Altered expression of G(q/11alpha) protein shapes mGlu1 and mGlu5 receptor-mediated single cell inositol 1,4,5-trisphosphate and Ca(2+) signaling

Abstract

The metabotropic glutamate (mGlu) receptors mGlu1 and mGlu5 mediate distinct inositol 1,4,5-trisphosphate (IP(3)) and Ca(2+) signaling patterns, governed in part by differential mechanisms of feedback regulation after activation. Single cell imaging has shown that mGlu1 receptors initiate sustained elevations in IP(3) and Ca(2+), which are sensitive to agonist concentration. In contrast, mGlu5 receptors are subject to cyclical PKC-dependent uncoupling and consequently mediate coincident IP(3) and Ca(2+) oscillations that are largely independent of agonist concentration. In this study, we investigated the contribution of G(q/11)alpha protein expression levels in shaping mGlu1/5 receptor-mediated IP(3) and Ca(2+) signals, using RNA interference (RNAi). RNAi-mediated knockdown of G(q/11)alpha almost abolished the single-cell increase in IP(3) caused by mGlu1 and mGlu5 receptor activation. For the mGlu1 receptor, this unmasked baseline Ca(2+) oscillations that persisted even at maximal agonist concentrations. mGlu5 receptor-activated Ca(2+) oscillations were still observed but were only initiated at high agonist concentrations. Recombinant overexpression of G(q)alpha enhanced IP(3) signals after mGlu1 and mGlu5 receptor activation. It is noteworthy that although mGlu5 receptor-mediated IP(3) and Ca(2+) oscillations in control cells were largely insensitive to agonist concentration, increasing G(q)alpha expression converted these oscillatory signatures to sustained plateau responses in a high proportion of cells. In addition to modulating temporal Ca(2+) signals, up- or down-regulation of G(q/11)alpha expression alters the threshold for the concentration of glutamate at which a measurable Ca(2+) signal could be detected. These experiments indicate that altering G(q/11)alpha expression levels differentially affects spatiotemporal aspects of IP(3) and Ca(2+) signaling mediated by the mGlu1 and mGlu5 receptors.