UC San Diego

Mechanisms involving heterotrimeric G proteins in the regulation of membrane trafficking are not well understood. Here, we reported G$$alpha$$s overexpression promotes ligand-dependent degradation of EGFR and G$$alpha$$s knockdown delays receptor degradation through interaction with RGS-PX1 and Hrs on early endosomes. These observations provide mechanistic insights into the function of G$$alpha$$s, RGS-PX1 and Hrs in endocytic sorting. To further understand RGS-PX1's role in EGFR trafficking and/or G$$alpha$$s signaling regulation in vivo, we generated RGS-PX1 knockout mice and found homozygous mice were embryonic lethal around midgestation. RGS-PX1- null embryos had significant overall growth retardation, defects in neural tube closure and blood vessel formation and dramatic changes in the organization of endocytic compartments, etc.. Taken together, our findings demonstrate an essential role for RGS-PX1in mouse development and provide new insights into its functions in the regulation of endocytosis dynamics. During our phylogenetic analysis of RGS-PX1, I noticed that C. elegans RGS-PX1 contained a 1000 aa uncharacterized N- terminus. I did a BLAST search on this 1000 aa and found its human homologue, called "unnamed protein product" along with homologues from other species. Among them, the Arabidopsis Thaliana homologue was named AtNOA1 (Arabidopsis Thaliana Nitric Oxide Associated, 1). Accordingly, we named the human homologue, hNOA1. We characterized hNOA1 as a large mitochondrial G protein, peripherally associated with the mitochondrial inner membrane facing the matrix. Overexpression and knockdown of hNOA1 led to changes in mitochondrial shape implying effects on mitochondrial dynamics. By carrying out IP-MS analysis of endogenous hNOA1, we found that hNOA1 interacts with Complex I of the electron transport chain and DAP3 (Death Associated Protein 3), a positive apoptosis regulator. Knockdown of hNOA1 reduces ̃ 20% mitochondrial O₂ consumption in a Complex I-dependent manner and renders cells more resistant to apoptotic stimuli. Thus, hNOA1 may potentially serve to link mitochondrial dynamics, respiration and apoptosis. It has been our interest to link RGS-PX1 with hNOA1. In this thesis, I provide further evidence that hNOA1 and RGS-PX1 interact with each other and some preliminary results leading to the hypothesis that the cytosolic hNOA1 promotes EGF receptor downregulation by sequestering RGS- PX1 from early endosomes to the cytosol.