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 stud ied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azid obenzoate (HSAB), iodinated, and used to visualize intracellular FGF-2 -affinity-labeled molecules after internalization at 37 degrees C. Iod inated HSAB-FGF-2 maintained the properties of natural FGF-2 such as a ffinity for heparin, binding to Bek and FIg receptors, interaction wit h high- and low-affinity binding sites, and reinitiating of DNA synthe sis in CCL39 cells. Affinity-labeling experiments at 4 degrees C with I-125-HSAB-FGF-2 led to the detection of several FGF-cell surface comp lexes with apparent molecular mass of 80, 100, 125, 150, 170-180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel e lectrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130-16 0 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with I-125-HSAB- FGF-2 at 4 degrees C and then washed, were shifted to 37 degrees C, ir radiation of the internalized labeled FGF-2 led to detection of a simi lar but fainted profile with one major specific band at 80 kDa. Hepari tinase II treatment of the cells reduced binding of I-125-HSAB-FGF-2 t o 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. (C) 199 6 Wiley-Liss, Inc.