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J. Biol. Chem., Vol. 276, Issue 6, 3778-3784, February 9, 2001
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From the Department of Cell Biology, University Medical Center
Utrecht and Institute of Biomembranes, 3584CX Utrecht,
The Netherlands
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-
Growth Hormone Receptor Ubiquitination Coincides with Recruitment
to Clathrin-coated Membrane Domains*
-cyclodextrin inhibited endocytosis at the stage of
coated vesicle formation. Growth hormone addition to
methyl--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.
*
This work was supported by Netherlands Organization for
Scientific Research Grant NWO-902-23-lg2) and European Union Network Grant ERBFMRXCT96-0026.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Cell Biology,
University Medical Center Utrecht and Institute of Biomembranes, Heidelberglaan 100, AZU-G02.525, 3584CX Utrecht, The Netherlands. Tel.:
31-30-250-6476; Fax: 31-30-254-1797; E-mail: strous@med.uu.nl.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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