Abstract 1393: The Guanine Exchange Factor 11 PDZ Protein Binds ABCA1 Positively Regulating Transporter Expression and Cholesterol Efflux via Rho Activation
ATP binding cassette transporter A1 (ABCA1) effluxes cellular cholesterol, which maintains plasma HDL levels and prevents cardiovascular disease. ABCA1 expression and efflux activity is highly regulated at transcriptional and posttranscriptional levels, although the molecular details of this posttranscriptional regulation remain unclear. We have previously shown that utrophin, as well as β1-syntrophin and β2-syntrophin, both PDZ proteins, interact with the C-terminus of ABCA1. These interactions increase cholesterol efflux by altering the cellular distribution of transporter and protecting it from degradation. Here we show that the guanine exchange factors 11 (GEF11), which have a PDZ domain and a RhoGEF/PH domain that facilitates the exchange of GDP for GTP and activate RhoA, can also interact with ABCA1. Using co-expression in 293 cells and immunoprecipitation assays the interaction of GEF11 with ABCA1 as full-length proteins was verified. Interestingly, co-expression of GEF11 with ABCA1 significantly increase the ABCA1 expression and cholesterol efflux, but GEF11 lacking the RhoGEF/PH domain, which is crucial for the GEF activity, lost the ability to stimulate ABCA1 protein levels and cholesterol efflux. This result suggests RhoA activity may modulate the ABCA1 expression and activity. To test this, the amount of activated RhoA was determined by precipitation with Rhotekin beads and immunoblotting using an anti-RhoA antibody. We found that RhoA was highly activated in GEF11-expressing 293 cells, and when ABCA1 and constitutively active RhoA (Q63L) was co-expressed the expression of ABCA1 was significantly increased. Conversely, the co-expression of a dominant negative form of RhoA (T19N) diminished ABCA1 expression, indicating RhoA positively regulates ABCA1 protein levels. It has been reported that the binding of apoA-I to ABCA1 prolonged its half-life by inhibiting a calpain-mediated degradation pathway. We confirmed this observation and show that apoA-I also activate RhoA in THP-1 cells. Together these data suggest a mechanism by which the binding of apoA-I to ABCA1 activates RhoA through the ABCA1/GEF11 complex with the resulting activated complex blocking the calpain-mediated degradation of ABCA1.