Regulation of cellular rabbit growth hormone (GH) receptor and GH-binding protein generation in vitro.
In rabbits and probably in man, GH-binding protein (GHBP) is generated from proteolysis of GH receptor (GHR). The present study describes the modulation of spontaneous release of GHBP into the culture medium in relation to cellular GH receptor (GHR) in Chinese hamster ovary cells transfected with rabbit GHR complementary DNA. Secretion of GHBP (approximately 50K protein) from these cells was dependent on time, percentage of FCS, temperature, and protein synthesis. GHBP was detected in the medium at 30 min, and a linear increase was observed over the next 4 h. GHBP release was reduced by low incubation temperature, suggesting that GHBP cleavage is an energy-requiring mechanism. N-Ethylmaleimide (500 microM for 30 min at 30 C) markedly increased GHBP secretion, matched by a corresponding decrease in GHR. However, the lack of effect of N-ethyl-maleimide observed at 4 C further confirms the temperature dependence of GHBP release. We have attempted to characterize the GHBP release protease with a number of recognized protease inhibitors. Benzamidine (10 mM) was the only protease inhibitor that reduced GHBP release; however, it also reduced the cellular GHR level. Cycloheximide (20 micrograms/ml) caused a parallel disappearance of cellular GHR and secreted GHBP with a half-life of about 50 min, but increased GHR messenger RNA expression (superinduction). Indeed, 4 h after removal of cycloheximide, GHR and GHBP were increased by 181% and 369%, respectively, compared to the control value. In summary, Chinese hamster ovary cells expressing rabbit GHR provide a useful cellular model system for studies on the mechanism of GHBP generation from GHR and its physiological importance.
Bick
T
T
Department of Pharmacology, Bruce Rappaport Faculty of Medicine, Technion, Israel.
Amit
T
T
Mansur
M
M
Bar-Am
O
O
Youdim
M B
MB
Hochberg
Z
Z
D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000096:biosynthesis*; D006224:Cricetinae; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D011480:Protease Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011817:Rabbits; D011986:Receptors, Somatotropin / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins; D014162:Transfection
D002352:Carrier Proteins; D011480:Protease Inhibitors; D011500:Protein Synthesis Inhibitors; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D005033:Ethylmaleimide; C051815:somatotropin-binding protein