Homer1c and CaSR interaction directing to the phosphorylation of AktS473

Islam KS1, Brennan SC1, Rybchyn MS2, Mason RS2 and Conigrave AD1

  1. University of Sydney, School of Life and Environmental Sciences.
  2. University of Sydney, Discipline of Physiology, School of Medical Sciences.

Homer proteins form multivalent complexes that bind proline-rich motifs in group 1 metabotropic glutamate receptors, thereby coupling these receptors in signalling complexes (1). We hypothesized that the Homer1c scaffolding protein could interact with the calcium-sensing receptor (CaSR) leading to phosphorylation of the downstream protein kinase Akt at the site S473 (AktS473). First we examined whether increases in extracellular Ca2+ concentration (Ca2+o) might induce phosphorylation of AktS473 in HEK-293 cells that stably express the CaSR (HEK-CaSR cells), a cell line that does not express the Homer1c protein endogenously. In the absence of Homer1c, activation of the CaSR by Ca2+o (0.1 mM to 8.0 mM) for 2-30 min had no effect on pAktS473 level as determined by Western Blotting. We next investigated the behaviour of HEK-CaSR cells that had been transiently transfected with Homer1c for 24-72 h and observed that in cells that had been transiently transfected with Homer1c for 48 h elevated Ca2+o induced phosphorylation of AktS473 in the range 0.1 to 5.0 mM, with a maximal increase of 3-fold at 3.0 mM compared to control ( Ca2+o 0.1 mM; p 0.05; n = 4). This effect was not observed in HEK-CaSR cells transfected with vector control (pcDNA3.1) or in control HEK-293 cells that did not express the CaSR. These experiments demonstrate that the interaction between the CaSR and Homer1c is necessary for phosphorylation of AktS473. We next examined the mechanism by which Homer1c interacts with CaSR, leading to the downstream phosphorylation of AktS47. We first showed that we can replicate the effects seen in HEK-CaSR cells, using HEK-293 cells transiently transfected with both Homer1c and the wild-type CaSR. In these cells, pAktS473 levels were increased by two-fold in cells exposed to 3.0 mM Ca2+o compared to 0.1 mM (p 0.05, n = 6). We next co-transfected Homer1c and a mutant CaSR truncated at residue 866, which lacks the entire C-terminus. The effect was preserved intact demonstrating that the CaSR C-terminus is not required (n = 4). Further experiments indicate that receptor intra-loops 2 and 3 are required for Ca2+o-dependent AktS473 phosphorylation. The present study demonstrates that Ca2+o-induced CaSR-mediated phosphorylation of AktS473 requires a novel interaction between a class C GPCR and Homer1c with potential implications for the role of the CaSR in bone formation.