Met-195 of the Cholecystokinin-A Receptor Interacts with the Sulfated Tyrosine of Cholecystokinin and Is Crucial for Receptor Transition to High Affinity State

Abstract

Sulfation of the tyrosine at the seventh position from the C terminus of cholecystokinin (CCK) is crucial for CCK binding to the CCK-A receptor. Using three-dimensional modeling, we identified methionine 195 of the CCK-A receptor as a putative amino acid in interaction with the aromatic ring of the sulfated tyrosine of CCK. We analyzed the role played by the two partners of this interaction. The exchange of Met-195 for a leucine caused a minor decrease (2.8-fold) on the affinity of the high affinity sites for sulfated CCK-9, a strong drop (73%) of their number, and a 30-fold decrease on the affinity of the low and very low affinity sites for sulfated CCK-9, with no change in their number. The mutation also caused a 54-fold decrease of the potency of the receptor to induce inositol phosphates production. The high affinity sites of the wild-type CCK-A receptor were highly selective (800-fold) toward sulfated versus nonsulfated CCK, whereas low and very low affinity sites were poorly selective (10-and 18-fold). In addition, the M195L mutant bound, and responded to, sulfated CCK analogues with decreased affinities and potencies, whereas it bound and responded to nonsulfated CCK identically to the wild-type receptor. Thus, Met-195 interacts with the aromatic ring of the sulfated tyrosine to correctly position the sulfated group of CCK in the binding site of the receptor. This interaction is essential for CCK-dependent transition of the CCK-A receptor to a high affinity state. Our data should represent an important step toward the identification of the residue(s) of the receptor in interaction with the sulfate moiety of CCK and the understanding of the molecular mechanisms that govern CCK-A receptor activation. The peptide cholecystokinin (CCK)1 is found throughout the gastrointestinal system and the central nervous system where it acts both as a hormone and a neurotransmitter (1). Posttranslational processing of CCK involves sulfation of the tyrosine at position seven from the C-terminal and ␣-amidation of the C-terminal phenylalanine residue (1). Studies using chemically synthesized fragments have shown that the C-terminal sulfated and amidated octapeptide Asp-Tyr(SO 3 H)-Met-GlyTrp-Met-Asp-Phe-NH 2 (Fig. 1) exhibits the full spectrum of biological activity. However, fragments as small as the C-terminal tetrapeptide Trp-Met-Asp-Phe-NH 2 , which CCK has in common with the related peptide gastrin, retain biological activity (2).The actions of CCK are mediated by membrane receptors that are divided into two subtypes, the CCK-A and the CCK-B/gastrin (3). The cloning of the cDNA coding for these receptors has shown that they belong to the superfamily of G protein-coupled receptors which are characterized by seven transmembrane domains connected by intracellular and extracellular loops with an extracellular N-terminal and intracellular C-terminal (4, 5). Both receptor subtypes can exist in several affinity states for sulfated CCK and have in common the functional coupling to phospholipase-C, presumably via binding to a G␣...

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

“…Trp-39 and Gln-40, located at the top of transmembrane helix I, were shown to interact directly with the N-terminal portion of CCK (14). , located in the second extracellular loop, were then shown to interact with the sulfated tyrosine (15,16). More recently, Arg-336 and Asn-333, at the top of helix VI, were demonstrated to pair with the Asp carboxylate and the Cterminal amide of CCK, respectively (17).…”

“…The resulting theoretical positioning of CCK into the CCK1R binding site was experimentally validated by two-dimensional site-directed mutagenesis. By doing so, Met-195-Arg-197, Arg-336, and Asn-333 were shown to belong the CCK1R binding site and to interact with the sulfated tyrosine of CCK, the Asp-8 carboxylate, and the C-terminal amide, respectively (15)(16)(17). The program package (Insight II, Discover, Homology, Biopolymer) from Molecular Simulations Inc. (San Diego, CA) was used for all the calculations.…”

“…In a first series of experiments, COS-7 cells expressing mutated receptors were assayed for binding of the non-peptide antagonist [ 3 H]SR-27,897. Indeed, binding of this antagonist, unlike that of an agonist, offers the advantage of allowing detection of CCK1R independent of the coupling state to G protein(s), thus yielding accurate expression levels of all mutants (15,17). Ligand binding data are summarized in Table I.…”

Identification of Tyrosine 189 and Asparagine 358 of the Cholecystokinin 2 Receptor in Direct Interaction with the Crucial C-Terminal Amide of Cholecystokinin by Molecular Modeling, Site-Directed Mutagenesis, and Structure/Affinity Studies

“…Despite the fact that sulfated and amidated CCK presents a similar high affinity for both receptors, our previous studies using mutagenesis and molecular modeling techniques brought several pieces of evidence for a different anchoring of CCK in the CCK1 and CCK2 receptors. Indeed, complementary substitutions in the ligand and in the receptor allowed us to demonstrate that two amino acids in the second extracellular loop of the CCK1R, Met195 and Arg197, interact with the tyrosine residue of CCK (Tyr3) whereas His207, located in the same loop in the CCK2R, interacts with Asp8, the penultimate residue of CCK (Gigoux et al, 1998(Gigoux et al, , 1999bSilvente-Poirot et al, 1999). In contrast, in the CCK1R, Asp8 of CCK was found in interaction with Arg336 (RL6.58), further confirming that different interactions are involved between CCK and the two subtypes (Gigoux et al, 1999a).…”

Structure of Cholecystokinin Receptor Binding Sites and Mechanism of Activation/Inactivation by Agonists/Antagonists

“…In a second step, experiments were performed by mutating candidate residue(s) of the receptor binding site which had been depicted in the 3-D model and analysing extensively effects of mutation(s). Several critical contacts between the CCK1R binding site and CCK were successively discovered: Met195 and Arg197 located in the second extracellular loop, were shown to interact with the sulfated tyrosine (Gigoux et al 1998(Gigoux et al & 1999b; Arg336 and Asn333 at the top of helix VI were demonstrated to pair with the Asp carboxylate and the Cterminal amide of CCK, respectively (Gigoux et al 1999a). More recently, search for residues of the receptor in interaction with Met/Nle and Phe of CCK led to description of a network of hydrophobic residues including Cys94, Met121, Val125, Phe218, Ile329, Phe330, Trp326, Ile352, Leu356 and Tyr360 all involved in the binding site for CCK and in the activation process of the CCK1R (Escrieut et al 2002) (fig.…”