Gp60 activation mediates albumin transcytosis in endothelial cells by tyrosine kinase-dependent pathway

被引:296
作者
Tiruppathi, C
Song, W
Bergenfeldt, M
Sass, P
Malik, AB
机构
[1] Department of Pharmacology, College of Medicine, University of Illinois, Chicago
[2] Dept. of Pharmacology, M/C 868, University of Illinois at Chicago, Chicago, IL 60612
关键词
D O I
10.1074/jbc.272.41.25968
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We investigated the function of gp60, an endothelial cell membrane 60-kDa albumin-binding protein localized in caveolae, and the mechanism of its activation in regulating endothelial permeability of albumin, Gp60 organization on the bovine pulmonary microvessel endothelial cell (BPMVEC) surface was punctate as shown by immunofluorescence using an anti-gp60 antibody (Ab) conjugated with bisfunctional, N-hydroxysuccinimidyl fluorophore (Cy3), Addition of a secondary Ab to anti-gp60 Ab-treated BPMVEC induced cross-linking of gp60 as evident by increased size of fluorescent particles and cell surface gp60 clustering, Gp60 cross-linking also produced 2-3-fold increases in the endothelial cell uptake and the luminal to abluminal permeability of I-125-albumin as well as the fluid-phase tracer, horseradish peroxidase, The increased transendothelial permeability of macromolecules was the result of transcytosis as it was not associated with an increase in the paracellular pathway, Incubation of anti-gp60 Ab with BPMVEC at 37 degrees C caused internalization of gp60, and thereby reduced the uptake of the macromolecules. Activation of gp60 by either albumin (the gp60 ligand) or gp60 crosslinking induced the phosphorylation of both gp60 and caveolin-1 (the major structural caveolar protein) on tyrosine residues, Gp60 activation also phosphorylated the Src family tyrosine kinases pp60(c-Src) and Fyn. The activated pp60(c-Src) and Fyn co-immunoprecipitated with caveolin-1 in BPMVEC membrane, Protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, prevented gp60-activated macromolecule uptake and transcytosis in a concentration-dependent manner, indicating the functional significance of the PTK pathway in activating albumin transcytosis, These findings indicate that activation of gp60 stimulates the Src PTK signaling pathway, and thus regulates the transcytosis of albumin across the endothelial cell monolayer.
引用
收藏
页码:25968 / 25975
页数:8
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