LIGAND-INDUCED INTERNALIZATION AND PHOSPHORYLATION-DEPENDENT DEGRADATION OF GROWTH-HORMONE RECEPTOR IN HUMAN IM-9 CELLS

The human growth hormone (hGH) induced a marked reduction in the numbe r of human growth hormone receptors (hGHR) within 60 min, as assessed by immunoblotting of the crude membrane fraction from human IM-9 cells , without an increase in soluble forms of hGHR. The disappearance of h GH-induced hGHR was markedly inhibited by reagents that raise the inte rnal pH of acidic organella and partially by protease inhibitors. Thes e results suggest that hGH stimulation results in degradation of inter nalized hGHRs, where proteases in acidic compartments such as lysosome s may be involved. The relationship between the hGH concentration and the number of residual cell surface hGHRs 60 min after hGH stimulation yielded a curve with an inverted bell shape showing maximum internali zation at 10 nM hGH. A similar relationship was shown in the hGHR degr adation. The fact that the ligands in excess gave reduced internalizat ion and degradation supports the idea that dimerization of hGHRs on th e cell surface through the bivalent ligand hGH is required for their i nternalization and subsequent degradation. Following hGH stimulation, several hGHR-associated proteins including JAK2 were phosphorylated. T hese phosphorylations were inhibited by pretreatment with a protein ki nase inhibitor, staurosporine. The hGHR internalization, however, was not markedly affected by the inhibitor. In contrast, the staurosporine inhibited the degradation of hGHR in a dose-dependent manner. These r esults suggest that staurosporine-sensitive phosphorylation is not req uired for the hGHR internalization, but the phosphorylation is involve d in the degradation of hGHR.