Essential role of RGS-PX1/sorting nexin 13 in mouse development and regulation of endocytosis dynamics.

RGS-PX1 (also known as sorting nexin 13) is a member of both the regulator of G protein signaling (RGS) and sorting nexin (SNX) protein families. Biochemical and cell culture studies have shown that RGS-PX1/SNX13 attenuates Galphas-mediated signaling through its RGS domain and regulates endocytic trafficking and degradation of the epidermal growth factor receptor. To understand the functions of RGS-PX1/SNX13 in vivo, we generated mice carrying targeted mutations of Snx13 and found that systemic Snx13-null mice were embryonic lethal around midgestation. Snx13-null embryos had significant overall growth retardation and defects in neural tube closure, blood vessel formation, and the formation of the placental labyrinthine layer. Moreover, the Snx13-null visceral yolk sac endoderm cells showed dramatic changes in the organization of endocytic compartments, abundant autophagic vacuoles, and abnormal localization of several endocytic markers, including megalin, a receptor for nutrients and proteins; ARH, a coat protein that binds megalin; LAMP2; and LC3. These changes suggest that Snx13-null embryos are defective in nutrient uptake and transport, which may contribute to the other developmental abnormalities observed. Taken together, our findings demonstrate an essential role for RGS-PX1/SNX13 in mouse development and provide previously undescribed insights into its cellular function in the regulation of endocytosis dynamics.

Regulation of epidermal growth factor receptor degradation by heterotrimeric Galphas 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 (Galphas) promotes ligand-dependent degradation of epidermal growth factor (EGF) receptors and Texas Red EGF, and knock-down of Galphas expression by RNA interference (RNAi) delays receptor degradation. We also show that Galphas 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. Galphas coimmunoprecipitates with Hrs and binds Hrs in pull-down assays. By immunofluorescence, exogenously expressed Galphas colocalizes with myc-Hrs and GFP-RGS-PX1 on early endosomes, and expression of either Hrs or RGS-PX1 increases the localization of Galphas on endosomes. Furthermore, knock-down of both Hrs and Galphas by double RNAi causes greater inhibition of EGF receptor degradation than knock-down of either protein alone, suggesting that Galphas and Hrs have cooperative effects on regulating EGF receptor degradation. These observations define a novel regulatory role for Galphas in EGF receptor degradation and provide mechanistic insights into the function of Galphas in endocytic sorting.