CONTEXT:Primary hyperparathyroidism, which occurs most commonly in patients with adenomatous disease of a single parathyroid gland, arises as a result of impaired extracellular Ca(2+) (Ca(2+)(o))-dependent feedback on PTH secretion, a process mediated by the calcium-sensing receptor (CaR).OBJECTIVE:Because the Ca(2+)(o) sensitivity of the CaR is positively modulated by L-amino acids, we decided to investigate whether the impaired feedback of PTH secretion in adenomatous parathyroid cells might arise from decreased sensitivity to L-amino acids.DESIGN:Samples of normal and adenomatous human parathyroid cells were prepared by collagenase treatment and then exposed in vitro to various concentrations of Ca(2+)(o) or the CaR-active amino acid, L-phenylalanine (L-Phe).SETTING AND PATIENTS:Excess normal parathyroid tissue was obtained from parathyroid autotransplants at the time of thyroid surgery. Samples of adenomatous tissue were obtained from histologically confirmed parathyroid adenomas.MAIN OUTCOME MEASURES:The primary measure was sensitivity of Ca(2+)(o)-dependent PTH secretion to the amino acid L-Phe. The secondary measure was sensitivity of Ca(2+)(o)-dependent intracellular Ca(2+) mobilization to L-Phe.RESULTS:Parathyroid adenomas exhibited reduced sensitivity to the CaR-active amino acid L-Phe, which affected both Ca(2+)(o)-dependent PTH secretion and Ca(2+)(o)-dependent intracellular Ca(2+) mobilization as a measure of CaR-dependent signaling in parathyroid cells.CONCLUSIONS:Impaired L-amino acid sensing by calcium-sensing receptors in adenomatous parathyroid cells contributes to the loss of feedback control of PTH secretion in primary hyperparathyroidism. The CaR's amino acid binding site may be exploited as a target in the medical treatment of primary and perhaps other forms of hyperparathyroidism.

The calcium-sensing receptor is a multimodal, multimetabolic sensor that mediates the feedback-dependent control of whole body calcium metabolism. Remarkably, in addition to its role in Ca(2+)(o) (extracellular Ca(2+)) sensing, the CaR (Ca(2+)-sensing receptor) also responds to L-amino acids. L-amino acids appear to activate, predominantly, a signalling pathway coupled with intracellular Ca(2+) mobilization, require a threshold concentration of Ca(2+)(o) for efficacy and sensitize the receptor to activation by Ca(2+)(o). Here, we review the evidence that the CaR, like other closely related members of the class 3 GPCR (G-protein-coupled receptor) family including GPRC6A, is a broad-spectrum amino acid-sensing receptor, consider the nature of the signalling response to amino acids and discuss its physiological significance.

The extracellular Ca2+-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 Ca2+-sensing receptor (CaR) Venus Flytrap domain, T145A and S170T, that specifically impair amino acid sensing, leaving Ca2+ sensing intact, as determined by receptor-dependent activation of intracellular Ca2+ 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 Ca2+ 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 Ca2+ and L-amino acid-sensing functions of the CaR can be dissociated.

We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by L-amino 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.

Parathyroid hormone (PTH) secretion is acutely regulated by the extracellular Ca2+-sensing receptor (CaR). Thus, Ca2+ ions, and to a lesser extent Mg2+ ions, have been viewed as the principal physiological regulators of PTH secretion. Herein we show that in physiological concentrations, L-amino acids acutely and reversibly activated the extracellular Ca2+-sensing receptor in normal human parathyroid cells and inhibited parathyroid hormone secretion. Individual L-amino acids, especially of the aromatic and aliphatic classes, as well as plasma-like amino acid mixtures, stereoselectively mobilized Ca2+ ions in normal human parathyroid cells in the presence but not the absence of the CaR agonists, extracellular Ca2+ (Ca-o(2+)), or spermine. The order of potency was L-Trp = L-Phe > L-His > L-Ala > L-Glu > L-Arg = L-Leu. CaR-active amino acids also acutely and reversibly suppressed PTH secretion at physiological ionized Ca2+ concentrations. At a Ca-o(2+) of 1.1 mM and an amino acid concentration of 1 mM, CaR-active amino acids (L-Phe = L-Trp > L-His = L-Ala), but not CaR-inactive amino acids (L-Leu and L-Arg), stereoselectively suppressed PTH secretion by up to 40%, similar to the effect of raising Ca-o(2+) to 1.2 mM. A physiologically relevant increase in the -fold concentration of the plasma-like amino acid mixture ( from 1 x to 2 x) also reversibly suppressed PTH secretion in the Ca-o(2+) concentration range 1.05 - 1.25 mM. In conclusion, L-amino acids acutely and reversibly activate endogenous CaRs and suppress PTH secretion at physiological concentrations. The results indicate that L-amino acids are physiological regulators of PTH secretion and thus whole body calcium metabolism.

We report a novel activating mutation (E604K) of the calcium-sensing receptor in a family with autosomal dominant hypocalcemia. Whereas all affected individuals exhibited marked hypocalcemia, some cases with untreated hypocalcemia exhibited seizures in infancy, whereas others were largely asymptomatic from birth into adulthood. The missense mutation E604K (G2182A; GenBank accession no. U20759), which affects an amino acid residue in the C terminus of the cysteine-rich domain of the extracellular head, cosegregated with hypocalcemia in all seven individuals for whom DNA was available. Two unaffected, normocalcemic members of the family did not exhibit the mutation. The molecular impact of the mutation on two key components of the signaling response was assessed in HEK-293 cells transiently transfected with cDNA corresponding to either the wild-type calcium-sensing receptor or the E604K mutation derived by site-directed mutagenesis. There was a significant leftward shift in the concentration response curves for the effects of extracellular Ca(2+) on both intracellular Ca(2+) mobilization (determined by aequorin luminescence) and MAPK activity (determined by luciferase expression). The C terminus of the cysteine-rich domain of the extracellular head may normally act to suppress receptor activity in the presence of low extracellular Ca(2+) concentrations.