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Originally published In Press as doi:10.1074/jbc.M202401200 on November 15, 2002

J. Biol. Chem., Vol. 278, Issue 10, 8706-8716, March 7, 2003
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Trafficking of TrkA-Green Fluorescent Protein Chimerae during Nerve Growth Factor-induced Differentiation*

Jérôme JullienDagger §, Vincent GuiliDagger §, Edmund A. Derrington, Jean-Luc Darlix, Louis F. Reichardt||**, and Brian B. RudkinDagger Dagger Dagger

From the Dagger  Differentiation and Cell Cycle Group, Laboratoire de Biologie Moleculaire et Cellulaire, UMR 5665 CNRS, Ecole Normale Supérieure de Lyon and  LaboRetro, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon Cedex 07, France and || Howard Hughes Medical Institute, University of California, San Francisco, California 94143

A chimera of the nerve growth factor (NGF) receptor, TrkA, and green fluorescent protein (GFP) was engineered by expressing GFP in phase with the carboxyl terminus of TrkA. TrkA-GFP becomes phosphorylated on tyrosine residues in response to NGF and is capable of initiating signaling cascades leading to prolonged MAPK activation and differentiation in PC12 nnr5 cells. TrkA constructs, progressively truncated in the carboxyl-terminal domain, were prepared as GFP chimerae in order to identify which part of the receptor intracellular domain is involved in its trafficking. Immunofluorescence observations show that TrkA-GFP is found mainly in cell surface membrane ruffles and in endosomes. Biochemical analysis indicated that the cytoplasmic domain of TrkA is not necessary for correct maturation and cell surface translocation of the receptor. An antibody against the extracellular domain of TrkA (RTA) was used as ligand to stimulate internalization and phosphorylation of TrkA. Co-localization studies with anti-phosphorylated TrkA antibodies support a role for such complexes in the propagation of signaling from the cell surface, resulting in the activation of TrkA in areas of the endosome devoid of receptor-ligand complexes. Confocal time-lapse analysis reveals that the TrkA-GFP chimera shows highly dynamic trafficking between the cell surface and internal locations. TrkA-positive vesicles were estimated to move 0.46 ± 0.09 µm/s anterograde and 0.48 ± 0.07 µm/s retrograde. This approach and the fidelity of the biochemical properties of the TrkA-GFP demonstrate that real-time visualization of trafficking of tyrosine kinase receptors in the presence or absence of the ligand is feasible.

* 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.

§ Supported by grants from the Ministère de l'Enseignement Supérieur et de la Recherche and fellowships from the Association for Research against Cancer.

** An investigator of the Howard Hughes Medical Institute; work in his laboratory was also supported by United States Public Health Service Grant NS 16033.

Dagger Dagger To whom correspondence should be addressed. Tel.: 334-72-72-81-96; Fax: 334-72-72-80-80; E-mail: bbrudkin@ens-lyon.fr.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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