Both calcium-sensing receptor intracellular C-terminal domains support homodimer signaling

Extracellular calcium-sensing receptors (CaSRs) are required for whole-body calcium homeostasis and operate as homodimers, shown structurally to bind only one heterotrimeric G protein at a time. Whether the intracellular domain (ICD) of the other, uncoupled monomer is functionally redundant or still required for optimal CaSR signaling remains unknown. Cotransfection of human embryonic kidney 293 cells with receptors containing both extracellular (CaSR₁^S170A) and intracellular (CaSR₂^F801A) mutations, which are nonfunctional when transfected individually, partially rescues function via trans-activation in CaSR₁^S170A:CaSR₂^F801A heterodimers. Further, mutation of an inhibitory, intracellular protein kinase C site T888 permits gain-of-function in CaSR. Therefore, we disabled phosphorylation of this residue (CaSR^T888A) by mutating one, and then both monomers in the CaSR₁^S170A:CaSR₂^F801Atrans-activation-enabled heterodimers. The extracellular Ca²⁺-induced intracellular Ca²⁺ mobilization elicited by CaSR₁^S170A:CaSR₂^F801A was significantly enhanced by disabling one T888 inhibitory phosphorylation site in either CaSR₁^S170A/T888A:CaSR₂^F801A or CaSR₁^S170A:CaSR₂^F801A/T888A, and further enhanced in cells in which both T888 sites had been removed in CaSR₁^S170A/T888A:CaSR₂^F801A/T888A. The results demonstrate that both ICDs of CaSR dimers contribute independently to receptor signaling. Further, in the presence of N-(3-[2-chlorophenyl]propyl)-(R)-alpha-methyl-3-methoxybenzylamine (NPS R-568; CaSR positive allosteric modulator), extracellular Ca²⁺-stimulated signaling from the nonfunctional CaSR^S170A mutant was rescued to wild-type levels by removal of the inhibitory T888 phosphorylation site in CaSR^S170A/T888A homodimers. Therefore, although CaSR^S170A destabilizes the closed (active) form of the Venus fly trap in wild-type homodimers, receptor function can be rescued by relieving intracellular C-terminal domain-dependent inhibition of signaling. SIGNIFICANCE STATEMENT: Despite the homodimeric calcium-sensing receptor coupling to only one G protein at a time, disinhibiting both intracellular domains elicits a greater increase in intracellular Ca²⁺ mobilization than disinhibiting only one, suggesting a positive functional contribution from both subunits.