Significance of the Calcium-Sensing Receptor’s Intra-loops and C-terminus in Ligand-Dependent Signalling

Abstract

The Ca2+o -sensing receptor (CaSR) is a Class C G-protein coupled receptor that responds to Ca2+o and L-amino acids to activate signalling pathways in a ligand-dependent manner. The residues that support ligand-biased signalling are largely unknown. In addition, variable phenotypes have been associated with the mutant, R220W. Thus, I tested whether common CaSR polymorphisms modify the function of R220W. We performed alanine scanning site-directed mutagenesis (SDM) of intraloops (iLs) -1, -2 and -3, and engineered truncations of the C-terminus. The functions of each mutant in response to Ca2+o and L-Phe were examined using IP1 accumulation, pERK1/2 and Ca2+i mobilisation readouts. HEK293 cells expressing the mutants I642A/K644A, P641A (iL-1), TA701-704, F706A (iL-2), R795A, L797A, P798A and F801A (iL-3), and F881X, K882X, and A884X (C-terminal truncations) markedly attenuated Ca2+o- stimulated IP1, Ca2+i mobilisation, and pERK1/2. By comparison, L-Phe- induced Ca2+i mobilisation responses were not affected by I642A, L703A and P798A. Furthermore, C-terminal truncations, V883X, A885X and R886X, exhibited sustained elevations in Ca2+i, that were unaffected by L-Phe, whereas T888X, and R890X exhibited sustained elevations in Ca2+i, that were enhanced by L-Phe. The results suggest residues in iL-1, -2, -3 and the C-terminus, support key elements of Ca2+o and L-Phe-mediated signalling. Moreover, E707A (iL -2) and N800A (iL -3) were identified as novel activating mutations. In addition, the function of HEK293 cells co- expressing the mutant R220W with either CaSR polymorphism, i.e. A986S, R990G, Q1011E, were assessed. R220W attenuated the Ca2+o -stimulated IP1 response while A986S, R990G, or Q1011E were comparable to WT. Moreover, co-expression of R220W with WT or A986S, R990G, or Q1011E, equally restored the impaired R220W response. The results indicate that these common polymorphisms have no effect on the function of R220W, for Ca2+o -stimulated PI-PLC activation.