Proteasomes are involved in the constitutive degradation of growth hormonereceptors

In the mouse Ba/F3-hGHR cell line, which stably expresses human growth horm one receptors (hGHRs), the hGHRs were rapidly degraded in the absence of th e 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 t han 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 inhib itors MG-132 and clasto-lactacystin beta -lactone, or the vacuolar H+-ATPas e inhibitor, bafilomycin A(1).hGH-enhanced degradation of hGHRs was also in hibited by MG-132, Moreover, MG-132 inhibited the internalization of hGHRs as assessed by I-125-hGH binding to the cell surfaces. Ubiquitinated hGHRs were detected in the cell lysate and increased by hGH-t reatment, Furthermore, MG-132 accumulated the ubiquitinated hGHRs induced b y hGH, However, the ratio of ubiquitinated hGHRs to un-ubiquitinated hGHRs was very small, even with treatment involving both hGH and MG-132. In the h GH-untreated cells, the ubiquitinated hGHRs were weakly detected. However, the ubiquitination of hGHR was not enhanced by MG-132 as a result of immuno blotting. Thus, the ubiquitination of hGHR is unlikely to be involved, at l east in the constitutive degradation. Taken together, both the proteasome pathway and endosome/lysosome pathway a re involved in the constitutive degradation of hGHRs, Our results also sugg est that ubiquitination of the hGHR itself is unlikely to be the trigger of the proteasome-dependent degradation.