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

## TL;DR

This study shows that both subunits of the calcium-sensing receptor contribute to signaling, even when only one is connected to a G protein.

## Contribution

The study reveals that both intracellular domains of the calcium-sensing receptor homodimer independently contribute to signaling.

## Key findings

- Disabling one T888 phosphorylation site in either subunit enhances CaSR signaling.
- Removing both T888 sites in the CaSR heterodimer further increases intracellular Ca2+ mobilization.
- A positive allosteric modulator rescues nonfunctional CaSR mutants by removing intracellular inhibition.

## Abstract

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 (CaSR1S170A) and intracellular (CaSR2F801A) mutations, which are nonfunctional when transfected individually, partially rescues function via trans-activation in CaSR1S170A:CaSR2F801A 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 (CaSRT888A) by mutating one, and then both monomers in the CaSR1S170A:CaSR2F801Atrans-activation–enabled heterodimers. The extracellular Ca2+–induced intracellular Ca2+ mobilization elicited by CaSR1S170A:CaSR2F801A was significantly enhanced by disabling one T888 inhibitory phosphorylation site in either CaSR1S170A/T888A:CaSR2F801A or CaSR1S170A:CaSR2F801A/T888A, and further enhanced in cells in which both T888 sites had been removed in CaSR1S170A/T888A:CaSR2F801A/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 CaSRS170A mutant was rescued to wild-type levels by removal of the inhibitory T888 phosphorylation site in CaSRS170A/T888A homodimers. Therefore, although CaSRS170A 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.

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.