The monoclonal anti-RanBPM antibody was a kind gift from Dr E

The monoclonal anti-RanBPM antibody was a kind gift from Dr E. L1 mutations, such as hypoplasia of the corticospinal tract and corpus callosum, hypoplasia of the cerebellar vermis and hydrocephalus (Dahme 1997; Cohen 1998; Fransen 1998). The extracellular domain of L1, composed of six Ig domains and five fibronectin type III repeats, is capable of binding to a variety of ligands, including L1 itself, other members of the Ig superfamily, integrins, neuropilin and several extracellular matrix components (for review, see Haspel and Grumet 2003). L1 can bind to ligands in to mediate adhesion or bind to ligands in to function as coreceptors. There is compelling evidence that the L1 cytoplasmic domain (L1CD) is crucial for L1 function. The L1CD is highly conserved and mutations in it cause Mental Retardation Aphasia Shuffling Flurazepam dihydrochloride Gait and Adducted Thumbs (MASA) syndrome (Fransen 1997). The L1CD is phosphorylated by several kinases and phosphorylation appears to regulate L1 function (Wong 1996). L1 is known to activate the extracellular signal-regulated kinase (ERK) pathway and it has been suggested that ERK activation is involved in L1-mediated neurite outgrowth and migration (Schaefer 1999; Schmid 2000). To date, three proteins, ankyrin, adaptor protein-2 (AP-2) and ezrin, have been shown to interact with the L1CD but their relationship to ERK activation is unclear. The ankyrin-binding site on the L1CD (amino acids 1204C1229) couples L1 to the underlying actin cytoskeleton. The interaction with ankyrin seems to mediate the stationary behavior of L1 and may play a critical role in the regulation of L1-mediated adhesion and migration (Gil 2003). L1 can bind to the clathrin adaptor AP-2 through the YRSL motif and this interaction is critical for Mouse monoclonal to CD19 clathrin-mediated L1 endocytosis (Kamiguchi 1998b). AP-2-mediated L1 endocytosis is critical for L1 recycling at the growth cone (Kamiguchi and Lemmon 2000), sorting of L1 to axons in dorsal root ganglion neurons and L1 transcytosis in hippocampal neurons (Kamiguchi and Lemmon 1998; Wisco 2003). The L1CD also binds to ezrin, a member of the ezrin, radixin and moesin family of membraneCcytoskeleton-linking proteins (Dickson 2002), through the YRSL motif and the juxtamembrane region (Cheng 2005). This interaction provides a link between L1 and the actin cytoskeleton and plays a critical role in the regulation of neurite branching (Cheng 2005). As no interactor with the L1CD has a clear relationship to ERK activation, we sought to identify additional L1 binding proteins by performing a yeast two-hybrid screen. We chose the last 28 amino acids of the L1CD as bait because we have previously shown that this region is phosphorylated (Schaefer 1999) but no protein interactions have been reported for this region. We identified RanBPM as an L1-interacting protein. RanBPM was originally cloned because it interacts with RAN, a Ras-like small GTPase, that functions as Flurazepam dihydrochloride a carrier in nuclearCcytoplasmic exchange (Nakamura 1998). Subsequently, a number of studies have identified RanBPM as a binding partner with several unrelated proteins, such as the hepatocyte growth factor (HGF) receptor Met (Wang 2004), integrin lymphocyte function-associated antigen-1 (LFA-1) (Denti 2004) and serine/threonine kinase Mirk/Dyrk (Zou 2003). RanBPM has also been shown to associate directly with the guanine nucleotide exchange factor Sos and to stimulate Ras/ERK (Wang 2002). It also regulates the transcriptional activity downstream of several receptors (Rao 2002; Wang 2002; Denti 2004). We have demonstrated that L1 and RanBPM interact both and The N-terminus of RanBPM was sufficient for the interaction with L1. In transfected cells, L1 and RanBPM colocalized in the plasma membrane and antibody-induced L1 patching caused redistribution of RanBPM with substantial colocalization with L1. Overexpression of the N-terminal fragment of RanBPM decreased L1-induced ERK activation by twofold in COS cells and partially inhibited L1-mediated neurite outgrowth in cerebellar neurons. These data suggest that RanBPM serves as an adaptor in L1-mediated signaling involved in neurite growth. Materials and methods Materials All cell culture Flurazepam dihydrochloride reagents were from Gibco (Carlsbad, CA, USA). The Nucleofector transfection kit was from Amaxa (Cologne, Germany). All chemicals were from Sigma (St Louis, MO, USA). Glutathione-Sepharose 4B beads, pGEX-4T-1 vector and anti-glutathione 2002). The anti-RanBPM antibody (5M) was a kind gift from Dr T Nishimito (Kyushu University, Japan). The monoclonal anti-RanBPM antibody was a kind gift from Dr E. Bianchi (Institut Pasteur, Paris, France). Yeast two-hybrid screen A cDNA encoding the last 28 amino acids of the L1CD was fused to sequences encoding the GAL4-DNA binding domain in.