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Abstract: FR-PO485

Calcium-Sensing Receptor Signal Modulation: Identification of CaRS875 as a Novel Phosphorlyation Site and the Contribution of Both Intracellular Domains to Homodimer Signaling

Session Information

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Binmahfouz, Lenah Saeed, The University of Manchester, Manchester, United Kingdom
  • Conigrave, Arthur D., University of Sydney, Sydney, New South Wales, Australia
  • Ward, Donald T,, The University of Manchester, Manchester, United Kingdom

Group or Team Name

  • Ward
Background

The calcium-sensing receptor (CaR) controls parathyroid hormone secretion and renal Ca2+ reabsorption and its function is inhibited by protein kinase C (PKC) phosphorylation on CaRT888. However, the equivalent PKC site in metabotropic glutamate receptor-5 aligns instead to CaRS875, not previously considered a PKC site. Thus, we examined whether a) CaRS875 represents a novel PKC site, b) mutation of CaRT888 can rescue loss-of-function (LOF) CaR mutants and c) whether one or both intracellular domains (ICD) of the CaR homodimer contribute to signaling.

Methods

The non-phosphorylatable mutation CaRS875A was introduced into both wild-type human CaR and CaRT888A to test whether this heightens signaling. Then CaRT888A was introduced into two loss-of-function (LOF) mutants, CaRS170A (extracellular domain, ECD) and CaRF801A (intracellular domain, ICD) to determine a) whether it can rescue their lost function and b) whether its addition to both monomers enhances the benefit. If so then this would indicate that both ICDs of the dimer contribute to signaling.

Results

In HEK-293 cells, CaRS875A exhibited significantly enhanced signaling vs wild-type CaR (EC50, 3.4 ± 0.2 mM WT vs. 1.6±0.1 S875A; P<0.001) while the phosphomimetic CaRS875D mutant reduced Ca2+o potency (4.7±0.2 mM S875D vs 3.4±0.2 WT; P<0.01). When combined with T888A, the Ca2+o responsiveness of the double mutant CaRS875A/T888A was further left-shifted relative to CaRT888A alone (2.1±0.4 S875A/T888A vs. 2.6±0.1 T888A; P<0.01) demonstrating the sites' additive effects.

Next, the LOF homodimers CaRS170A (ECD) and CaRF801A (ICD) exhibited no Ca2+o responsiveness up to 40mM, whereas co-mutation with T888A significantly rescued their Ca2+o responsiveness. It is known that heterodimerisation of CaRS170A and CaRF801A permits partial rescue of their function. However, additional co-mutation with T888A not only enhanced the rescue but gave maximal rescue when present on both monomers.

Conclusion

Thus, CaRS875 is a novel PKC site that, together with CaRT888, shapes the CaR signalling that underpins Ca2+o homeostasis. Furthermore, removal of inhibitory signaling at CaRT888 can rescue both ECD and ICD loss-of-function mutants, and, both ICDs of the dimer contribute to CaR signalling.

Funding

  • Government Support - Non-U.S.