Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C25
Identification of Calcium-Sensing ReceptorS875 as a novel phosphorylation site contributing to the feedback regulation of receptor activity.
K. L. Campion1, K. L. Bailey1, S. L. Davies1, A. D. Conigrave2, D. T. Ward1
1. Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom. 2. School of Molecular Bioscience, University of Sydney, Sydney, New South Wales, Australia.
Extracellular calcium (Ca2+o) homeostasis is controlled by the action of the Ca2+-sensing receptor (CaR) on parathyroid hormone secretion and renal Ca2+ reabsorption. CaR contains 5 intracellular serine/threonine residues predicted to be protein kinase C (PKC) phosphorylation sequences. Previously we have shown that one of these, CaRT888, represents the key phosphorylation site responsible for protein kinase C-mediated inhibition of CaR-elicited Ca2+i mobilisation in vitro [1,2] and PTH secretion in vivo . Importantly though, we found that CaRT888 cannot be the sole determinant of the PKC response and previous work suggests that the other 4 known PKC sites play little or no role . For the structurally homologous metabotropic glutamate receptor (mGluR) 5, the equivalent PKC site is Ser-839 which aligns in CaR not with CaRT888 but with CaRS875, which has not been considered a putative PKC site before. Thus, here we examined the effect on CaR responsiveness of mutating CaRS875 to alanine (non-phosphorylatable) or aspartate (phosphomimetic) in either wild-type (WT) CaR or a CaRT888A mutant (QuikChange) expressed in Fura2-loaded HEK-293 cells. For CaRS875A, the Ca2+o concentration-dependency was significantly left-shifted (EC50, 2.7±0.1 vs. WT, 3.4±0.1 mM; P<0.05 by ANOVA, N=6) while for CaRS875D, the relationship was shifted to the right (4.2±0.3 vs. WT, 3.4±0.1 mM; P<0.05). Thus, CaRS875 most likely represents a phosphorylation site similar in action to CaRT888 (EC50 for CaRT888A also 2.7±0.1). Next, we showed that the Ca2+o responsiveness of the double mutant CaRS875A/T888A was left-shifted to an even greater extent than for CaRT888A alone (EC50, 2.2±0.1 vs. 2.7±0.1 respectively; P<0.01) demonstrating the additive effects of the two mutations. Next, in mGluR5 the neighbouring threonine-840 residue is permissive for mGluR5S839 phosphorylation and thus we also investigated the responsiveness of the mutants CaRT876A and CaRT876D. For CaRT876A, the Ca2+o concentration-dependency was no different from WT (EC50, 3.8±0.4 vs. WT, 3.4±0.1 mM) whereas for CaRS875D, the relationship was shifted markedly to the right (4.9±0.3; P<0.05 vs. WT). This suggests that CaRT876 is not normally a phosphorylation site but that its phosphomimetic mutation is also inhibitory for CaR-mediated Ca2+i mobilisation. Therefore, CaRS875 is most likely a previously unrecognised PKC phosphorylation site that, together with CaRT888, acts to shape CaR signalling and maintain the CaR-mediated control of Ca2+o homeostasis.
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