Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+ homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+ and PO43- ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ ions stabilize the active state, PO43- ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.DOI: http://dx.doi.org/10.7554/eLife.13662.001

Extracellular phosphate regulates its own renal excretion by eliciting concentration-dependent secretion of parathyroid hormone (PTH). However, the phosphate-sensing mechanism remains unknown and requires elucidation for understanding the aetiology of secondary hyperparathyroidism in chronic kidney disease (CKD). The calcium-sensing receptor (CaSR) is the main controller of PTH secretion and here we show that raising phosphate concentration within the pathophysiologic range for CKD significantly inhibits CaSR activity via non-competitive antagonism. Mutation of residue R62 in anion binding site-1 abolishes phosphate-induced inhibition of CaSR. Further, pathophysiologic phosphate concentrations elicit rapid and reversible increases in PTH secretion from freshly-isolated human parathyroid cells consistent with a receptor-mediated action. The same effect is seen in wild-type murine parathyroid glands, but not in CaSR knockout glands. By sensing moderate changes in extracellular phosphate concentration, the CaSR represents a phosphate sensor in the parathyroid gland, explaining the stimulatory effect of phosphate on PTH secretion.

Molecular analysis of the CaR indicates that it is composed of several functional domains that are conserved across related members of subgroup C. The N-terminal Venus Fly Trap domains of many of these receptors recognize amino acids (especially glutamate) or the amino acid analog, ␥-aminobutyric acid. More recent work indicates that several cloned members of the family are also broad-spectrum amino acid sensors. In mammals, these include the CaR, which is allosterically activated by aromatic, aliphatic, and polar amino acids as well as plasma-like amino acid mixtures (6) and a heterodimeric amino acid taste receptor, which has broad selectivity for aliphatic, polar, and charged amino acids but not aromatic amino acids (7). In human embryonic kidney (HEK)293 cells that stably express the human CaR, L-amino acids markedly enhanced the sensitivity of the receptor to Ca 2ϩ and other cationic agonists including spermine and Gd 3ϩ (6). Because the CaR mediates the acute control of PTH secretion, the finding that it is allosterically activated by L-amino acids raises the possibility that PTH secretion is acutely regulated not only by adjustments in extracellular Ca 2ϩ o but also by physiological changes in amino acid concentration. We have tested and confirmed this hypothesis in the current study on normal human parathyroid cells. The data indicate that Lamino acids and plasma-like mixtures of L-amino acids allosterically activate endogenous parathyroid CaRs contributing to the control of intracellular signaling pathways and PTH secretion. In the presence of physiological concentrations of extracellular Ca 2ϩ , L-amino acids (also at physiological concentrations) stereoselectively activated Ca 2ϩ mobilization and inhibited PTH secretion. The data support the view that fluctuations in serum amino acid levels acting via the CaR acutely regulate PTH secretion and thus whole body calcium metabolism.

We previously demonstrated that the human calciumsensing receptor (CaR) is allosterically activated by Lamino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814 -4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1-599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.The extracellular Ca 2ϩ -sensing receptor (CaR) 1 plays a key role in the regulation of whole body calcium metabolism. In keeping with this, the CaR-null mouse exhibits loss of feedback control of parathyroid hormone secretion, hyperparathyroidism, and a metabolic bone disease (2). Furthermore, inactivating and activating mutations of the receptor in humans have been shown to induce various disorders of calcium metabolism (for review, see Ref. 3).Although the CaR is a key molecular regulator of whole body calcium metabolism, it presents something of a conundrum. It is widely expressed in mammalian tissues, including tissues such as the brain, that are not clearly involved in calcium metabolism. Furthermore, its closest relatives in molecular terms are members of sub-group C of the G protein-coupled receptors, receptors for specific amino acids such as L-glutamate (mGlus) and glutamate analogs, e.g. GABA. The large extracellular heads of these receptors are related to nutrientsensing, bacterial periplasmic-binding proteins (4). The finding that the CaR is allosterically activated by a broad spectrum of L-amino acids, including aromatics such as L-Phe and L-Trp and aliphatic and polar amino acids such as L-Ala and L-Ser, has the effect of drawing it closer functionally to other members of subgroup C (1). However, the site of amino acid binding has been unclear. A si...

The extracellular Ca 2؉ -sensing receptor is activated allosterically by L-amino acids, and recent molecular analysis indicates that amino acids are likely to bind in the receptor's Venus flytrap domain. In the current study we set out to identify residues in the VFT domain that specifically support amino acid binding and/or amino acid-dependent receptor activation. Herein we describe two mutations of the Ca 2؉ -sensing receptor (CaR) Venus Flytrap domain, T145A and S170T, that specifically impair amino acid sensing, leaving Ca 2؉ sensing intact, as determined by receptor-dependent activation of intracellular Ca 2؉ mobilization in fura-2-loaded HEK293 cells. With respect to the wild-type CaR, T145A and S170T exhibited reduced sensitivity to L-Phe, and T145A also exhibited markedly impaired L/D selectivity. When combined, the double mutant T145A/S170T exhibited normal or near-normal sensitivity to extracellular Ca 2؉ but was resistant to L-Phe at concentrations up to 100 mM. We conclude that T145A/S170T selectively disables L-amino acid sensing and that the Ca 2؉ and Lamino acid-sensing functions of the CaR can be dissociated.