Basic fibroblast growth factor (FGF-2) is addressed to caveolae after binding to the plasma membrane of BHK cells.
bFGF endocytosis in BHK cells was examined by electron microscopy using a conjugate of recombinant human bFGF and digoxigenin (bFGF-DIG). This probe keeps the biological activity of non-labeled bFGF and can be readily detected with anti-digoxigenin antibodies (Gleizes et al., Anal. Biochem. 219, 360-367 (1994)). Time-course studies of bFGF-DIG endocytosis were performed by incubating BHK cells at 4 degrees C in the presence of first 20 ng/ml bFGF-DIG and then antidigoxigenin antibodies adsorbed onto 10-nm gold particles. A semi-quantitative study revealed that caveolae were the main endocytic pathway of bFGF-DIG in these cells, whereas clathrin-coated pits were scarcely labeled. After occurring in caveolae, bFGF-DIG was sequentially detected in tubulovesicular early endosomes, multivesicular late endosomes, and lysosomes. Under the same conditions, low density lipoprotein (LDL)-gold was seen entering the cell exclusively through clathrin-coated pits. However, LDL-gold and bFGF-DIG were colocalized, at least in part, in common endosomal structures. Pretreatment of the cells with phosphatidylinositol-phospholipase C reduced the proportion of membrane-bound bFGF-DIG in caveolae, but did not inhibit bFGF-DIG presence in caveolae. These data suggest that bFGF enters into BHK cells through caveolae and is then shuttled into a degradative pathway similar to that of LDL.
Laboratoire de Biologie Moléculaire Eucaryote, CNRS UPR 9006, Toulouse/France.
P E
PE
Gleizes
J
J
Noaillac-Depeyre
M A
MA
Dupont
N
N
Gas
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000648:ultrastructure; D006224:Cricetinae; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004730:Endothelium, Vascular; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism* / Q000648:ultrastructure; D007668:Kidney; D008077:Lipoproteins, LDL / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D011994:Recombinant Proteins; D013997:Time Factors
D008077:Lipoproteins, LDL; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase