Calcium Sensing Receptor-Homer1 mediated activation of protein kinase Akt: reconstitution and mechanism

Abstract

We recently showed that the scaffolding protein Homer1 is required for Ca2+o-stimulated phosphorylation of Akt-S473 (pAkt-S473) in osteoblasts, and reconstituted the signalling pathway in HEK-CaSR cells by transfecting them with Homer1c (Rybchyn et al., 2019). We have now examined whether the CaSR C-terminus is required for Ca2+o-stimulated pAkt-S473. In particular, we investigated whether Ca2+o-stimulated pAkt-S473 occurred in HEK-293 cells transfected with the WT CaSR, CaSR 909X or CaSR 866X in the absence or presence of co-transfected Homer1c. We first confirmed that elevated Ca2+o stimulated pAkt-S473 in HEK-293 cells that were transiently transfected with the CaSR and Homer1c. As Ca2+o increased to 3.0 mM from a baseline of 0.1 mM, the fold-change reached a maximum of 2.65 ± 0.5 and the EC50 for Ca2+o was 1.14  0.2 mM (n = 4). The results demonstrate that Ca2+o-stimulated pAktS473 requires the presence of both Homer1c and the WT CaSR. We next investigated whether the CaSR truncation mutant 909X could support Ca2+o-stimulated pAkt-S473 in the absence or presence of Homer1c and obtained very similar results. i.e., Homer1c was required for Ca2+o-stimulated pAkt-S473. Finally, we tested whether the proximal segment of the CaSR C-terminus (between residues 863-908) might be required for Homer1-dependent activation of pAkt-S473. Interestingly, HEK-293 cells transiently transfected with CaSR 866X, which removes the entire C-terminus with the exception of residues 863-865, also supported Ca2+o-stimulated pAkt-S473 in the presence of Homer1c. We next tested whether cells transfected with CaSR 866X might exhibit Ca2+o-stimulated pAkt-S473 even in the absence of Homer1c. Surprisingly, we observed Homer1-independent Ca2+o-stimulated pAkt-S473 under these circumstances and the fold-change with respect to baseline was 2.37  0.2. The findings demonstrate that the proximal C-terminus of the CaSR acts as an inhibitory domain for Ca2+o-stimulated pAkt-S473 and that Homer1 disinhibits the signalling pathway.