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 th e 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 (sim ilar to 50K protein) from these cells was dependent on time, percentag e 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 mu M for 30 min at 30 C) markedly increased GHBP secretion, match ed by a corresponding decrease in GHR. However, the lack of effect of N-ethylmaleimide observed at 4 C further confirms the temperature depe ndence of GHBP release. We have attempted to characterize the GHBP rel ease protease with a number of recognized protease inhibitors. Benzami dine (10 mM) was the only protease inhibitor that reduced GHBP release ; however, it also reduced the cellular GHR level. Cycloheximide (20 m u g/ml) caused a parallel disappearance of cellular GHR and secreted G HBP with a half-life of about 50 min, but increased GHR messenger RNA expression (superinduction). Indeed, 4 h after removal of cycloheximid e, GHR and GHBP were increased by 181% and 369%, respectively, compare d to the control value. In summary, Chinese hamster ovary cells expres sing rabbit GHR provide a useful cellular model system for studies on the mechanism of GHBP generation from GHR and its physiological import ance.