Phosphorylation of Calcium-Sensing Receptor Intracellular Domain in Signal Modulation

Abstract

The calcium-sensing receptor (CaR) is the key controller of mammalian extracellular calcium (Ca2+o) homeostasis and acts by suppressing parathyroid hormone (PTH) secretion and renal Ca2+ reabsorption. Physiological CaR signalling involves coupling to multiple heterotrimeric G proteins, including Gαq/11, Gαi/o and Gα12/13. This thesis confirms the central role of Gαq/11 in CaR signalling, not only for intracellular Ca2+ (Ca2+i) mobilisation but also as a mediator of ERK phosphorylation, CaRT888 phosphorylation, actin polymerisation and morphological changes in CaR-transfected HEK-293 (CaR-HEK) cells. By contrast, the Gα12 protein had no apparent role in CaR-induced Ca2+i mobilisation or morphological changes. It was also found that the inositol 1,4,5-trisphosphate receptor is the sole source of cytosolic Ca2+ for CaR-induced Ca2+i mobilisation in CaR-HEK cells. A major determinant of CaR responsiveness is the phosphorylation status of Thr-888, whereby increased CaRT888 phosphorylation suppresses receptor activity (thus permitting PTH secretion), while protein kinase C (PKC) inhibition enhances CaR activity (thus reducing PTH secretion). The clinical mutation CaRT888M is not susceptible to this inhibitory feedback and is, therefore, gain-of-function, resulting in tonic PTH suppression (autosomal dominant hypocalcaemia). Previous work from this laboratory found that phosphorylation of the mature 160-kDa CaRT888 is mediated primarily via PKCα and can be prevented by overnight pretreatment with the phorbol ester PMA to downregulate PKC. However, little is known about the effect of phosphorylation on the immature 140-kDa CaRT888 and whether this affects CaR maturation, forward trafficking or signalling. While chronic PMA pretreatment prevented 160-kDa CaRT888 phosphorylation as expected, there was an unexpected, concomitant increase in 140-kDa CaRT888 phosphorylation, which was still mediated by PKCα despite its substantial downregulation. Since PKC inhibition (which decreases phosphorylation of both 140 and 160-kDa proteins) enhances CaR-induced Ca2+i mobilisation more than for chronic PMA pretreatment (with its opposite effects on the two proteins), the PKCα-mediated phosphorylation of the 140-kDa CaRT888 appeared inhibitory to receptor responsiveness. In apparent opposition to these findings, CaR membrane localisation was enhanced by chronic PMA pretreatment but inhibited by PKC inhibition. Meanwhile, CaRT888 phosphorylation is not the sole inhibitory determinant of the PKC response since Ca2+i mobilisation was still enhanced by PKC inhibition in CaRT888A mutant cells. Mutational analysis revealed that CaRS875 represents the missing inhibitory phosphorylation site in the CaR intracellular domain (ICD). Indeed, the loss of both sites (CaRS875A/T888A) rendered the receptor insensitive to PKC inhibition, and when removed from a CaR which also lacked its extracellular domain (ECD), the result was a receptor maximally active even under control conditions. Finally, I investigated the contribution of both ICD subunits to CaR signalling, using a trans-activation heterodimeric model. Removal of the inhibitory CaRT888 site enhanced the heterodimeric signalling elicited by CaRS170A (an ECD mutant) and CaRF801A (an ICD mutant); mutants that elicit no signal when expressed alone. Crucially, when present on both of the heterodimer proteins, CaRT888A produced a greater rescue than when expressed on only one of the monomers suggesting that both CaR ICDs contribute to signalling simultaneously. Overall, these data demonstrate that CaR activity is determined not only by its extracellular agonists and allosteric modulators but also by integration with other intracellular signals that affect its phosphorylation status. These findings further our understanding of the fundamental physiology of Ca2+o homeostasis and may aid in the development of potential therapies for hyperparathyroid disorders.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Donald Ward (Supervisor) & Xin Wang (Supervisor)