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 number 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 hGH-induced hGHR was markedly inhibited by reagents that raise the internal pH of acidic organella and partially by protease inhibitors. These results suggest that hGH stimulation results in degradation of internalized hGHRs, where proteases in acidic compartments such as lysosomes 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 internalization at 10 nM hGH. A similar relationship was shown in the hGHR degradation. The fact that the ligands in excess gave reduced internalization and degradation supports the idea that dimerization of hGHRs on the cell surface through the bivalent ligand hGH is required for their internalization and subsequent degradation. Following hGH stimulation, several hGHR-associated proteins including JAK2 were phosphorylated. These phosphorylations were inhibited by pretreatment with a protein kinase 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 results suggest that staurosporine-sensitive phosphorylation is not required for the hGHR internalization, but the phosphorylation is involved in the degradation of hGHR.
Saito
Y
Y
Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Tokyo, Japan.
Teshima
R
R
Yamazaki
T
T
Ikebuchi
H
H
Sawada
J
J
D000470:Alkaloids / Q000494:pharmacology; D002460:Cell Line; D013006:Growth Hormone / Q000494:pharmacology*; D006801:Humans; D007163:Immunosorbent Techniques; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D011986:Receptors, Somatotropin / Q000378:metabolism*; D019311:Staurosporine
D000470:Alkaloids; D010710:Phosphates; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D011986:Receptors, Somatotropin; D013006:Growth Hormone; D011494:Protein Kinases; D019311:Staurosporine