BASIC FIBROBLAST GROWTH-FACTOR (FGF-2) INTERNALIZATION THROUGH THE HEPARAN-SULFATE PROTEOGLYCANS-MEDIATED PATHWAY - AN ULTRASTRUCTURAL APPROACH

Biochemical studies have shown that basic fibroblast growth factor (bF GF or FGF-2) is internalized by two pathways, after binding to either FGF tyrosine kinase receptors or to heparan sulfate proteoglycans (HSP G). To get insights on the HSPG-mediated pathway, we have examined by electron microscopy the intracellular route of bFGF-HRP, a monovalent conjugate of bFGF and horseradish peroxidase which was found to bind t o HSPG only and was detectable by electron microscopy. bFGF-HRP associ ation to adult bovine aortic endothelial (ABAE) cells or baby hamster kidney (BHK) cells was inhibited by a high molar excess of native bFGF , a 2 M NaCl wash at neutral pH, heparin and heparan sulfate, but not by chondroitin 4-sulfate or chondroitin 6-sulfate. bFGF-HRP was not ab le to displace [I-125]bFGF from its high-affinity binding sites, and t he dissociation constant of its binding to ABAE cells was estimated at 3 nM. Timecourse experiments were performed to follow bFGF-HRP endocy tosis in ABAE cells. bFGF-HRP was found to enter the cell after bindin g to the plasma membrane or extracellular matrix. On the cell surface, the probe accumulated in noncoated flask-shaped invaginations and in caveolae rather than in clathrin-coated pits. Immediately after endocy tosis, bFGF-HRP was detected in pleiomorphic tubulovesicular and tubul ovesicular early endosomes. Multivesicular bodies contained diaminoben zidine (DAB) precipitate after 5 to 15 min, but lysosomes were not lab eled before 1 h, indicating a delayed transfer from late endosomes to lysosomes. Labeling was never detected in the nucleus, even after inte nsification of the DAB reaction product by silver-gold enhancement. Si milar endocytic pathways and intracellular locations were observed in other endothelial and non-endothelial cell types. These results sugges t that bFGF associated to HSPG can enter the cell via several pathways and follows mainly a degradative route.