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)
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