Endothelial Cell-Surface gp60 Activates Vesicle Formation and Trafficking via Gi-Coupled Src Kinase Signaling Pathway

• Published in: The Journal of cell biology 2000-09, Vol.150 (5), p.1057-1069
• Main Authors: Minshall, Richard D., Tiruppathi, Chinnaswamy, Vogel, Stephen M., Niles, Walter D., Gilchrist, Annette, Hamm, Heidi E., Malik, Asrar B.
• Format: Article
• Language: English
• Subjects: Albumins; Cell membranes; Cells; Cellular biology; Delta cells; Dyes; Endocytosis; Endothelial cells; Fluorescence; Original; Plasma; Proteins; Toxins; Whooping cough
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.150.5.1057

We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein Gi, and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial125I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11-amino acid Gα icarboxyl-terminal peptide inhibited endothelial125I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 over-expression resulted in the sequestration of Gα iwith the caveolin-1, whereas dn-Src inhibited Gα ibinding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60 with caveolin-1, followed by the activation of the downstream Gi-coupled Src kinase signaling pathway.