Abstract
CD36, a multi-ligand plasma membrane receptor, has been implicated in immunity, metabolism and angiogenesis. We have recently demonstrated that CD36 nanoclustering at the plasma membrane is key to the initiation of CD36 signaling. In endothelial cells (ECs), the binding of thrombospondin-1 (TSP-1, an endogenous extracellular matrix anti-angiogenic factor) to CD36 nanoclusters activates an associated Src family kinase, Fyn, leading to ECs apoptosis, hence, inhibiting angiogenesis. We are interested in elucidating the mechanisms of CD36-Fyn enrichment and the role of lipid nanodomains and actin cytoskeleton during TSP-1 induced signaling in ECs. We hypothesize that lipid nanodomains play a role in bringing together CD36-Fyn to F-actin regions through adaptor molecules which forms a signaling platform. Using microscopy methods on HeLa cells co-transfected with Fyn and various fluorescent lipid biosensors and stained for F-actin (Phalloidin-AF647), we determined that Fyn is enriched on F-actin area at sites of phosphatidylinositol 4,5-bisphosphate enrichment (PIP2). During TSP-1 stimulation on Human Microvascular Endothelial Cells (HMEC), the CD36-Fyn-F-actin enrichment shift to domains containing PI(3,4,5)P3, suggesting a role for the phosphoinositide 3-kinase in signaling. The role of this kinase is further investigated using inhibitor targeting PI3-Kinase subunits for signal transduction. Additionally, we will be using rapamycin dimerization system to understand the role of these lipid nanodomains in CD36-Fyn signaling. Furthermore, to characterize the adaptor molecules involved in connecting F-actin to lipid nanodomains and/or CD36 nanoclusters, we are conducting APEX2 proximity labelling and fractionation approaches followed by mass spectrometry (MS) analysis. The MS screen will further narrow down proteins that are biotinylated, adjacent to CD36 and enriched in F-actin. Altogether, our investigation will provide insights into understanding the activity of plasma membrane receptor nanoclustering and signaling.
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