A Double Mutation in the Extracellular Ca2+-sensing Receptor's Venus Flytrap Domain That Selectively Disables L-Amino Acid Sensing

Royal North Shore Hospital, Sydney, New South Wales, Australia
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2005; 280(32):29067-72. DOI: 10.1074/jbc.M500002200
Source: PubMed


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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    • ") and polyamines (spermine > spermidine > putrescine) (Quinn et al., 1997), and by Ca 2+ at a transmembrane domain site (Ray and Northup, 2002; Mun et al., 2004). Allosteric interactions with endogenous modulators (amino acids, protons, glutathione) or drugs (calcimimetics or calcilytics) can occur at sites within the venus flytrap or transmembrane domains (Hu et al., 2000; Miedlich et al., 2004; Mun et al., 2005). While the orthosteric ligand Ca 2+ is the dominant regulator of CaSR signalling in organs involved in systemic Ca 2+ homeostasis, including the parathyroid, kidney and bone, multiple studies suggest that allosteric activation may dominate in certain tissues and/or physiological states. "
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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.
    Full-text · Article · Apr 2011 · British Journal of Pharmacology
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    • "Analysis of CaR/mGlu-1 chimeric receptors and a headless CaR construct expressed in HEK293 cells localized the L-amino acid binding site to the VFT domain (Mun et al., 2004) indicating that the L-glutamate binding site in mGlu-1 is conserved in the CaR as a broad-spectrum L-amino acid binding site (Conigrave and Hampson, 2006). Consistent with this idea, the double mutant T145A/S170T markedly impaired L-Phe sensitivity but had little or no effect on Ca 2+ osensing in CaR-expressing HEK293 cells (Mun et al., 2005). As noted above, this site may be closely associated with a moderately high affinity Ca 2+ binding site located in the hinge region (Huang et al., 2009) providing a potential explanation for the positive interactions between Ca 2+ and L-amino acids (Conigrave et al., 2007). "
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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).
    Preview · Article · Feb 2010 · British Journal of Pharmacology
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    • "Mutation studies have predicted several Ca 2 binding sites in the cleft of the VFT (Brä uner-Osborne et al., 1999; Huang et al., 2007), but Ca 2 could also potentially bind between the two bilobed VFT domains, stabilizing the closed conformation, as has been shown for Gd 3 in mGlu 1 (Fig. 1) (Tsuchiya et al., 2002). Mun et al. (2005) identified two mutations , T145A and S170T, that specifically impair amino acid sensing but leave Ca 2 sensing intact. Others have also identified the three serines Ser169 to Ser171 as important for amino acid binding (Zhang et al., 2002b; Lee et al., 2007). "
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    ABSTRACT: A number of highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized within the last few years. It is noteworthy that many of these receptors are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids and are expressed in taste tissue, the gastrointestinal tract, endocrine glands, adipose tissue, and/or kidney. These receptors thus hold the potential to act as sensors of food intake, regulating, for example, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue. The promiscuous tendency in ligand recognition of these receptors is in contrast to the typical specific interaction with one physiological agonist seen for most receptors, which challenges the classic "lock-and-key" concept. We here review the molecular mechanisms of nutrient sensing of the calcium-sensing receptor, the G protein-coupled receptor family C, group 6, subtype A (GPRC6A), and the taste1 receptor T1R1/T1R3, which are sensing L-alpha-amino acids, the carbohydrate-sensing T1R2/T1R3 receptor, the proteolytic degradation product sensor GPR93 (also termed GPR92), and the free fatty acid (FFA) sensing receptors FFA1, FFA2, FFA3, GPR84, and GPR120. The involvement of the individual receptors in sensing of food intake has been validated to different degrees because of limited availability of specific pharmacological tools and/or receptor knockout mice. However, as a group, the receptors represent potential drug targets, to treat, for example, type II diabetes by mimicking food intake by potent agonists or positive allosteric modulators. The ligand-receptor interactions of the promiscuous receptors of organic nutrients thus remain an interesting subject of emerging functional importance.
    Preview · Article · Jul 2009 · Molecular pharmacology
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