BASIC FIBROBLAST GROWTH-FACTOR (FGF-2) IS ADDRESSED TO CAVEOLAE AFTERBINDING TO THE PLASMA-MEMBRANE OF BHK CELLS

bFGF endocytosis in BHK cells was examined by electron microscopy usin g a conjugate of recombinant human bFGF and digoxigenin (bFGF-DIG). Th is probe keeps the biological activity of non-labeled bFGF and can be readily detected with anti-digoxigenin antibodies (Gleizes et al., Ana l, Biochem, 219, 360-357 (1994)). Time-course studies of bFGF-DIG endo cytosis were performed by incubating BHK cells at 4 degrees C in the p resence of first 20 ng/ml bFGF-DIG and then antidigoxigenin antibodies absorbed onto 10-nm gold particles. A semiquantitative study revealed that caveolae were the main endocytic pathway of bFGF-DIG in these ce lls, whereas clathrin-coated pits were scarcely labeled. After occurri ng in caveolae, bFGF-DIG was sequentially detected in tubulovesicular early endosomes, multivesicular late endosomes, and lysosomes. Under t he same conditions, low density lipoprotein (LDL)-gold was seen enteri ng the cell exclusively, through clathrin-coated pits. However, LDL-go ld and bFGF-DIG were colocalized, at least in part, in common endosoma l structures. Pretreatment of the cells with phosphatidylinositol-phos pholipase C reduced the proportion of membrane-bound bFGF-DIG in caveo lae, but did not inhibit bFGF-DIG presence in caveolae. These data sug gest that bFGF enters into BHK cells through caveolae and is then shut tled into a degradative pathway similar to that of LDL.