Abstract P239: Identification Of Binding Sites For Phosphodiesterase 5 (PDE5) On Rho-related BTB Domain Containing 1 (RhoBTB1) As A Guide To Identifying Novel RhoBTB1-Binding Partners

Abstract

In-depth studies of molecular pathways have provided insights to explore therapeutic targets for treating hypertension as a complex disease. We previously identified PDE5 as an interacting partner with RhoBTB1 protein in aortic smooth muscle cells (SMCs). RhoBTB1 regulates PDE5 activity and therefore intracellular cGMP levels, and restoration of RhoBTB1 expression in RhoBTB1-deficient states results in improvement of cardiovascular status in a mouse model of hypertension. Here we identified the specific regions of RhoBTB1 that are responsible for the recruitment of PDE5. Our goal is to use this as a guide to identify additional RhoBTB1 interacting proteins which play important roles in cardiovascular function in the hope their identification may open novel therapeutic avenues against hypertension and associated pathologies. We hypothesize that RhoBTB1 acts as an adaptor for Cullin-3 E3 ring ubiquitin ligase complex, where Cullin-3 functions as “scaffold” which delivers PDE5 (and other targets) to Cullin-3 complex followed by ubiquitination and proteasomal degradation. We generated several truncations in full length RhoBTB1, splitting it into its GTPase, BTB1, BTB2, Carboxyl-terminal (CT), BTB1-BTB2, and BTB1-BTB2-CT domains. To check the preferred binding interface for PDE5, we co-transfected HEK293 cells with epitope tagged PDE5 and separately tagged truncations of RhoBTB1, and analyzed the interaction between PDE5 and RhoBTB1 domains using co-immunoprecipitation assays. Our result shows that BTB1-BTB2-C domain of RhoBTB1 is the preferred binding region for PDE5. The BTB1-BTB2 domain lacking the CT was unable to bind to PDE5. Next, we will utilize stable isotope labeling by amino acids in cell culture (SILAC) followed by proteomic profiling to identify additional targets and/or adaptors involved in the binding of PDE5 and RhoBTB1. Similarly, we will tag specific domains of RhoBTB1 using the ascorbate peroxidase (APEX2) fusion system followed by proteomic profiling.