Increased Receptor Stimulation Elicits Differential Calcium-sensing ReceptorT888 Dephosphorylation

McCormick, Wanda D; Atkinson-Dell, Rebecca; Campion, Karis; Mun, Hee-Chang; Conigrave, Arthur D.; Ward, Donald T.

doi:10.1074/jbc.m109.071084

Cited by 33 publications

(37 citation statements)

References 37 publications

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“…9,10 Indeed, mild acidosis (pH o 7.2) significantly inhibited Ca renders the CaR inactive), variations in pH o between 7.2 and 7.6 had no effect on actin polymerization. This finding indicates that changing pH o does not otherwise affect baseline signaling.…”

Section: Resultsmentioning

confidence: 99%

“…Normal human parathyroid cells were obtained by collagenase-digestion after neck surgery 9 (procedures performed under institutional ethical guidelines and with patients' written informed consent). PTH levels 9 ), and 1 mg/ml BSA.…”

Section: Parathyroid Gland Preparation and Pth Secretion Assaymentioning

confidence: 99%

“…Bovine parathyroid cells (abattoir-sourced) were obtained by collagenase-digestion 9 and cultured on collagen-coated coverslips. Normal human parathyroid cells were obtained by collagenase-digestion after neck surgery 9 (procedures performed under institutional ethical guidelines and with patients' written informed consent).…”

Section: Parathyroid Gland Preparation and Pth Secretion Assaymentioning

confidence: 99%

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Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism

Campion

McCormick

Warwicker

et al. 2015

Journal of the American Society of Nephrology

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The calcium-sensing receptor (CaR) modulates renal calcium reabsorption and parathyroid hormone (PTH) secretion and is involved in the etiology of secondary hyperparathyroidism in CKD. Supraphysiologic changes in extracellular pH (pH o ) modulate CaR responsiveness in HEK-293 (CaR-HEK) cells. Therefore, because acidosis and alkalosis are associated with altered PTH secretion in vivo, we examined whether pathophysiologic changes in pH o can significantly alter CaR responsiveness in both heterologous and endogenous expression systems and whether this affects PTH secretion. In both CaR-HEK and isolated bovine parathyroid cells, decreasing pH o from 7.4 to 7.2 rapidly inhibited CaR-induced intracellular calcium (Ca 2+ i ) mobilization, whereas raising pH o to 7.6 potentiated responsiveness to extracellular calcium (Ca i mobilization. Intracellular pH was unaffected by acute 0.4-unit pH o changes, and the presence of physiologic albumin concentrations failed to attenuate the pH o -mediated effects. None of the individual point mutations created at histidine or cysteine residues in the extracellular domain of CaR attenuated pH o sensitivity. Finally, pathophysiologic pH o elevation reversibly suppressed PTH secretion from perifused human parathyroid cells, and acidosis transiently increased PTH secretion. Therefore, pathophysiologic pH o changes can modulate CaR responsiveness in HEK-293 and parathyroid cells independently of extracellular histidine residues. Specifically, pathophysiologic acidification inhibits CaR activity, thus permitting PTH secretion, whereas alkalinization potentiates CaR activity to suppress PTH secretion. These findings suggest that acid-base disturbances may affect the CaR-mediated control of parathyroid function and calcium metabolism in vivo.

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Section: Resultsmentioning

confidence: 99%

Section: Parathyroid Gland Preparation and Pth Secretion Assaymentioning

confidence: 99%

Section: Parathyroid Gland Preparation and Pth Secretion Assaymentioning

confidence: 99%

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Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism

Campion

McCormick

Warwicker

et al. 2015

Journal of the American Society of Nephrology

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“…Such a mechanism could explain how the C-terminal truncation mutant R866X abolished Ca 2+ i mobilization. G 12 has also been reported to activate protein phosphatase 2A [29], an enzyme that supports Ca 2+ o -stimulated dephosphorylation of CaSR residue T888 and, in turn, promotes stable elevations in Ca 2+ i [30]. Preliminary experiments investigating the effects of pretreatment with pertussis toxin and of acute exposure to the broad-spectrum phosphodiesterase (PDE) inhibitor IBMX demonstrated that the suppressive effect of Ca 2+ o on forskolin-induced elevations in cAMP levels was dependent on G i/o -mediated inhibition of adenylyl cyclase and not PDE.…”

Section: Discussionmentioning

confidence: 99%

Roles of intraloops‐2 and ‐3 and the proximal C‐terminus in signalling pathway selection from the human calcium‐sensing receptor

et al. 2014

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Edited by Ned ManteiKeywords: Calcium-sensing receptor G-protein coupled receptor Biased signalling Phosphatidylinositol-specific phospholipase-C Adenosine 3 0 ,5 0 -cyclic monophosphate ERK 1/2 a b s t r a c tThe calcium-sensing receptor (CaSR) couples to signalling pathways via intracellular loops 2 and 3, and the C-terminus. However, the requirements for signalling are largely undefined. We investigated the impacts of selected point mutations in iL-2 (F706A) and iL-3 (L797A and E803A), and a truncation of the C-terminus (R866X) on extracellular Ca 2+ (Ca 2+ o )-stimulated phosphatidylinositolspecific phospholipase-C (PI-PLC) and various other signalling responses. CaSR-mediated activation of PI-PLC was markedly attenuated in all four mutants and similar suppressions were observed for Ca 2+ o -stimulated ERK 1/2 phosphorylation. Ca 2+ o -stimulated intracellular Ca 2+ (Ca 2+ i ) mobilization, however, was relatively preserved for the iL-2 and iL-3 mutants and suppression of adenylyl cyclase was unaffected by either E803A or R866X. The CaSR selects for specific signalling pathways via the proximal C-terminus and key residues in iL-2, iL-3.

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“…2; wt, 3.0G0.1 mM; T888M, 2.5G0.2 mM; P!0.05 by unpaired t-test of log EC 50 values). Western blotting revealed similar expression of protein bands consistent with the wellrecognized 140 kDa core-glycosylated and 160 kDa mature receptors (7,(12)(13)(14)(15)20) in both sets of transfected cells (Fig. 2C).…”

Section: T888m Mutation In Hek-293 Cellsmentioning

confidence: 71%

A novel mutation of the primary protein kinase C phosphorylation site in the calcium-sensing receptor causes autosomal dominant hypocalcemia

Lazarus

Pretorius²,

Khafagi³

et al. 2011

European Journal of Endocrinology

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Objective: The calcium-sensing receptor (CASR) is a key controller of calcium homeostasis by regulating parathyroid hormone (PTH) secretion and renal calcium reabsorption. CASR T888 is a protein kinase C (PKC) phosphorylation site in the receptor's intracellular domain that has previously been identified as a critical negative regulator of CASR downstream signaling in vitro, but whose importance in vivo is unknown. Case report: The proband presented with mild symptomatic hypocalcemia following treatment for nephrotic syndrome due to minimal change glomerulonephropathy. Laboratory tests revealed inappropriately normal PTH concentrations and relative hypercalciuria typical of autosomal dominant hypocalcemia. His asymptomatic father had similar laboratory test results. Design and methods: The CASR gene was sequenced. To investigate the molecular consequences of CASR T888M mutation, site-directed mutagenesis was used to modify the wild-type (wt)-CASR gene, with the resulting mutant being transfected transiently into HEK-293 cells. Results: A novel CASR missense mutation, T888M, was identified in both cases. The CASR T888M mutant exhibited enhanced sensitivity to extracellular calcium concentration, both for intracellular calcium ðCa 2C i Þ mobilization and for ERK phosphorylation, despite having unaltered levels of cell surface expression. Furthermore, CASR T888M elicited sustained Ca 2C i mobilization rather than high frequency Ca 2C i oscillations, and, unlike the wt-CASR, the response was resistant to acute inhibition by the PKC activator, phorbol 12-myristate 13-acetate. Conclusions: The clinical and functional data provide the first genotype-phenotype correlation for a mutation at T888, indicating its critical physiological importance in CASR signaling. Thus, CASR T888 represents a functionally important, inhibitory phosphorylation site that contributes to the control of PTH secretion.

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Increased Receptor Stimulation Elicits Differential Calcium-sensing ReceptorT888 Dephosphorylation

Cited by 33 publications

References 37 publications

Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism

Pathophysiologic Changes in Extracellular pH Modulate Parathyroid Calcium-Sensing Receptor Activity and Secretion via a Histidine-Independent Mechanism

Roles of intraloops‐2 and ‐3 and the proximal C‐terminus in signalling pathway selection from the human calcium‐sensing receptor

A novel mutation of the primary protein kinase C phosphorylation site in the calcium-sensing receptor causes autosomal dominant hypocalcemia

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