Three Adjacent Serines in the Extracellular Domains of the CaR Are Required for l-Amino Acid-mediated Potentiation of Receptor Function

Abstract

The extracellular calcium (Ca In this study, we examined whether conserved amino acid residues involved in the binding of glutamate to metabotropic glutamate receptors (mGluRs) also participate in the potentiation of the activity of CaR by L-phenylalanine. Ser-170 corresponding to Thr-188 in rat mGluR1a appears to be important for the modulating actions of phenylalanine. In the presence of phenylalanine, a mutant CaR with a single mutation S170A showed no significant decrease in its EC 50 for stimulation by Ca o metabolism. It has been documented that these two homeostatic systems are intimately related. For instance, a reduction in protein intake below the normal level results in secondary hyperparathyroidism in the context of normocalcemia (4), and high dietary protein intake induces elevated urinary calcium excretion (5, 6).The CaR is a G protein-coupled receptor in the same subfamily C as the metabotropic glutamate receptors, mGluRs1-8 (7-9). The family C receptors all possess unusually large 500 -600-residue extracellular amino-terminal domains (ECDs), possibly with a bilobed Venus flytrap structure similar to that found in bacterial periplasmic binding proteins for nutrients (10, 11). The family C receptors interact with their physiological agonists through their ECDs (11-13).For the rat homolog of mGluR1a, the key residues involved in the binding of glutamate to its ECD have been identified through the determination of its crystal structure when it is complexed with glutamate (11). Ser-164, Ser-165, Thr-188, and Glu-292 form hydrogen bonds with the ␣-carboxyl group in glutamate. Five residues, Ser-186, Thr-188, Asp-208, Tyr-236, and Asp-318, form additional hydrogen bonds with the ␣-amino group of glutamate. Of these residues, Ser-165, Thr-188, Asp-208, Tyr-236, and Asp-318 in the mGluR1a are conserved in the CaR, corresponding to Ser-147, Ser-170, Asp-190, Tyr-218, and Glu-297, respectively. The ␥-carboxyl group in glutamate forms one hydrogen bond each with Lys-409 and Arg-323 in mGluR1a; however, these two positively charged amino acid residues are not conserved in the CaR. Mutations S165A and T188A have been reported to impair glutamate binding and resultant activation of mGluR1a (10), whereas the functional significance of other residues in the glutamate binding site of the mGluRs have not been reported.Previous studies of the CaR have shown that substitutions of amino acids such as Ser-147 and Ser-170, which correspond to Ser-165 and Thr-188 in mGluR1a, markedly reduce activation of the receptor by Ca 2ϩ o (13). In addition, the mutations of

Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium

“…Here, we have also identified and characterized an L-Phebinding pocket formed by residues Leu 51 based on the structure of mGluR1␣ (53). S170T has been reported by different groups to interfere with the L-Phe sensing ability of CaSR (51,53).…”

“…As shown in Fig. 1 (51)(52)(53). This L-Phe-binding site is also located within the hinge region of the ECD of CaSR with a relatively localized configuration during MD simulations.…”

Homology Modeling of the Transmembrane Domain of the Human Calcium Sensing Receptor and Localization of an Allosteric Binding Site

“…The phenylalkylamines are of particular interest, because both allosteric agonists (calcimimetics) and antagonists (calcilytics) have been identified, typified by NPS R-467 or R-568 (calcimimetics) (9) and NPS 2143 (calcilytic) (11). The putative binding site(s) for amino acids and potentially for peptides (including poly-L-arginine, protamine, ␤-amyloid) have been identified within the extracellular agonist binding domain by homology with metabotropic glutamate receptors, and have been localized to a triple serine motif, Ser-169 -171 (12). Mutant CaRs, which are no longer modulated by amino acids, still exhibit altered potency of Ca 2ϩ in the presence of phenylalkylamines (13), confirming that the phenylalkylamines interact at a distinct site.…”

Comment moduler l’activité du récepteur du calcium extracellulaire ?

“…Les acides aminés aromatiques (phénylalanine, tyrosine…) de forme L, à des concentrations compatibles avec celles trouvées dans le sang (2 mM à 5 mM), augmentent la sensibilité du Ca 2+ pour le CaR [14]. Leur site d'action sur le CaR pourrait être voisin de celui des ions Ca 2+ (Figure2B) et ferait notamment intervenir la sérine 170 [15]. Ces observations pourraient en partie expliquer l'influence de la consommation quotidienne de protéines sur l'homéostasie calcique, et l'hypercalcémie souvent associée aux régimes hyperprotidiques.…”