Pe. Gleizes et al., BASIC FIBROBLAST GROWTH-FACTOR (FGF-2) IS ADDRESSED TO CAVEOLAE AFTERBINDING TO THE PLASMA-MEMBRANE OF BHK CELLS, European journal of cell biology, 71(2), 1996, pp. 144-153
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.