Abstract 5483: Deficiency Of The Stimulatory G Protein alpha-subunit Gs (alpha) Leads To Reduced ApoA-I Mediated Cellular Lipid Efflux and Impaired ABCA1-dependent Nascent HDL Biogenesis Pathway
It has been suggested that initial apoA-I binding to ABCA1 is coupled to the activation of signal transduction pathways, allowing excess cholesterol removal from tissues. However, the role of the heterotrimeric G protein alpha-subunit (Gs alpha) in this pathway remains uncertain. We obtained evidence that exposure of human fibroblasts or CHO cells overexpressing ABCA1 to apoA-I increased significantly intracellular cAMP production (1.5-fold). Furthermore, mutations of ABCA1 associated with Tangier disease (TD) (C1477R, 2203X and 2145X) severely reduced this response. Interestingly, ABCA1 was found to be associated with Gs alpha in human fibroblasts as assessed by co-immunoprecipitation with Gs alpha antibody. To determine whether Gs alpha is implicated in the apoA-I/ABCA1-mediated efflux system, we used Gs alpha-null embryonic fibroblasts derived from mice homozygous for disruption of Gnas exon 2 (GnasE2-/E2- MEFs) and examined cellular lipid efflux. We found that GnasE2-/E2- MEFs exhibited decreased cholesterol and phospholipid efflux to apoA-I compared to normal cells. Furthermore, induction of ABCA1 in both normal and GnasE2-/E2- MEFs by LXR agonist (22OH/9CRA) increased ABCA1 expression, and stimulated lipid efflux to apoA-I. However, stimulation with 22OH/9CRA or 8-Br-cAMP did not correct the lipid efflux defect in GnasE2-/E2-MEFs. At the same time, 125I-apoA-I binding was found significantly decreased in GnasE2-/E2-MEFs. As expected, both lipid efflux and 125I-apoA-I binding were found severely decreased in ABCA1−/− MEF negative control cells. Finally, 125I-apoA-I incubated with normal MEF generated nascent alpha-LpA-I with particle size of 9 to 17 nm. However, 125I-apoA-I incubated with GnasE2-/E2- MEFs or ABCA1−/− MEFs was unable to form such particles. Our results provide a biochemical basis for the HDL biogenesis pathway, involving both ABCA1 and the heterotrimeric G protein alpha-subunit Gs alpha (and/or its variant XLas, which is also ablated in GnasE2-/E2- MEFs). This supports the concept that ABCA1 transduces signals from apoA-I by activating the heterotrimeric G protein alpha-subunit signaling cascade with subsequent regulation of cellular lipid homeostasis and nascent HDL genesis.