Original Article

Endothelial Cell-surface gp60 Activates Vesicle Formation and Trafficking via G_i-coupled Src Kinase Signaling Pathway

Correspondence to: Asrar B. Malik, Department of Pharmacology, University of Illinois, College of Medicine, 835 South Wolcott Avenue (M/C 868), Chicago, IL 60612. Tel:(312) 996-7635 Fax:(312) 996-1225 E-mail:abmalik{at}uic.ed.

We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein G_i, 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 transendothelial ¹²⁵I-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 G_i carboxyl-terminal peptide inhibited endothelial ¹²⁵I-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 overexpression resulted in the sequestration of G_i with the caveolin-1, whereas dn-Src inhibited G_i binding 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 G_i-coupled Src kinase signaling pathway.

Key Words: transcytosis, endocytosis, caveolae, microvascular endothelial cells, albumin permeability

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