Endothelial cell-surface gp60 activates vesicle formation and trafficking via G(i)-coupled Src kinase signaling pathway

We tested the hypothesis that the albumin-docking protein gp60, which is lo calized in caveolae, couples to the heterotrimeric GTP binding protein G(i) , and thereby activates plasmalemmal vesicle formation and the directed mig ration of vesicles in endothelial cells (ECs). We used the water-soluble st yryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyri dinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp6 0 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they releas ed 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 fluorescen ce. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab an d secondary Ab) in EC grown on microporous filters increased transendotheli al I-125-albumin permeability without altering liquid permeability (hydraul ic conductivity), thus, indicating the dissociation of hydraulic conductivi ty from the albumin permeability pathway. The findings that the sterol bind ing agent, filipin, prevented gp60-activated vesicle formation and that cav eolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin o f endocytic vesicles. Pertussis toxin pretreatment and expression of the do minant negative construct encoding an 11-amino acid G(alpha i) carboxyl-ter minal peptide inhibited endothelial I-125-albumin endocytosis and vesicle f ormation induced by gp60 activation. Expression of dominant negative Src (d n-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activ ated endocytosis. Caveolin-1 overexpression resulted in the sequestration o f G(alpha i) with the caveolin-1, whereas dn-Src inhibited G(alpha i) bindi ng to caveolin-1,Thus, vesicle formation induced by gp60 and migration of v esicles to the basolateral membrane requires the interaction of gp60 with c aveolin-1, followed by the activation of the downstream G(i)-coupled Src ki nase signaling pathway.