In search of the cellular site of growth hormone (GH)-binding protein cleavage from the rabbit GH receptor.
Transfection of Chinese hamster ovary cells with rabbit GH receptor (GHR) complementary DNA resulted in high expression of cellular GHR as well as markedly time- and temperature-dependent secretion of soluble GH-binding protein (GHBP) into the culture medium. In the present study, these cells were used as an in vitro model system to examine GHBP secretion in relation to GHR internalization, degradation, recycling, and biosynthesis. Incubation for 20 h with the lysosomotropic agents NH4Cl and monensin inhibited GH internalization and reduced cell surface GHR, whereas no significant effect on the level of secreted GHBP was observed. Cytochalasin B, a microfilament-disrupting agent, reduced the GHR level, but GHBP was not affected. Colchicine, a microfilament depolymerization agent, had no effect on the GHR level; however, it stimulated GHBP secretion approximately 2-fold. Brefeldin A (5 micrograms/ml), a transport blocker, incubated for 15-180 min resulted in a time-dependent decline in GHR, whereas no significant modulation effect on GHBP was apparent. The capacity of these cells to synthesize and incorporate GHR at the plasma membrane in relation to the generation of soluble GHBP was obtained by destruction of cell surface GHR by mild trypsinization and subsequently monitoring the rate of recovered GHR and GHBP. The rate of reappearance of GHR and GHBP was rapid, being observed within 1 h, whereas full recovery occurred within 2 and 3 h, respectively. The recovery was completely blocked by cycloheximide and brefeldin A. NH4Cl and monensin reduced GHR restoration by about 50%, but the recovery of GHBP was not affected. These data emphasize the importance of lysosomes and vesicular traffic in the regulation of secreted GHBP that might be derived from the internalized GHR and may provide insight into a better understanding of the cleavage process of GHBP from GHR.
Amit
T
T
Department of Pharmacology, Bruce Rappaport Faculty of Medicine, Technion, Israel.
Bick
T
T
Youdim
M B
MB
Hochberg
Z
Z
D000818:Animals; D020126:Brefeldin A; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology; D004734:Energy Metabolism; D008247:Lysosomes / Q000502:physiology; D011817:Rabbits; D011986:Receptors, Somatotropin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D002352:Carrier Proteins; D003517:Cyclopentanes; D011986:Receptors, Somatotropin; D020126:Brefeldin A; D014357:Trypsin; C051815:somatotropin-binding protein