title: Intersectin-2L regulates caveola endocytosis secondary to Cdc42-mediated actin polymerization.
abstract: Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PH(ITSN-2L)) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The "catalytically dead" DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PH(ITSN-2L)-transfected cells compared with control and 1 microM jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane.
authors.affiliation: Department of Pharmacology, Rush University Medical Center, Chicago, Illinois 60612, USA.
authors.forename: Irene K
authors.initials: IK
authors.lastname: Klein
authors.forename: Dan N
authors.initials: DN
authors.lastname: Predescu
authors.forename: Tiffany
authors.initials: T
authors.lastname: Sharma
authors.forename: Ivana
authors.initials: I
authors.lastname: Knezevic
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
authors.forename: Sanda
authors.initials: S
authors.lastname: Predescu
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000235:genetics / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D021941:Caveolae / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D017434:Protein Structure, Tertiary / Q000502:physiology; D011554:Pseudopodia / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051316:Wiskott-Aldrich Syndrome Protein / Q000235:genetics / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D033942:Adaptor Proteins, Vesicular Transport; C509776:ITSN2 protein, human; D034741:RNA, Small Interfering; C495767:WAS protein, human; D051316:Wiskott-Aldrich Syndrome Protein; D020764:cdc42 GTP-Binding Protein