GP60 ACTIVATION MEDIATES ALBUMIN TRANSCYTOSIS IN ENDOTHELIAL-CELLS BYTYROSINE KINASE-DEPENDENT PATHWAY

We investigated the function of gp60, an endothelial cell membrane 60- kDa albumin-binding protein localized in caveolae, and the mechanism o f its activation in regulating endothelial permeability of albumin, Gp 60 organization on the bovine pulmonary microvessel endothelial cell ( BPMVEC) surface was punctate as shown by immunofluorescence using an a nti-gp60 antibody (Ab) conjugated with bisfunctional, N-hydroxysuccini midyl fluorophore (Cy3), Addition of a secondary Ab to anti-gp60 Ab-tr eated BPMVEC induced cross-linking of gp60 as evident by increased siz e of fluorescent particles and cell surface gp60 clustering, Gp60 cros s-linking also produced 2-3-fold increases in the endothelial cell upt ake and the luminal to abluminal permeability of I-125-albumin as well as the fluid-phase tracer, horseradish peroxidase, The increased tran sendothelial permeability of macromolecules was the result of transcyt osis as it was not associated with an increase in the paracellular pat hway, Incubation of anti-gp60 Ab with BPMVEC at 37 degrees C caused in ternalization of gp60, and thereby reduced the uptake of the macromole cules. 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 acti vation 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) inhibito rs, herbimycin A and genistein, prevented gp60-activated macromolecule uptake and transcytosis in a concentration-dependent manner, indicati ng the functional significance of the PTK pathway in activating albumi n transcytosis, These findings indicate that activation of gp60 stimul ates the Src PTK signaling pathway, and thus regulates the transcytosi s of albumin across the endothelial cell monolayer.