Hee-Chang Mun

A double mutation in the extracellular Ca2+-sensing receptor's venus flytrap domain that selectively disables L-amino acid sensing.

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 receptors 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.

Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation

The calcium-sensing receptor (CaR) mediates feedback control of Ca2+o (extracellular Ca2+) concentration. Although the mechanisms are not fully understood, the CaR couples to several important intracellular signalling enzymes, including PI-PLC (phosphoinositide-specific phospholipase C), leading to Ca2+i (intracellular Ca2+) mobilization, and ERK1/2 (extracellular-signal-regulated kinase 1/2). In addition to Ca2+o, the CaR is activated allosterically by several subclasses of L-amino acids, including the aromatics L-phenylalanine and L-tryptophan. These amino acids enhance the Ca2+o-sensitivity of Ca2+i mobilization in CaR-expressing HEK-293 (human embryonic kidney) cells and normal human parathyroid cells. Furthermore, on a background of a physiological fasting serum L-amino acid mixture, they induce a small, but physiologically significant, enhancement of Ca2+o-dependent suppression of PTH (parathyroid hormone) secretion. The impact of amino acids on CaR-stimulated ERK1/2, however, has not been determined. In the present study, we examined the effects of L-amino acids on Ca2+o-stimulated ERK1/2 phosphorylation as determined by Western blotting and a newly developed quantitative assay (SureFire). L-Amino acids induced a small, but significant, enhancement of Ca2+o-stimulated ERK1/2. In CaR-expressing HEK-293 cells, 10 mM L-phenylalanine lowered the EC50 for Ca2+o from approx. 2.3 to 2.0 mM in the Western blot assay and from 3.4 to 2.9 mM in the SureFire assay. The effect was stereoselective (L>D), and another aromatic amino acid, L-tryptophan, was also effective. The effects of amino acids were investigated further in HEK-293 cells that expressed the CaR mutant S169T. L-Phenylalanine normalized the EC50 for Ca2+o-stimulated Ca2+i mobilization from approx. 12 mM to 5.0 mM and ERK1/2 phosphorylation from approx. 4.6 mM to 2.6 mM. Taken together, the data indicate that L-phenylalanine and other amino acids enhance the Ca2+o-sensitivity of CaR-stimulated ERK1/2 phosphorylation; however, the effect is comparatively small and operates in the form of a fine-tuning mechanism.

Physiological significance of L-amino acid sensing by extracellular Ca2+-sensing receptors

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.

Allosteric Modulation of the Calcium-sensing Receptor by γ-Glutamyl Peptides INHIBITION OF PTH SECRETION, SUPPRESSION OF INTRACELLULAR cAMP LEVELS, AND A COMMON MECHANISM OF ACTION WITH l-AMINO ACIDS

γ-Glutamyl peptides were identified previously as novel positive allosteric modulators of Ca2+o-dependent intracellular Ca2+ mobilization in HEK-293 cells that bind in the calcium-sensing receptor VFT domain. In the current study, we investigated whether γ-glutamyl-tripeptides including γ-Glu-Cys-Gly (glutathione) and its analogs S-methylglutathione and S-propylglutathione, or dipeptides including γ-Glu-Ala and γ-Glu-Cys are positive allosteric modulators of Ca2+o-dependent Ca2+i mobilization and PTH secretion from normal human parathyroid cells as well as Ca2+o-dependent suppression of intracellular cAMP levels in calcium-sensing receptor (CaR)-expressing HEK-293 cells. In addition, we compared the effects of the potent γ-glutamyl peptide S-methylglutathione, and the amino acid l-Phe on HEK-293 cells that stably expressed either the wild-type CaR or the double mutant T145A/S170T, which exhibits selectively impaired responses to l-amino acids. We find that γ-glutamyl peptides are potent positive allosteric modulators of the CaR that promote Ca2+o-dependent Ca2+i mobilization, suppress intracellular cAMP levels and inhibit PTH secretion from normal human parathyroid cells. Furthermore, we find that the double mutant T145A/S170T exhibits markedly impaired Ca2+i mobilization and cAMP suppression responses to S-methylglutathione as well as l-Phe indicating that γ-glutamyl peptides and l-amino acids activate the CaR via a common mechanism.γ-Glutamyl peptides were identified previously as novel positive allosteric modulators of Ca(2+)(o)-dependent intracellular Ca(2+) mobilization in HEK-293 cells that bind in the calcium-sensing receptor VFT domain. In the current study, we investigated whether γ-glutamyl-tripeptides including γ-Glu-Cys-Gly (glutathione) and its analogs S-methylglutathione and S-propylglutathione, or dipeptides including γ-Glu-Ala and γ-Glu-Cys are positive allosteric modulators of Ca(2+)(o)-dependent Ca(2+)(i) mobilization and PTH secretion from normal human parathyroid cells as well as Ca(2+)(o)-dependent suppression of intracellular cAMP levels in calcium-sensing receptor (CaR)-expressing HEK-293 cells. In addition, we compared the effects of the potent γ-glutamyl peptide S-methylglutathione, and the amino acid L-Phe on HEK-293 cells that stably expressed either the wild-type CaR or the double mutant T145A/S170T, which exhibits selectively impaired responses to L-amino acids. We find that γ-glutamyl peptides are potent positive allosteric modulators of the CaR that promote Ca(2+)(o)-dependent Ca(2+)(i) mobilization, suppress intracellular cAMP levels and inhibit PTH secretion from normal human parathyroid cells. Furthermore, we find that the double mutant T145A/S170T exhibits markedly impaired Ca(2+)(i) mobilization and cAMP suppression responses to S-methylglutathione as well as L-Phe indicating that γ-glutamyl peptides and L-amino acids activate the CaR via a common mechanism.

Structural mechanism of ligand activation in human calcium-sensing receptor

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

Aromatic l-Amino Acids Activate the Calcium-Sensing Receptor

The calcium-sensing receptor (CaR) is recognized as a member of class 3 of the G-protein coupled receptor superfamily. Members of this subgroup, which have large N-terminal extracellular domains, include receptors that respond specifically to the amino acid glutamate; receptors that respond to the glutamate analogue, gamma-amino butyric acid; and several receptors that act as broad-spectrum amino acid sensors. The CaR is one of these broad-spectrum amino acid sensors that, along with several other members of the subgroup, also responds to extracellular Ca2+. In this mini-review, we consider evidence that the CaR is a sensor of aromatic amino acids, that it has broad-spectrum amino acid sensing properties, that it provides an amino acid binding site in its extracellular N-terminal Venus Fly Trap domain, and that amino acids have a physiological impact on systems in which the CaR is expressed.

ISOLATION BREEDS NAIVETY: ISLAND LIVING ROBS AUSTRALIAN VARANID LIZARDS OF TOAD-TOXIN IMMUNITY VIA FOUR-BASE-PAIR MUTATION

Since their introduction to the toad‐free Australian continent cane toads (Bufo marinus) have caused a dramatic increase in naïve varanid mortality when these large lizards attempt to feed on this toxic amphibian. In contrast Asian–African varanids, which have coevolved with toads, are resistant to toad toxin. Toad toxins, such as Bufalin target the H1‐H2 domain of the α1 subunit of the sodium‐potassium‐ATPase enzyme. Sequencing of this domain revealed identical nucleotide sequences in four Asian as well as in three African varanids, and identical sequences in all 11 Australian varanids. However, compared to the Asian–African varanids, the Australian varanids showed four‐base‐pair substitutions, resulting in the alteration in three of the 12 amino acids representing the H1‐H2 domain. The phenotypic effect of the substitutions was investigated in human embryonic kidney (HEK) 293 cells stably transfected with the Australian and the Asian–African H1‐H2 domains. The transfections resulted in an approximate 3000‐fold reduction in resistance to Bufalin in the Australian HEK293 cells compared to the Asian–African HEK293 cells, demonstrating the critical role of this minor mutation in providing Bufalin resistance. Our study hence presents a clear link between genotype and phenotype, a critical step in understanding the evolution of phenotypic diversity.

Calcium-sensing receptor-dependent activation of CREB phosphorylation in HEK293 cells and human parathyroid cells

In addition to its acute effects on hormone secretion, epithelial transport, and shape change, the calcium-sensing receptor (CaSR) modulates the expression of genes that control cell survival, proliferation, and differentiation as well as the synthesis of peptide hormones and enzymes. In the present study, we investigated the impacts of a CaSR agonist and several CaSR modulators on phosphorylation of transcription factor CREB residue Ser(133) in CaSR-expressing HEK293 (HEK-CaSR) cells and human adenomatous parathyroid cells. Elevated Ca(2+)o concentration had no effect on CREB phosphorylation (p-CREB) in control HEK293 cells but stimulated p-CREB in both HEK-CaSR cells and human parathyroid cells. In addition, p-CREB was stimulated by the positive modulator cinacalcet and inhibited by the negative modulator NPS 2143 in both CaSR-expressing cell types. Two positive modulators that bind in the receptors Venus Fly Trap domain, l-phenylalanine and S-methylglutathione, had no effect on p-CREB in HEK-CaSR cells, demonstrating the existence of pronounced signaling bias. Analysis of the signaling pathways using specific inhibitors demonstrated that phosphoinositide-specific phospholipase C and conventional protein kinase C isoforms make major contributions to Ca(2+)o-induced p-CREB in both cell-types, suggesting key roles for Gq/11. In addition, in parathyroid cells but not HEK-CaSR cells, activation of p-CREB was dependent on Gi/o, demonstrating the existence of cell type-specific signaling.

Receptor Expression Modulates Calcium-Sensing Receptor Mediated Intracellular Ca2+ Mobilization

Calcium-sensing receptors (CaSRs) are class C G protein-coupled receptors that respond to physiological activators, including extracellular Ca2+ (Cao2+) and L-amino acids as well as the pharmaceutical calcimimetic, cinacalcet. Unlike Cao2+, which is an orthosteric agonist, L-amino acids and cinacalcet are positive allosteric modulators. CaSR expression levels vary considerably between tissues, but the physiological significance of these differences in expression for the effects of its activators is unknown. To investigate the impact of receptor expression on CaSR-mediated signaling we used a tetracycline-inducible expression system and focused on intracellular Ca2+ (Cai2+) responses in single cells and considered both population and single-cell behavior. Increased receptor expression positively modulated CaSR-mediated Cai2+ mobilization in response to elevated Cao2+, the amino acid L-phenylalanine, or the calcimimetic cinacalcet. It lowered threshold concentrations for the initiation of Cai2+ oscillations and for their transformation to sustained Cai2+ elevations, and it increased the proportions of responding cells. It also positively modulated the frequency of Cai2+ oscillations with the order of effectiveness: cinacalcet equal to or greater than Cao2+ greater than L-phenylalanine. The results indicate that receptor expression modulates key characteristics of the Cai2+ response at the single-cell level as well as the amplitude of whole-tissue CaSR-mediated responses by recruiting quiescent cells into the active pool of responding cells. By lowering the threshold concentrations for Cao2+- and L-amino acid-induced responses below the physiological levels of these nutrients in plasma, mechanisms that up-regulate receptor expression can control tissue function in the absence of dynamic changes in ligand concentration.

Adenomatous Human Parathyroid Cells Exhibit Impaired Sensitivity to l-Amino Acids

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 CaRs amino acid binding site may be exploited as a target in the medical treatment of primary and perhaps other forms of hyperparathyroidism.