Cellular processing of growth hormone in IM-9 cells: evidence for exocytosis of internalized hormone

• Published in: Endocrinology (Philadelphia) Vol. 130; no. 4; p. 2037
• Main Authors: Ilondo, M M, Vanderschueren-Lodeweyckx, M, Courtoy, P J, De Meyts, P
• Format: Journal Article
• Language: English
• Published: United States 01.04.1992
• Subjects: Cell Line; Computer Simulation; Electrophoresis, Polyacrylamide Gel; Exocytosis; Growth Hormone - metabolism; Humans; Lymphocytes - metabolism; Receptors, Somatotropin - metabolism
• ISSN: 0013-7227
• DOI: 10.1210/en.130.4.2037

IM-9 cells extensively internalize [125I]human (h) GH at physiological temperatures, yet little is known regarding the final destination of internalized hormone and its receptor. We studied this by first binding [125I]hGH to the cell surface at 4 C, and then following its fate during a subsequent incubation at 30 C in isotope-free medium. Cell-associated radioactivity decreased with time at 30 C, with a biphasic pattern suggestive of a rapid (but minor) and a slow component. The kinetics of the latter were critically influenced by NH4Cl and were abolished at 20 C. Intracellular (acid-resistant) [125I]hGH first increased with time at 30 C until it reached a maximum after 1 h, then declined continuously upon prolonged incubation. The radioactivity released by the cells was recovered in the medium as both trichloroacetic acid-precipitable material and trichloroacetic acid-soluble fragments. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, one major band migrating with an estimated mol wt of 22,000 was identified, presumably corresponding to intact [125I]hGH. These data suggest exocytosis of intact hormone via recycling endosomes and degradation in the lysosomes, respectively. Computer modelling was consistent with two intracellular compartments acting partially in series and probably corresponding to these two organelles. When analyzed by computer curve fitting, this model accurately described the kinetics of [125I]hGH internalization. So, receptor-mediated endocytosis and subsequent exocytosis are part of the GH pathway in IM-9 cells. In as much as they reflect pathways of GH receptors, these processes contribute to receptor down-regulation and could provide an explanation for release into the medium of the high affinity GH-binding protein.