The calcium-sensing receptor (CaSR) forms homodimers that may couple to only a single heterotrimeric G protein at any one time. Whether the intracellular domain (ICD) of the uncoupled protomer remains functionally active is unknown.
To investigate this, we used a transactivation model in HEK-293 cells in which the loss of-function mutants CaSRS170A (extracellular domain) and CaSRF801A (intracellular domain) were co-expressed to generate partially functional heterodimers. We then introduced the gain-of-function mutation T888A, which prevents inhibitory PKC-mediated phosphorylation, into one or both protomers. By comparing signalling responses in heterodimers containing T888A in either one or both ICDs, we assessed whether both ICDs contribute to receptor activity despite the apparent restriction to single G protein coupling.
Extracellular Ca2+-induced intracellular Ca2+ mobilisation, measured using Fura-2, was significantly enhanced when T888 phosphorylation was prevented in either protomer. Importantly, signalling increased further when T888A was present in both protomers (CaSRS170A/T888A:CaSRF801A/T888A), indicating that each ICD contributes independently to receptor signalling.
Although CaSRS170A homodimers were non-functional at extracellular Ca2+ concentrations up to at least 40 mM, combining the T888A mutation with the calcimimetic R-568 restored receptor activity to wild-type levels. These findings suggest that inhibition of T888 phosphorylation can help overcome the conformational defect imposed by the S170A mutation.
We also examined extracellular domain-deleted ("headless") receptors (Rho-C-hCaSR). Preventing T888 phosphorylation significantly enhanced signalling, while additional mutation of the inhibitory phosphorylation site S875 (Rho-C-hCaSRT888A/S875A) produced near-maximal activity even under basal conditions (0.5 mM Ca2+/Mg2+).
Together, these results demonstrate a critical role for ICD phosphorylation in regulating CaSR activity. Furthermore, the greater signalling observed when both protomer ICDs are disinhibited indicates that each ICD makes an independent positive contribution to receptor signalling, despite only one G protein being coupled at a time.