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J. Biol. Chem., Vol. 277, Issue 41, 38700-38708, October 11, 2002
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From the A growing body of evidence indicates a close
relationship between tyrosine kinase receptor trafficking and
signaling. Biochemical and molecular analyses of the expression, fate,
and kinetics of membrane trafficking of the nerve growth factor (NGF)
receptor TrkA were performed in PC12 cells. Pulse-chase experiments
indicate that TrkA is synthesized as a 110-kDa
N-glycosylated precursor that leads to the mature 140-kDa
form of the receptor with a half-life of conversion of ~24 ± 0.5 min. Neuraminidase digestion shows that modification of the
carbohydrate moiety of the receptor by sialylation occurs
during maturation. The 140-kDa form is rapidly translocated to the cell
surface as assessed by cell surface biotinylation performed on intact
PC12 cells. Mature receptor half-life is ~138 ± 4 min and is
shortened to 86 ± 8 min by NGF treatment. Flow cytometric
analysis indicates that NGF induces clearing of this receptor from the
cell surface within minutes of treatment. The addition of NGF decreases
the half-life of cell surface gp140TrkA from
100 to 35 min and leads to enhanced lysosomal degradation of the
receptor. The process of NGF-induced TrkA internalization is clearly
affected by interfering with ligand binding to p75NTR. An
analysis of receptor activation kinetics also shows that receptor
signaling primarily takes place from an intracellular location.
Together, these data show that the primary effect of NGF treatment is a
p75NTR-modulated decrease in TrkA transit time at the cell surface.
Molecular Kinetics of Nerve Growth Factor Receptor
Trafficking and Activation*
§¶,§¶,
**, and
Differentiation and Cell Cycle Group,
Laboratoire de Biologie Moleculaire et Cellulaire, CNRS UMR 5665, Ecole
Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon
Cedex 07, France and the Howard Hughes Medical Institute,
University of California, San Francisco, California 94143
*
This work was supported by grants from the Ligue Nationale
Contre le Cancer, the committee of the Ligue from the Rhône, the Rhône-Alpes Region, the Association for Research against Cancer (ARC), and the Fondation de France.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.:
334-7272-8196; Fax: 334-7272-8080; E-mail: bbrudkin@ens-lyon.fr.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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