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J. Biol. Chem., Vol. 281, Issue 39, 28584-28595, September 29, 2006

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Human Receptors Patched and Smoothened Partially Transduce Hedgehog Signal When Expressed in Drosophila Cells^*

Matthieu De Rivoyre¹, Laurent Ruel¹, Markku Varjosalo^¶, Agnès Loubat^||, Michel Bidet, Pascal Thérond², and Isabelle Mus-Veteau³

From the Laboratoire de Physiologie Cellulaire et Moléculaire, CNRS Unité Mixte de Recherche (UMR) 6548, Université de Nice-Sophia Antipolis, Parc Valrose 06108 Nice Cedex 2, France, Institut of Signaling, Developmental Biology, and Cancer Research, CNRS UMR 6543, Centre de Biochimie, Parc Valrose, 06108 Nice Cedex 02, France, ^¶Biomedicum, Rm. B502b, Haartman Institute, Department of Virology, P.O. Box 63 (Haartmaninkatu 8), University of Helsinki, Helsinki FIN-00014, Finland, and ^||INSERM U364, Ave. de Valombrose, Faculte de Medecine, 06107 Nice Cedex 02, France

In humans, dysfunctions of the Hedgehog receptors Patched and Smoothened are responsible for numerous pathologies. However, signaling mechanisms involving these receptors are less well characterized in mammals than in Drosophila. To obtain structure-function relationship information on human Patched and Smoothened, we expressed these human receptors in Drosophila Schneider 2 cells. We show here that, as its Drosophila counterpart, human Patched is able to repress the signaling pathway in the absence of Hedgehog ligand. In response to Hedgehog, human Patched is able to release Drosophila Smoothened inhibition, suggesting that human Patched is expressed in a functional state in Drosophila cells. We also provide experiments showing that human Smo, when expressed in Schneider cells, is able to bind the alkaloid cyclopamine, suggesting that it is expressed in a native conformational state. Furthermore, contrary to Drosophila Smoothened, human Smoothened does not interact with the kinesin Costal 2 and thus is unable to transduce the Hedgehog signal. Moreover, cell surface fluorescent labeling suggest that human Smoothened is enriched at the Schneider 2 plasma membrane in response to Hedgehog. These results suggest that human Smoothened is expressed in a functional state in Drosophila cells, where it undergoes a regulation of its localization comparable with its Drosophila homologue. Thus, we propose that the upstream part of the Hedgehog pathway involving Hedgehog interaction with Patched, regulation of Smoothened by Patched, and Smoothened enrichment at the plasma membrane is highly conserved between Drosophila and humans; in contrast, signaling downstream of Smoothened is different.

Received for publication, December 5, 2005 , and in revised form, July 5, 2006.

^* This work was supported by grants from the "Federation pour la Recherche sur le Cerveau" and the Centre National de la Recherche Scientifique (to I. M.-V. and P. T.), by the Ligue Nationale Contre le Cancer (to P. T.), and by the European PCRD6 program (to I. M.-V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ These two authors contributed equally to this work.

² To whom correspondence may be addressed. Tel.: 33-4-92-07-64-46; Fax: 33-4-92-07-64-32; E-mail: Pascal.THEROND{at}unice.fr. ³ To whom correspondence may be addressed. Tel.: 33-4-92-07-68-71; Fax: 33-4-92-07-68-50; E-mail: Isabelle.Mus-Veteau{at}unice.fr.

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