Regulation of epidermal growth factor receptor degradation by heterotrimeric G alpha s protein

Heterotrimeric G proteins have been implicated in the regulation of membrane trafficking, but the mechanisms involved are not well understood. Here, we report that overexpression of the stimulatory G protein subunit (Gas) promotes ligand-dependent degradation of epidermal growth factor (EGF) receptors and Texas Red EGF, and knock-down of Gas expression by RNA interference (RNAi) delays receptor degradation. We also show that Gas and its GTPase activating protein (GAP), RGS-PX1, interact with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a critical component of the endosomal sorting machinery. Gas coimmunoprecipitates with Hrs and binds Hrs in pull-down assays. By immunofluorescence, exogenously expressed Gas colocalizes with myc-Hrs and GFP-RGS-PX1 on early endosomes, and expression of either Hrs or RGS-PX1 increases the localization of Gas on endosomes. Furthermore, knock-down of both Hrs and Gas by double RNAi causes greater inhibition of EGF receptor degradation than knock-down of either protein alone, suggesting that Gas and Hrs have cooperative effects on regulating EGF receptor degradation. These observations define a novel regulatory role for Gas in EGF receptor degradation and provide mechanistic insights into the function of Gas in endocytic sorting.