EVIDENCE THAT BOTH RECEPTOR-SULFATE AND HEPARAN-SULFATE PROTEOGLYCAN-BOUND BASIC FIBROBLAST GROWTH-FACTOR ARE INTERNALIZED BY CULTURED IMMATURE LEYDIG-CELLS

The present studies examined how I-125-labeled basic fibroblast growth factor (bFGF) bound to high affinity receptors and with lower affinity to heparan sulfate proteoglycans (HSPG) of cultured immature rat Leydig cells was processed. Following incubation for 2 h at 4 degrees C with I-125-bFGF, cells were washed to remove unbound radioactivity. Fresh medium was added, and cells were incubated at 4 degrees and/or 37 degrees C. At time zero and at specific intervals over the next 6 h, the incubation medium was saved and cells washed to quantitate I-125-bFGF released into the medium, associated with HSPG of the cell surface or extracellular matrix (radioactivity released by washing cells with 2 M NaCl, pH 7.4), associated with cell surface receptors (radioactivity released by washing cells with 2 M NaCl, pH 4.0) or internalized (radioactivity resistant to high salt and acid washes, and solubilized with 0.5 M NaOH). Radioactivity released into the initial medium and the pooled washes was further divided into a trichloroacetic acid (TCA)-precipitated form (radioactivity precipitated by 10% TCA) and a TCA-soluble form (radioactivity remaining in the TCA supernatant). I-125-bFGF associated with both HSPG and surface receptors declined progressively during the first 4 h of incubation before stabilizing when cells were transferred to 37 degrees C. These declines were associated with a corresponding increase in intracellular I-125-bFGF. These changes were blocked by maintaining cells at 4 degrees C. The majority of internalized I-125-bFGF appeared to originate from the HSPG-bound fraction as there was a greater decline in HSPG-associated radioactivity and most of the increase in internalized radioactivity could be blocked by the inclusion of 10 mu g/ml heparin (which mainly blocks I-125-bFGF binding to HSPG but not to high affinity receptors) during the initial incubation with I-125-bFGF for 2 h at 4 degrees C. Furthermore, HSPG-mediated internalization appeared to have two components: the major fraction was blocked by the inclusion of 10 mu g/ml heparin, while a heparin-resistant fraction, appeared to be closely linked both quantitatively and temporarily to receptor-mediated internalization. A minor fraction of internalized I-125-bFGF waS metabolized in lysosomes, as the inclusion of 50 mu M chloroquine during the 6 h incubation at 37 degrees C inhibited most of the increase in TCA-soluble radioactivity appearing in the incubation medium. Exposure of cultured cells to increasing concentrations of bFGF (0.1-10 ng/ml) for similar to 16 h decreased bFGF receptor levels in a dose-dependent manner, suggesting that internalization, in part, is associated with down-regulation of bFGF receptors.