Doublecortin Interacts with  Subunits of Clathrin Adaptor Complexes in the Developing Nervous System Gae ¨lle Friocourt,* Philippe Chafey,* Pierre Billuart,* Annette Koulakoff, † Marie-Claude Vinet,* Bruce T. Schaar, ‡ Susan K. McConnell, ‡ Fiona Francis,* and Jamel Chelly* ,1 *Laboratoire de Ge ´ne ´tique et Physiopathologie des retards mentaux, ICGM, INSERM, CHU Cochin, 24, rue du Faubourg Saint Jacques, 75014 Paris, France; † Laboratoire de Biochimie cellulaire, CNRS UPR 9065, Colle `ge de France, 11 Place M. Berthelot, 75005 Paris, France; and ‡ Department of Biological Sciences, Stanford University, Stanford, California 94305 Doublecortin is a microtubule-associated protein re- quired for normal corticogenesis in the developing brain. We carried out a yeast two-hybrid screen to identify in- teracting proteins. One of the isolated clones encodes the 1 subunit of the adaptor complex AP-1 involved in clath- rin-dependent protein sorting. We found that Doublecor- tin also interacts in yeast with 2 from the AP-2 complex. Mutagenesis and pull-down experiments showed that these interactions were mediated through a tyrosine- based sorting signal (YLPL) in the C-terminal part of Dou- blecortin. The functional relevance of these interactions was suggested by the coimmunoprecipitation of Dou- blecortin with AP-1 and AP-2 from mouse brain extracts. This interaction was further supported by RNA in situ hybridization and immunofluorescence studies. Taken to- gether these data indicate that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in vivo and are consistent with a potential involvement of Doublecor- tin in protein sorting or vesicular trafficking. INTRODUCTION During embryonic development of the mammalian brain, neuronal migration is one of the key processes that leads to the formation of the cerebral cortex. Neu- rons migrate long distances from the neuroepithelium, where they arise, to the cortical plate where they form the complex laminar structures of the cortex. Postmi- totic neurons exit from the neuroepithelium in waves, then migrate tangentially, or radially, along glial fibres to the cortical plate. They settle in six layers, which are formed sequentially from the deepest to the most su- perficial (“inside-out”) so that each new wave of neu- rons migrates past the previously formed ones (Mc- Connell, 1995; Caviness et al., 1997). The molecular events that drive migrating neuronal cells to their final destinations as well as the environmental cues that regulate their spatial position are not yet fully under- stood. However, the genetic analysis of mammalian neuronal migration disorders suggests the involvement of several pathways, which can be more and more considered as complementary. Mutations in LISI (Reiner et al., 1993), doublecortin (des Portes et al., 1998; Gleeson et al., 1998), cdk5 (Oshima et al., 1996), p35 (Chae et al., 1997), filamin 1 (Fox et al., 1998), reelin (D’Arcangelo et al., 1995), mdab1 (Howell et al., 1997; Ware et al., 1997; Sheldon et al., 1997), VLDLR and ApoER2 (Trommsdorff et al., 1999) have been found to lead to specific phenotypes in which corticogenesis is disrupted. The strikingly similar phenotype of lissen- cephaly (Pinard et al., 1994) between humans with LIS1 mutations and those with doublecortin mutations along with findings demonstrating that both proteins are mi- crotubule-associated proteins (MAPs) (Sapir et al., 1997; Francis et al., 1999; Gleeson et al., 1999; Horesh et al., 1999) suggest that LIS1 and Doublecortin may function through similar mechanisms in neuronal migration. Moreover, recent studies showed that reelin, mDab1, ApoER2, and VLDLR are potentially involved in the 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (33) 1 44 41 24 21. E-mail: chelly@icgm.cochin. inserm.fr. Molecular and Cellular Neuroscience 18, 307–319 (2001) doi:10.1006/mcne.2001.1022, available online at http://www.idealibrary.com on MCN 1044-7431/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 307