Kidney Week

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

• Bone and Mineral Metabolism: Basic
  October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Bone and Mineral Metabolism

• 401 Bone and Mineral Metabolism: Basic

Authors

• Binmahfouz, Lenah Saeed, The University of Manchester, Manchester, United Kingdom

Lenah Saeed Binmahfouz,

• Conigrave, Arthur D., University of Sydney, Sydney, New South Wales, Australia

Arthur D. Conigrave,

• Ward, Donald T,, The University of Manchester, Manchester, United Kingdom

Donald T, Ward,

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 CaR^T888. However, the equivalent PKC site in metabotropic glutamate receptor-5 aligns instead to CaR^S875, not previously considered a PKC site. Thus, we examined whether a) CaR^S875 represents a novel PKC site, b) mutation of CaR^T888 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 CaR^S875A was introduced into both wild-type human CaR and CaR^T888A to test whether this heightens signaling. Then CaR^T888A was introduced into two loss-of-function (LOF) mutants, CaR^S170A (extracellular domain, ECD) and CaR^F801A (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, CaR^S875A 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 CaR^S875D 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 CaR^S875A/T888A was further left-shifted relative to CaR^T888A 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 CaR^S170A (ECD) and CaR^F801A (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 CaR^S170A and CaR^F801A 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, CaR^S875 is a novel PKC site that, together with CaR^T888, shapes the CaR signalling that underpins Ca²⁺_o homeostasis. Furthermore, removal of inhibitory signaling at CaR^T888 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.