title: Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells.
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.
authors: From the School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, Sichuan 610054, China, the Departments of Anesthesiology.
authors: Ying
authors: Y
authors: Jiang
authors: Pharmacology, and.
authors: Maria S
authors: MS
authors: Sverdlov
authors: Pharmacology, and.
authors: Peter T
authors: PT
authors: Toth
authors: Pharmacology, and Medicine, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, and.
authors: Long Shuang
authors: LS
authors: Huang
authors: the Departments of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas 77030.
authors: Guangwei
authors: G
authors: Du
authors: From the School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, Sichuan 610054, China.
authors: Yiyao
authors: Y
authors: Liu
authors: Pharmacology, and Medicine, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, and.
authors: Viswanathan
authors: V
authors: Natarajan
authors: the Departments of Anesthesiology, Pharmacology, and rminsh@uic.edu.
authors: Richard D
authors: RD
authors: Minshall
mesh_terms: D001693:Biological Transport, Active / Q000502:physiology; D021941:Caveolae / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D010712:Phosphatidic Acids / Q000096:biosynthesis* / Q000235:genetics; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D020725:ral GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D010712:Phosphatidic Acids; C105606:phospholipase D2; D010739:Phospholipase D; C492198:RALA protein, human; D020725:ral GTP-Binding Proteins
keywords: Ras protein; caveolin; endocytosis; filamin; trafficking