A double mutation in the extracellular Ca2+-sensing receptor's Venus flytrap domain that selectively disables L-amino acid sensing
ABSTRACT 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 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.
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ABSTRACT: Calcium-sensing receptors (CaSR) are integral to regulation of systemic Ca(2+) homeostasis. Altered expression levels or mutations in CaSR cause Ca(2+) handling diseases. CaSR is regulated by both endogenous allosteric modulators and allosteric drugs, including the first Food and Drug Administration-approved allosteric agonist, Cinacalcet HCl (Sensipar®). Recent studies suggest that allosteric modulators not only alter function of plasma membrane-localized CaSR, but regulate CaSR stability at the endoplasmic reticulum. This brief review summarizes our current understanding of the role of membrane-permeant allosteric agonists in cotranslational stabilization of CaSR, and highlights additional, indirect, signalling-dependent role(s) for membrane-impermeant allosteric drugs. Overall, these studies suggest that allosteric drugs act at multiple cellular organelles to control receptor abundance and hence function, and that drug hydrophobicity can bias the relative contributions of plasma membrane and intracellular organelles to CaSR abundance and signalling.British Journal of Pharmacology 04/2011; 165(6):1670-7. DOI:10.1111/j.1476-5381.2011.01403.x · 4.99 Impact Factor
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ABSTRACT: In addition to its role in PI-PLC signalling, the C-terminal domain interacts either directly or indirectly with intracellular proteins that modulate or mediate receptor trafficking, subcellular localization and downstream signalling pathways (Huang and Miller, 2007). Interactions with inwardly rectifying K+ channels, Kir4.1 and Kir4.2, for example, provide a mechanism by which alterations in Ca2+o modulate renal salt and water transport (Huang et al., 2007a). Binding to filamin-A, on the other hand, establishes a link to the actin cytoskeleton to direct receptors to specific subcellular compartments for the creation of signalling scaffolds (Awata et al., 2001; Hjalm et al., 2001; Zhang and Breitwieser, 2005). Filamin may mediate, for example, the CaR's interactions with caveolin, thereby targeting the receptor to plasma membrane caveolae (Kifor et al., 1998). In addition, the association between the CaR and filamin is required for coupling between the receptor and ERK 1/2 (Awata et al., 2001; Hjalm et al., 2001) and may permit G12/13 control of small G-proteins including Rho, upstream of PI-4 kinase and phospholipase D (Pi et al., 2002; Rey et al., 2005). Two filamin-A binding sites have been reported: a high affinity site located in the approximate region 960–990 (Awata et al., 2001; Hjalm et al., 2001; Zhang and Breitwieser, 2005) and a lower affinity, membrane proximal binding site that appears to contribute to Ca2+o-induced ERK1/2 activation (Zhang and Breitwieser, 2005).British Journal of Pharmacology 02/2010; 159(5):1039-50. DOI:10.1111/j.1476-5381.2009.00603.x · 4.99 Impact Factor
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ABSTRACT: Family C of human G-protein-coupled receptors (GPCRs) is constituted by eight metabotropic glutamate receptors, two gamma-aminobutyric acid type B (GABA(B1-2)) subunits forming the heterodimeric GABA(B) receptor, the calcium-sensing receptor, three taste1 receptors (T1R1-3), a promiscuous L-alpha;-amino acid receptor G-protein-coupled receptor family C, group 6, subtype A (GPRC6A) and seven orphan receptors. Aside from the orphan receptors, the family C GPCRs are dimeric receptors characterized by a large extracellular Venus flytrap domain which bind the endogenous agonists. Except from the GABA(B1-2) and T1R2-3 receptor, all receptors are either activated or positively modulated by amino acids. In this review, we outline mutational, biophysical and structural studies which have elucidated the interaction of the amino acids with the Venus flytrap domains, molecular mechanisms of receptor selectivity and the initial steps in receptor activation.British Journal of Pharmacology 04/2009; 156(6):869-84. DOI:10.1111/j.1476-5381.2008.00078.x · 4.99 Impact Factor