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J Biol Chem, Vol. 275, Issue 1, 557-564, January 7, 2000
From the Department of Pathology, Division of Cell Biology and
Immunology, University of Utah, Salt Lake City, Utah 84132
Ligands that bind to the epidermal growth factor
(EGF) receptor are initially synthesized as integral membrane proteins
that are released from the cell surface by regulated proteolysis. To study the role of the membrane-anchoring domain in ligand release, we
made two artificial ligands. The first possessed the membrane-anchoring domain from EGF whereas the second had the corresponding domain from
heparin binding EGF-like growth factor (HB-EGF). Both ligands lacked
amino-terminal extensions, and were epitope-tagged at the carboxyl
terminus. Following stable expression in human mammary epithelial
cells, their cellular localization and rate of proteolytic release were
examined. We found that constructs with the membrane-anchoring domain
from EGF were found primarily at the cell surface and displayed a
relatively high rate of constitutive release. Constructs with the
HB-EGF membrane-anchoring domain displayed a higher internalized fraction and a very low rate of constitutive release. The two ligand
constructs also displayed different patterns of stimulated release.
Proteolysis of the chimera with the HB-EGF membrane-anchoring domain
was stimulated by activation of protein kinase C, but release of EGF
from constructs with the EGF membrane-anchoring domain was insensitive
to this. Calcium ionophores, calmodulin antagonists, and tyrosine
phosphatase inhibitors stimulated the release of both ligands.
Furthermore, the release of the two constructs showed different
sensitivity to metalloprotease inhibitors. Despite a large
fold-increase in ligand proteolysis following cell stimulation, only a
small fraction of total cell-associated ligand was released per hour.
Our results show that the membrane-anchoring domain of EGF-like ligands
can specify both their localization and proteolytic processing. The
structures of the membrane-anchoring region of this class of ligands
can thus regulate their activity.
Trafficking and Proteolytic Release of Epidermal Growth
Factor Receptor Ligands Are Modulated by Their Membrane-anchoring
Domains*
*
This work was supported by Natonal Science Foundation Grant
BES-9727145 and National Institutes of Health Grant PO1-HD28528.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. Tel.: 801-581-5967;
Fax: 801-581-4517; E-mail: Wiley@path.med.utah.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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