Internalization and Limited Processing of Basic Fibroblast Growth Factor on Chinese Hamster Lung Fibroblasts

Abstract

Using either acidic (pH 2.5) or trypsic treatments, we demonstrated that ¹²⁵I-labeled basic Fibroblast Growth Factor (¹²⁵I-bFGF) was submitted to an internalization process on responsive Chinese hamster lung fibroblasts (CCL39) at 37°C. Various experiments based on the measurement of cell-associated radioactivity, as well as on research of degradated products of ¹²⁵I-bFGF in cellular supernatants, showed that most of the internalized radioactivity remained intracellularly located after up to 5 hr of incubation. Analyses of this radioactivity by NaDodSO₄-PAGE revealed the presence of labeled peptides issued from the limited processing of the native ¹²⁵I-bFGF form (17 kD) and whose molecular weights were estimated to be 9 and 6 kD. Kinetic experiments indicated that proteolysis of the ¹²⁵I-bFGF began early on incubation (< 30 min) and led to a prolonged preservation of the 9-and 6-kD peptides which were still detectable after 13 hr of incubation. Preincubation of the cells with different lysosomotropic agents completely inhibited the proteolysis, indicating that this event occurred probably in an intracellular acidic compartment. Two enzyme inhibitors, leupeptin and N-α-tosyl-L-lysine chloromethyl ketone (TLCK), were also shown to interfere with the formation of both 9-and 6-kD peptides, thus suggesting a way to control the appearance of these fragments, and hence to determine their potential intracellular role.

Key Words:

• basic Fibroblast Growth Factor
• enzyme inhibitors
• internalization