Growth Hormone Receptor Ubiquitination Coincides with Recruitment to Clathrin-coated Membrane Domains

Peter Van Kerkhof,Martin Sachse,Judith Klumperman,Ger J Strous

doi:10.1074/jbc.m007326200

Peter Van Kerkhof, Martin Sachse + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m007326200

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Abstract

Endocytosis of the growth hormone receptor (GHR) depends on a functional ubiquitin conjugation system. A 10-amino acid residue motif within the GHR cytosolic tail (the ubiquitin-dependent endocytosis motif) is involved in both GHR ubiquitination and endocytosis. As shown previously, ubiquitination of the receptor itself is not required. In this paper ubiquitination of the GHR was used as a tool to address the question of at which stage the ubiquitin conjugation system acts in the process of GHR endocytosis. If potassium depletion was used to interfere with early stages of coated pit formation, both GHR endocytosis and ubiquitination were inhibited. Treatment of cells with methyl-beta-cyclodextrin inhibited endocytosis at the stage of coated vesicle formation. Growth hormone addition to methyl-beta-cyclodextrin-treated cells resulted in an accumulation of ubiquitinated GHR at the cell surface. Using immunoelectron microscopy, the GHR was localized in flattened clathrin-coated membranes. In addition, when clathrin-mediated endocytosis was inhibited in HeLa cells expressing a temperature-sensitive dynamin mutant, ubiquitinated GHR accumulated at the cell surface. Together, these data show that the GHR is ubiquitinated at the plasma membrane, before endocytosis occurs, and indicate that the resident time of the GHR at the cell surface is regulated by the ubiquitin conjugation system together with the endocytic machinery.

Highlights

Clathrin-mediated endocytosis involves the formation of clathrin-coated vesicles from coated pits at the plasma membrane
When clathrin-mediated endocytosis was inhibited in HeLa cells expressing a temperature-sensitive dynamin mutant, ubiquitinated growth hormone receptor (GHR) accumulated at the cell surface
In this study two independent methods were used to inhibit clathrin-mediated endocytosis at the level of clathrin-coated vesicle formation. Both methods inhibited the endocytosis of the GHR, resulting in an accumulation of ubiquitinated receptors at the cell surface

Summary

EXPERIMENTAL PROCEDURES

Materials and Antibodies—Antibody mAb5 recognizing the lumenal part of the GHR was from AGEN Inc. (Parsippany, NJ). For transfection experiments subconfluent cultures of tTA-HeLa cells were washed with phosphate-buffered saline (PBS), detached with trypsin-EDTA, plated on 60-mm dishes, and incubated at the permissive temperature of 32 °C in the absence of tetracycline for 24 h. Cells were grown in 12-well cluster plates, washed with MEM␣ supplemented with 20 mM Hepes, pH 7.4, and 0.1% bovine serum albumin (BSA), and incubated in a water bath. For GHR immunoprecipitations, cells were lysed on ice in 0.3 ml of lysis mix containing 1% Triton X-100, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 2 ␮M carbobenzoxy-L-leucyl-Lleucyl-L-leucinal, and 100 mM phenylmethylsulfonyl fluoride in PBS. For anti-GH immunoprecipitations of GH-GHR complexes, the cells were lysed on ice in a lysis mix containing 1% Triton X-100, 150 mM NaCl, 10% glycerol, 50 mM Tris-HCl, pH 8.0, 10 mM N-ethylmaleimide, and various inhibitors. The frequency of each category was expressed as a percentage of the total number of clathrin-coated structures at the plasma membrane

RESULTS

GHR Ubiquitination Occurs in Coated Pits

DISCUSSION