (Applied Biosystems, Foster City, CA). These mutants were characterized previously for their ability to bind and to respond to CCK (Langer et al, 2005).…”

“…The effect of mutation of four reference amino acids (Blaker et al, 1998;Gales et al, 2003a;Langer et al, 2005) of the CCK binding site on the affinity of synthetic ligands was evaluated for docking refinement. Mutation factors, which account for shifts of affinity, varied for the different nonpeptide ligands and were generally lower than for CCK-4 ( Table 1).…”

“…CCK2R.CCK-4 (CCK-4: Boc-Trp-Met-Asp-Phe-CONH 2 ) complex model was built on the basis of the CCK2R.CCK9 complex model published previously (Langer et al, 2005) by deleting the first five residues, adding Boc-group and refining as described previously.…”

“…These interspecies differences toward synthetic ligands were shown to be caused by sequence variations within the binding site of the CCK2R, thus pointing out the high importance of in vivo and in vitro tests with human protein targets before clinical evaluation of the molecules in humans . Availability of synthetic ligands presenting distinct pharmacological profiles, together with data on the structure of the human CCK2R binding site such as those recently obtained (Langer et al, 2005), offers a unique opportunity to investigate the molecular basis for receptor activation or blocking and to further test the feasibility of "rational drug design or improvement".…”