Endothelial cell-surface gp60 activates vesicle formation and trafficking via Gi-coupled Src kinase signaling pathway

被引:224
作者
Minshall, RD
Tiruppathi, C
Vogel, SM
Niles, WD
Gilchrist, A
Hamm, HE
Malik, AB
机构
[1] Univ Illinois, Coll Med, Dept Pharmacol, Chicago, IL 60612 USA
[2] Northwestern Univ, Sch Med, Dept Mol Biol & Pharmacol, Chicago, IL 60611 USA
关键词
transcytosis; endocytosis; caveolae; microvascular endothelial cells; albumin permeability;
D O I
10.1083/jcb.150.5.1057
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
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 I-125-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(alpha i) carboxyl-terminal peptide inhibited endothelial I-125-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(alpha i) with the caveolin-1, whereas dn-Src inhibited G(alpha 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.
引用
收藏
页码:1057 / 1069
页数:13
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