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 (bFGF or FGF-2) is internalized by two pathways, after binding to either FGF tyrosine kinase receptors or to heparan sulfate proteoglycans (HSPG). 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 to HSPG only and was detectable by electron microscopy. bFGF-HRP association 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 able to displace [125I]bFGF from its high-affinity binding sites, and the dissociation constant of its binding to ABAE cells was estimated at 3 nM. Time-course experiments were performed to follow bFGF-HRP endocytosis in ABAE cells. bFGF-HRP was found to enter the cell after binding 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 endocytosis, bFGF-HRP was detected in pleiomorphic tubulovesicular and tubulocisternal early endosomes. Multivesicular bodies contained diaminobenzidine (DAB) precipitate after 5 to 15 min, but lysosomes were not labeled before 1 h, indicating a delayed transfer from late endosomes to lysosomes. Labeling was never detected in the nucleus, even after intensification of the DAB reaction product by silver-gold enhancement. Similar endocytic pathways and intracellular locations were observed in other endothelial and non-endothelial cell types. These results suggest that bFGF associated to HSPG can enter the cell via several pathways and follows mainly a degradative route.
Gleizes
P E
PE
Laboratoire de Biologie Moléculaire Eucaryote, CNRS UPR 9006, Toulouse/France.
Noaillac-Depeyre
J
J
Amalric
F
F
Gas
N
N
D000818:Animals; D002417:Cattle; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry; D008854:Microscopy, Electron; D011509:Proteoglycans / Q000378:metabolism*
D016222:Fibroblast Growth Factor 2; D006497:Heparan Sulfate; D006735:Horseradish Peroxidase; D011509:Proteoglycans