Work

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

Year: 2025

Type: article

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 (CaSR 1 S170A ) and intracellular (CaSR 2 F801A ) mutations, which are nonfunctional when transfected individually, partially rescues function via trans -activation in CaSR 1 S170A :CaSR 2 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 CaSR 1 S170A :CaSR 2 F801A trans -activation–enabled heterodimers. The extracellular Ca 2+ –induced intracellular Ca 2+ mobilization elicited by CaSR 1 S170A :CaSR 2 F801A was significantly enhanced by disabling one T888 inhibitory phosphorylation site in either CaSR 1 S170A/T888A :CaSR 2 F801A or CaSR 1 S170A :CaSR 2 F801A/T888A , and further enhanced in cells in which both T888 sites had been removed in CaSR 1 S170A/T888A :CaSR 2 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 Ca 2+ –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 Ca 2+ mobilization than disinhibiting only one, suggesting a positive functional contribution from both subunits.

Source: Molecular Pharmacology

DOI: 10.1016/j.molpha.2025.100077

Authors: Lenah S. Binmahfouz, Mahvash A. Goolam, Eleanor Barker, Arthur D. Conigrave, Donald T. Ward

Institutions: King Abdulaziz University, The University of Sydney, University of Manchester

Language: English

Topic: Receptor Mechanisms and Signaling

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,600

Funders: Kidney Research UK, King Abdulaziz University