title: Gbetagamma activation of Src induces caveolae-mediated endocytosis in endothelial cells.
abstract: Caveolae-mediated endocytosis in endothelial cells is stimulated by the binding of albumin to gp60, a specific albumin-binding protein localized in caveolae. The activation of gp60 induces its cell surface clustering and association with caveolin-1, the caveolar-scaffolding protein. This interaction leads to G(i)-induced Src kinase activation, which in turn signals dynamin-2-mediated fission and directed migration of caveolae-derived vesicles from apical to basal membrane. In this study, we investigated the possible role of the Gbetagamma heterodimer in signaling G(i)-induced Src activation and subsequent caveolae-mediated endocytosis. We observed using rat lung microvascular endothelial cells that expression of the C terminus of beta-adrenergic receptor kinase (ct-betaARK), an inhibitor Gbetagamma signaling, prevented gp60-dependent Src activation as well as caveolae-mediated endocytosis and transcellular transport of albumin and uptake of cholera toxin subunit B, a specific marker of caveolae internalization. Expression of ct-betaARK also prevented Src-mediated tyrosine phosphorylation of caveolin-1 and dynamin-2 and the resultant phosphorylation-dependent association of dynamin-2 and caveolin-1. Also, the direct activation of Gbetagamma using a specific cell-permeant activating peptide (myristoylated-SIRKALNILGYPDYD) simulated the effects of gp60 in inducing Src activation, caveolin-1, and dynamin-2 phosphorylation as well as caveolae-mediated endocytosis of cholera toxin subunit B. The myristoylated-SIRKALNILGYPDYD peptide-induced responses were inhibited by the expression of ct-betaARK. Taken together, our results demonstrate that Gbetagamma activation of Src signals caveolae-mediated endocytosis and transendothelial albumin transport via transcytosis.
authors.affiliation: Pharmacology and Anesthesiology, University of Illinois, College of Medicine, Chicago, Illinois 60612, USA.
authors.forename: Ayesha N
authors.initials: AN
authors.lastname: Shajahan
authors.forename: Chinnaswamy
authors.initials: C
authors.lastname: Tiruppathi
authors.forename: Alan V
authors.initials: AV
authors.lastname: Smrcka
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
authors.forename: Richard D
authors.initials: RD
authors.lastname: Minshall
mesh_terms: D000418:Albumins / Q000378:metabolism; D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002478:Cells, Cultured; D034285:Dynamin II / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D008168:Lung / Q000098:blood supply; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015290:Second Messenger Systems / Q000502:physiology; D012795:Sialoglycoproteins / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D000418:Albumins; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D005456:Fluorescent Dyes; D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; C445259:beta-adrenergic receptor kinase inhibitory peptide; C065610:sialoglycoprotein gp60; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D034285:Dynamin II