Inhibition of the calcium-sensing receptor by extracellular phosphate ions and by intracellular phosphorylation

Abstract

As both a sensor of extracellular calcium (Ca²⁺ _o) concentration and a key controller of Ca²⁺ _o homeostasis, one of the most interesting properties of the calcium-sensing receptor (CaR) is its sensitivity to, and modulation by, ions and small ligands other than Ca²⁺. There is emerging evidence that extracellular phosphate can act as a partial, non-competitive CaR antagonist to modulate parathyroid hormone (PTH) secretion, thus permitting the CaR to integrate mineral homeostasis more broadly. Interestingly, phosphorylation of certain intracellular CaR residues can also inhibit CaR responsiveness. Thus, negatively charged phosphate can decrease CaR activity both extracellularly (via association with arginine) and intracellularly (via covalent phosphorylation).

Keywords: calcium-sensing receptor; parathyroid hormone; phosphate-sensing; receptor phosphorylation; secondary hyperparathyroidism.

FIGURE 1

Identification of ionic binding sites in the CaR extracellular domain. Panel (A) Sequence alignment and structural superposition of the currently available CaR ECD models in the active conformation. The Zhang et al. (2016) model (5FBK) is shown in blue (left) and the Geng et al. (2016) model (5K5S) is shown in pink (right). In the middle, an alignment of both models reveals an almost identical CaR ECD structure with minor differences. Pymol root mean square deviation (RMSD) 0.5, after 5 cycles of iteration. Panel (B) Active conformation of the CaR ECD with reported ligand binding sites. The Zhang model (5FBH) is shown as the left monomer, with the Geng model (5K5S) on the right. The Zhang model describes three Ca²⁺-binding sites, one in the upper domain and two in the lower domain facing the interface between monomers. At the same locations, the Geng model describes two Ca²⁺-binding sites, but two additional Ca²⁺-binding sites in the cleft between the upper and lower domains. Both models identified a common L-amino acid binding site and a common anion binding site, both located at the cleft between the upper and lower domains. In addition, the Geng model includes an anion binding site located in the lower domain. The ligand binding sites highlighted in boxes are those found in both crystal models. L-AA, L-amino acid.

FIGURE 2

Schematic representation of CaR inhibition by extracellular phosphate ions and by intracellular, covalent phosphorylation. The binding-sites of the (activating) Ca²⁺ and (inhibitory) phosphate (PO₄) ions shown here are approximate, though their 4:1 ratio is consistent with both the crystal models and functional Hill coefficients. Sustained phosphorylation (P) of ICD residues CaR^S875 and CaR^T888 supresses Ca²⁺ _i mobilisation. Episodic dephosphorylation of CaR^T888 (at least) permits Ca²⁺ _i oscillations, while continuous dephosphorylation of these sites elicits enhanced, sustained Ca²⁺ _i mobilisation. The two monomers of the CaR homodimer are shown either as orange or blue. G_q, G protein-q/11; PLC, phospholipase C; IP₃R, IP₃ receptor; ER, Ca²⁺ stores of the endoplasmic reticulum; PKC, protein kinase C. The CaR domains shown are the venus flytrap (VFT), cysteine-rich domain (CRD), transmembrane domain (TMD) and intracellular domain (ICD).