Activation of Family C G-protein-coupled Receptors by the Tripeptide Glutathione

Abstract

The Family C G-protein-coupled receptors include the metabotropic glutamate receptors, the ␥-aminobutyric acid, type B (GABA B ) receptor, the calcium-sensing receptor (CaSR), which participates in the regulation of calcium homeostasis in the body, and a diverse group of sensory receptors that encompass the amino acidactivated fish 5.24 chemosensory receptor, the mammalian T1R taste receptors, and the V2R pheromone receptors. A common feature of Family C receptors is the presence of an amino acid binding site. In this study, a preliminary in silico analysis of the size and shape of the amino acid binding pocket in selected Family C receptors suggested that some members of this family could accommodate larger ligands such as peptides. Subsequent screening and docking experiments identified GSH as a potential ligand or coligand at the fish 5.24 receptor and the rat CaSR. These in silico predictions were confirmed using an [ 3 H]GSH radioligand binding assay and a fluorescence-based functional assay performed on wildtype and chimeric receptors. Glutathione was shown to act as an orthosteric agonist at the 5.24 receptor and as a potent enhancer of calcium-induced activation of the CaSR. Within the mammalian receptors, this effect was specific to the CaSR because GSH neither directly activated nor potentiated other Family C receptors including GPRC6A (the putative mammalian homolog of the fish 5.24 receptor), the metabotropic glutamate receptors, or the GABA B receptor. Our findings reveal a potential new role for GSH and suggest that this peptide may act as an endogenous modulator of the CaSR in the parathyroid gland where this receptor is known to control the release of parathyroid hormone, and in other tissues such as the brain and gastrointestinal tract where the role of the calcium receptor appears to subserve other, as yet unknown, physiological functions.Free amino acids act within the large extracellular ligand binding domains of Family C G-protein-coupled receptors as either the direct acting orthosteric agonists or as allosteric modulators. Glutamate, and the glutamate metabolite GABA, are the endogenous ligands at the metabotropic glutamate receptors (mGluRs) 3 and the GABA B receptor, respectively. In contrast to the restricted activation of the mGluRs and the GABA B receptor by glutamate and GABA, other Family C receptors can be activated by multiple amino acids. The fish 5.24 chemosensory receptor, which is expressed in several sensory organs and is thought to play a role in feeding and navigation, can be activated by most amino acids (1). A similar, albeit non-identical, spectrum of amino acid activation is also an intrinsic property of GPRC6A, the putative mammalian homolog of the 5.24 receptor (2, 3), and the T1R1/T1R3 heteromeric taste receptor (4).Amino acids also allosterically regulate the activity of the calciumsensing receptor (CaSR), another member of the Family C receptors. Although calcium is considered the primary endogenous ligand, calcium-induced responses mediated by the CaSR are enhance...

Involvement of the Calcium-sensing Receptor in Human Taste Perception

“…Among the class C G-protein-coupled receptors, the CaSR has the highest homology with GPRC6A. It has been reported that a chimeric receptor consisting of the goldfish 5.24 (mammalian GPRC6A homolog) outer membrane domain and the mouse GPRC6A trans-and intramembrane domains is activated by GSH but that neither a chimeric receptor consisting of the GPRC6A outer membrane domain and 5.24 trans-and intramembrane domains nor full-length GPRC6A itself is functionally active (28). Wellendorph et al (30) reported that GPRC6A is expressed in rat taste bud-containing epithelia.…”

“…Wang et al (28) reported that GSH does not directly activate the CaSR but potentiates calcium-induced responses. They showed that GSH does not activate the CaSR in an assay buffer containing 0.5 mM calcium, but activation occurred under similar conditions when the assay buffer was supplemented with an additional 0.3 mM calcium administered simultaneously.…”

Allosteric Modulation of the Calcium-sensing Receptor by γ-Glutamyl Peptides

“…S1). The results indicate that ␥-glutamyl peptides and S-methylgluta- (14) and glutathione (21) binding sites to the VFT domain and we previously identified T145A/S170T as a double mutant form of the VFT domain with near-normal Ca 2ϩ o -sensitivity but markedly impaired L-amino acid sensing (15). In the current study, we first confirmed that this mutant exhibited normal or near-normal Ca 2ϩ o sensitivity in the absence of L-Phe or S-methylglutathione (Fig.…”

“…In addition, the CaR binds socalled type-II calcimimetics, which are typically uncharged organic compounds that act as positive allosteric modulators. Two main sites for the binding of positive allosteric modulators have been identified: one in the heptahelical domain for type-II calcimimetics such as the phenylalkylamines NPS R-467 and cinacalcet (30,32); and one or more in the VFT domain for L-amino acids such as L-Phe or L-Trp (14) and glutathione and analogs (21). Previous work suggested that the L-amino acid binding site is selectively disabled by the double mutant T145A/S170T (15).…”

“…The larger side-chain binding surfaces in this subgroup of receptors appear to explain, at least in part, their notable promiscuity for various sub-classes of amino acids (reviews: Refs. 19,20) and has led to the successful prediction that small peptides that retain ␣-amino and ␣-carboxylate functional groups at their N termini, including the tri-peptide glutathione would bind and activate the CaR expressed in HEK-293 cells (21). Building on this observation, ␥-glutamyl peptides were recently found to be potent taste enhancers (22) raising the possibility that they may be physiologically im-portant receptor modulators in various tissues in which the CaR is expressed.…”