Abstract
Caveolae are the primary route for internalization and transendothelial transport of macromolecules, such as insulin and albumin. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and subsequent recruitment of dynamin-2 and filamin A (FilA), which facilitate vesicle fission and trafficking, respectively. Here, we tested the role of RalA and phospholipase D (PLD) signaling in the regulation of caveolae-mediated endocytosis and trafficking. The addition of albumin to human lung microvascular endothelial cells induced the activation of RalA within minutes, and siRNA-mediated down-regulation of RalA abolished fluorescent BSA uptake. Co-immunoprecipitation studies revealed that albumin induced the association between RalA, Cav-1, and FilA; however, RalA knockdown with siRNA did not affect FilA recruitment to Cav-1, suggesting that RalA was not required for FilA and Cav-1 complex formation. Rather, RalA probably facilitates caveolae-mediated endocytosis by activating downstream effectors. PLD2 was shown to be activated by RalA, and inhibition of PLD2 abolished Alexa-488-BSA uptake, indicating that phosphatidic acid (PA) generated by PLD2 may facilitate caveolae-mediated endocytosis. Furthermore, using a PA biosensor, GFP-PASS, we observed that BSA induced an increase in PA co-localization with Cav-1-RFP, which could be blocked by a dominant negative PLD2 mutant. Total internal reflection fluorescence microscopy studies of Cav-1-RFP also showed that fusion of caveolae with the basal plasma membrane was dependent on PLD2 activity. Thus, our results suggest that the small GTPase RalA plays an important role in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA but by stimulating PLD2-mediated generation of phosphatidic acid.
Highlights
Caveolae-mediated transcellular transport of macromolecules through endothelial cells (ECs)2 lining blood vessels is a highly selective and regulated process [1]
RalA Is Activated by Albumin in human lung microvascular endothelial cells (HLMVECs)—To test whether RalA is activated upon the addition of albumin to stimulate caveolae-mediated endocytosis, we assessed RalA-GTP/ RalBP1 binding
RalA Is Associated with Cargo-loaded Caveolae in Endothelial Cells—To investigate whether RalA is localized on caveolae, we examined RalA distribution in density gradient fractions of HLMVECs
Summary
Caveolae-mediated transcellular transport of macromolecules through endothelial cells (ECs) lining blood vessels is a highly selective and regulated process [1]. Cdc, a small GTPase of the Rho family, was detected in caveolae [9], where it is thought to control caveolae-mediated endocytosis by regulating actin polymerization and interactions between the actin cytoskeleton and intersectin, a scaffolding protein required for efficient fission and internalization of caveolae (10 –12) Another small GTPase, Rab, which is known to participate in endocytosis by regulating vesicle docking and fusion, directly binds to caveolin-1, and this interaction increases Rab activity [13]. We have previously shown that actin-binding protein FilA is recruited to caveolae following activation of Src kinase and Srcdependent phosphorylation of caveolin-1 and that FilA is required for endocytosis of albumin and trafficking of caveolae [6] Another group demonstrated that FilA interacts with active RalA [17]. We observed that PLD2-mediated production of PA downstream of RalA was involved in the regulation of caveolae fusion and fission events, arguably by promoting changes in membrane curvature and actin cytoskeleton reorganization
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