Proteasomes are involved in the constitutive degradation of growth hormone receptors.
In the mouse Ba/F3-hGHR cell line, which stably expresses human growth hormone receptors (hGHRs), the hGHRs were rapidly degraded in the absence of the ligand. Human growth hormone-binding protein (hGH-BP), a soluble form of hGHR, was released from Ba/F3-hGHR cells, but the hGH-BP release was less than 1% of total hGHRs in the cells. Therefore, the hGH-BP release does not markedly contribute to hGHR degradation in Ba/F3-hGHR cells. The constitutive degradation of hGHRs was inhibited by the proteasome inhibitors MG-132 and clasto-lactacystin beta-lactone, or the vacuolar H+-ATPase inhibitor, bafilomycin A1. hGH-enhanced degradation of hGHRs was also inhibited by MG-132. Moreover, MG-132 inhibited the internalization of hGHRs as assessed by 125I-hGH binding to the cell surfaces. Ubiquitinated hGHRs were detected in the cell lysate and increased by hGH-treatment. Furthermore, MG-132 accumulated the ubiquitinated hGHRs induced by hGH. However, the ratio of ubiquitinated hGHRs to unubiquitinated hGHRs was very small, even with treatment involving both hGH and MG-132. In the hGH-untreated cells, the ubiquitinated hGHRs were weakly detected. However, the ubiquitination of hGHR was not enhanced by MG-132 as a result of immunoblotting. Thus, the ubiquitination of hGHR is unlikely to be involved, at least in the constitutive degradation. Taken together, both the proteasome pathway and endosome/lysosome pathway are involved in the constitutive degradation of hGHRs. Our results also suggest that ubiquitination of the hGHR itself is unlikely to be the trigger of the proteasome-dependent degradation.
Takagi
K
K
Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Setagaya, Tokyo, Japan.
Saito
Y
Y
Sawada
J I
JI
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000502:physiology*; D006801:Humans; D051379:Mice; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism; D011986:Receptors, Somatotropin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D009097:Multienzyme Complexes; D011500:Protein Synthesis Inhibitors; D011956:Receptors, Cell Surface; D011986:Receptors, Somatotropin; D025801:Ubiquitin; D003513:Cycloheximide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex