Evidence for Distinct Cation and Calcimimetic Compound (NPS 568) Recognition Domains in the Transmembrane Regions of the Human Ca2+ Receptor

Ray, Kausik K.; Northup, John K.

doi:10.1074/jbc.m202113200

Cited by 84 publications

(70 citation statements)

References 30 publications

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“…The finding that R-467 retains activity in the headless receptor is consistent with the idea that the transmembrane domain region of CaR contains binding sites for type-II calcimimetics (10) as well as Ca 2ϩ o (12). However, R-467 exhibited differential potencies on the control and headless constructs.…”

Section: Effects Of L-amino Acids and Nps R-467 On A Headlesssupporting

confidence: 84%

The Venus Fly Trap Domain of the Extracellular Ca²⁺-sensing Receptor Is Required for l-Amino Acid Sensing

et al. 2004

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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...

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Section: Effects Of L-amino Acids and Nps R-467 On A Headlesssupporting

confidence: 84%

The Venus Fly Trap Domain of the Extracellular Ca²⁺-sensing Receptor Is Required for l-Amino Acid Sensing

et al. 2004

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“…Different functional studies have shown that the hCaR is activated by Ca 2ϩ ions with a Hill coefficient greater than 3 and multiple Ca 2ϩ ions are assumed to interact at several sites in both the ECD and 7TMD (14,16). Whereas the specific residues of contact for the Ca 2ϩ -binding sites within the ECD and 7TMD are unknown, data obtained by mutagenesis and chimeric receptor constructions indicate that the binding sites for the calcimimetic and calcilytic allosteric compounds identified thus far * This work was supported in part by the Intramural Research Program of the National Institutes of Health, NIDCD.…”

Section: A Member Of the Family-3 G-protein-coupled Receptors (Gpcr) mentioning

confidence: 99%

Calindol, a Positive Allosteric Modulator of the Human Ca2+ Receptor, Activates an Extracellular Ligand-binding Domain-deleted Rhodopsin-like Seven-transmembrane Structure in the Absence of Ca2+

Ray

Tisdale

Dodd

et al. 2005

Journal of Biological Chemistry

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“…Ray and Northup (27) recently reported that HEK-293 cells transiently transfected with the identical mutant, Rho-ChCaR/5A, show poor activation by metal ions and organic polycations in the presence of NPS-R568 but retain responsiveness to the combination of [Ca 2ϩ ] o and poly-L-arginine. They interpreted these results as evidence for a role of the five acidic residues in exo-loop 2 in CaR response to [Ca 2ϩ ] o and to NPS R-568, but because they failed to provide evidence for cell surface expression of the Rho-C-hCaR/5A mutant, this interpretation may be incorrect.…”

Section: Immunocytochemistry Of Cells Expressing Rho-c-hcar/5amentioning

confidence: 99%

Identification of Acidic Residues in the Extracellular Loops of the Seven-transmembrane Domain of the Human Ca2+ Receptor Critical for Response to Ca2+ and a Positive Allosteric Modulator

Reyes‐Cruz

Chen

et al. 2002

Journal of Biological Chemistry

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We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca 2؉ receptor (hCaR) in receptor activation by Ca 2؉ and in response to a positive allosteric modulator, NPS R-568. Both in the context of the full-length receptor and of a truncated receptor lacking the extracellular domain (Rho-C-hCaR), we mutated each acidic residue to alanine, singly and in combination, and tested the effect on expression of the receptor, on activation by Ca 2؉ , and on NPS R-568 augmentation of sensitivity to Ca 2؉ . Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp 758 , Glu 759 , and Glu 767 , increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca 2؉ . Mutation of all five acidic residues in exo-loop 2, whether in the full-length receptor or in Rho-C-hCaR, impaired cell surface expression of the mutant receptor and thereby largely abolished response to Ca 2؉ . Mutation of Glu 837 in exo-loop 3 to alanine did not alter Ca 2؉ sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu 837 drastically reduced sensitivity to NPS R-568. Our data point to a key role of three specific acidic residues in exo-loop 2 in hCaR activation and to Glu 837 at the junction between exo-loop 3 and transmembrane helix seven in response to NPS R-568. We speculate on the basis of these results that the three acidic residues we identified in exo-loop 2 help maintain an inactive conformation of the seven-transmembrane domain of the hCaR.The G protein-coupled [Ca 2ϩ ] o receptor (CaR) 1 plays a central role in the regulation of [Ca 2ϩ ] o homeostasis (1, 2). [Ca 2ϩ ] o activates the CaR in the parathyroid, thereby inhibiting parathyroid hormone secretion, and in the kidney, causing increased urinary calcium excretion. The physiological importance of the CaR in determining the level at which [Ca 2ϩ ] o is set in vivo has been documented by the identification of inactivating mutations in the CaR gene as the cause of familial hypocalciuric hypercalcemia and activating mutations as the cause of autosomal dominant hypocalcemia (3, 4). Naturally occurring CaR mutations identified in subjects with autosomal dominant hypocalcemia generally cause increased CaR sensitivity to [Ca 2ϩ ] o rather than causing constitutive activation (5).The CaR belongs to a unique subfamily, family 3, of G protein-coupled receptors (GPCR) with an unusually large N-terminal, extracellular domain (ECD) comprised of Venus's-flytrap (VFT) and cysteine-rich domains, in addition to the seventransmembrane domain (7TM) characteristic of all GPCR (6). Studies with chimeric family 3 GPCR (7-10) and the threedimensional structure of the metabotropic glutamate type 1 receptor (mGluR1) determined by x-ray crystallography (11) show that the VFT is the site of agonist binding in family 3 GPCR. The precise site(s) for binding to the CaR, however, have not been identified.The activation of family ...

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Evidence for Distinct Cation and Calcimimetic Compound (NPS 568) Recognition Domains in the Transmembrane Regions of the Human Ca2+ Receptor

Cited by 84 publications

References 30 publications

The Venus Fly Trap Domain of the Extracellular Ca²⁺-sensing Receptor Is Required for l-Amino Acid Sensing

The Venus Fly Trap Domain of the Extracellular Ca²⁺-sensing Receptor Is Required for l-Amino Acid Sensing

Calindol, a Positive Allosteric Modulator of the Human Ca2+ Receptor, Activates an Extracellular Ligand-binding Domain-deleted Rhodopsin-like Seven-transmembrane Structure in the Absence of Ca2+

Identification of Acidic Residues in the Extracellular Loops of the Seven-transmembrane Domain of the Human Ca2+ Receptor Critical for Response to Ca2+ and a Positive Allosteric Modulator

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Evidence for Distinct Cation and Calcimimetic Compound (NPS 568) Recognition Domains in the Transmembrane Regions of the Human Ca2+ Receptor

Cited by 84 publications

References 30 publications

The Venus Fly Trap Domain of the Extracellular Ca2+-sensing Receptor Is Required for l-Amino Acid Sensing

The Venus Fly Trap Domain of the Extracellular Ca2+-sensing Receptor Is Required for l-Amino Acid Sensing

Calindol, a Positive Allosteric Modulator of the Human Ca2+ Receptor, Activates an Extracellular Ligand-binding Domain-deleted Rhodopsin-like Seven-transmembrane Structure in the Absence of Ca2+

Identification of Acidic Residues in the Extracellular Loops of the Seven-transmembrane Domain of the Human Ca2+ Receptor Critical for Response to Ca2+ and a Positive Allosteric Modulator

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The Venus Fly Trap Domain of the Extracellular Ca²⁺-sensing Receptor Is Required for l-Amino Acid Sensing

The Venus Fly Trap Domain of the Extracellular Ca²⁺-sensing Receptor Is Required for l-Amino Acid Sensing