Cell Internalization of the Third Helix of the Antennapedia Homeodomain Is Receptor-independent

doi:10.1074/jbc.271.30.18188

Cell Internalization of the Third Helix of the Antennapedia Homeodomain Is Receptor-independent*

From the ^‡ CNRS URA 1414, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France and
^¶ CNRS URA 493, Chimie Organique et Biologique, Université Pierre et Marie Curie, 4 Place Jussieu, 75005 Paris, France

^∥ To whom correspondence should be addressed. Tel.: 33-1-44-32-37-12; Fax: 33-1-44-32-39-88; E-mail: prochian{at}wotan.ens.fr

Abstract

We have recently reported that a 16-amino acid long polypeptide corresponding to the third helix of the DNA binding domain (homeodomain) of Antennapedia, a Drosophila transcription factor, is internalized by cells in culture (Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) J. Biol. Chem. 269, 10444-10450). The capture of the homeodomain and of its third helix at temperatures below 10°C raised the problem of the mechanism of internalization. The present demonstration, that a reverse helix and a helix composed of D-enantiomers still translocate across biological membranes at 4 and 37°C strongly suggests that the third helix of the homeodomain is internalized by a receptor-independent mechanism. The finding that introducing 1 or 3 prolines in the structure does not hamper internalization also demonstrates that the α-helical structure is not necessary. The data presented are compatible with a translocation process based on the establishment of direct interactions with the membrane phospholipids. The third helix of the homeodomain has been used successfully to address biologically active substances to the cytoplasm and nucleus of cells in culture (Théodore, L., Derossi, D., Chassaing, G., Llirbat, B., Kubes, M., Jordan, P., Chneiweiss, H., Godement, P., and Prochiantz, A. (1995) J. Neurosci. 15, 7158-7167). Therefore, in addition to their physiological implications (Prochiantz, A., and Théodore, L. (1995) BioEssays 17, 39-45), the present results open the way to the molecular design of cellular vectors.

Footnotes

↵^§ The two first authors have contributed equally to the work.
↵* This study was supported by CNRS, Ecole Normale Supérieure and grants from Agence Nationale de la Recherche sur le SIDA and Fondation pour la Recherche Médicale (Sidaction). 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.
↵¹ The abbreviations used are:

AntpHD

Antennapedia homeodomain

HPLC

high performance liquid chromatography

PBS

phosphate-buffered saline

ELISA

enzyme-linked immunosorbent assay

iso

isocratic.
↵² G. Rossi, M. Volovitch, and A. Prochiantz, unpublished observations.
↵³ D. Derossi, M. Thieffry, and A. Prochiantz, unpublished results.
↵⁴ J.-P. Berlose, D. Derossi, A. Brunissen, O. Convert, and G. Chassaing, manuscript in preparation.
- Received November 28, 1995.
- Revision received March 28, 1996.