Visualization of several binding sites for basic fibroblast growth factor (FGF-2) on fibroblasts by photoaffinity labeling: evidence for intracellular complexes.
The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37 degrees C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Fig receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4 degrees C with 125I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170-180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130-160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with 125I-HSAB-FGF-2 at 4 degrees C and then washed, were shifted to 37 degrees C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of 125I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex.
Gannoun-Zaki
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Laboratoire de Recherche sur la Croissance Cellulaire, la Réparation et la Régénération Tissulaires, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, MESR 415, Université de Paris XII, France.
Pieri
I
I
Badet
J
J
Barritault
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D
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001665:Binding Sites; D001692:Biological Transport; D001921:Brain; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents; D004247:DNA / Q000096:biosynthesis; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000737:chemistry* / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000378:metabolism; D011133:Polysaccharide-Lyases; D011509:Proteoglycans / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis* / Q000378:metabolism; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor / Q000032:analysis* / Q000378:metabolism; D013388:Succinimides
D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D004247:DNA; D016222:Fibroblast Growth Factor 2; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011133:Polysaccharide-Lyases; D011509:Proteoglycans; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor; D013388:Succinimides; C029534:hydroxysuccinimidyl-4-azidobenzoate; C021349:heparitinsulfate lyase; C019358:disuccinimidyl suberate