Homer 2 tunes G protein–coupled receptors stimulus intensity by regulating RGS proteins and PLCβ GAP activities

Abstract

Homers are scaffolding proteins that bind G protein–coupled receptors (GPCRs), inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), ryanodine receptors, and TRP channels. However, their role in Ca2+ signaling in vivo is not known. Characterization of Ca2+ signaling in pancreatic acinar cells from Homer2−/− and Homer3−/− mice showed that Homer 3 has no discernible role in Ca2+ signaling in these cells. In contrast, we found that Homer 2 tunes intensity of Ca2+ signaling by GPCRs to regulate the frequency of [Ca2+]i oscillations. Thus, deletion of Homer 2 increased stimulus intensity by increasing the potency for agonists acting on various GPCRs to activate PLCβ and evoke Ca2+ release and oscillations. This was not due to aberrant localization of IP3Rs in cellular microdomains or IP3R channel activity. Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit Ca2+ signaling in vivo. Moreover, Homer 2 preferentially bound to PLCβ in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLCβ in an in vitro reconstitution system, with minimal effect on PLCβ-mediated PIP2 hydrolysis. These findings describe a novel, unexpected function of Homer proteins, demonstrate that RGS proteins and PLCβ GAP activities are regulated functions, and provide a molecular mechanism for tuning signal intensity generated by GPCRs and, thus, the characteristics of [Ca2+]i oscillations.

Homer proteins in Ca2+ signaling by excitable and non-excitable cells

“…1B-1D, which shows that Homer1 and Homer2 are restricted to the apical pole, whereas Homer3 is restricted to the basal pole. Importantly, Ca 2+ signaling proteins are also enriched at the apical pole and show complete co-localization with Homer1 and Homer2, but not with Homer3 [30]. These findings implicate Homer1 and Homer2, but not Homer3, in regulation of Ca 2+ signaling in these cells.…”

“…As indicated above, all Homer isoforms co-localize with mGluRs in the PSD and Homer expression is enriched in dendrites [26][27][28][29]. The localization of the Homer isoforms was further examined in the polarized pancreatic acinar cells and was found to be isoform-specific [30]. This is illustrated in Fig.…”

“…These studies rely on deletion of one or more Homer genes in mice and suggest that the Homers are not essential for targeting, localization or retention of Ca 2+ signaling complexes in cellular microdomains [25,30]. Several lines of evidence support this conclusion.…”

“…Several lines of evidence support this conclusion. First, deletion of the Homers enhances, rather than disrupts, Ca 2+ signaling [30]. Second, deletion of the individual Homer isoforms does not affect localization of Ca 2+ signaling proteins in the apical pole of polarized secretory cells [25,30].…”

“…1D) suggests that it may not control the activity of Ca 2+ signaling complexes. Indeed, preliminary studies showed that deletion of Homer3 does not affect the overall GPCR-mediated Ca 2+ signaling in pancreatic acinar cells [30]. Therefore, the function of Homer3 will not be considered further in this review.…”