Role of Albumin‐induced RalA Activation and PLD2‐mediated Phosphatidic Acid Production in Caveolae‐mediated Endocytosis and Trafficking in Endothelial Cells

Abstract

Caveolae are the primary route for internalization and transcellular transport of macromolecules through the endothelium. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and recruitment of dynamin-2 and filamin A (FilA). Here, we test the hypothesis that activation of caveolae-mediated endocytosis requires RalA. Addition of albumin to human lung microvascular endothelial cells (HLMVECs) induced the activation RalA within minutes and siRNA-mediated downregulation of RalA abolished fluorescent BSA uptake. Co-IP studies revealed albumin induced the association of RalA, Cav-1 and FilA, however, RalA siRNA did not affect FilA recruitment to Cav-1 suggesting RalA was not required for complex formation. PLD is known to be activated by RalA, and PLD inhibitor 1-butanol abolished Alexa 488-BSA uptake. Using the PA biosensor mRFP-PASS, studies will determine whether the generation PA in Cav-1-YFP or PLD2-GFP positive membrane microdomains and whether PA production and trafficking of Cav-1-YFP coated vesicles can be blocked by RalA siRNA, 1-butanol, and PLD2 siRNA. Results thus far suggest the small GTPase RalA is important in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA, but rather by promoting PLD2-mediated generation of PA. Supported by NIH P01 HL60678 (RM), P01 HL98050 (VN), and the China Scholarship Council (YJ, YL)