Semaphorin3A-induced receptor endocytosis during axon guidance responses is mediated by L1 CAM

Abstract

During axon navigation, Semaphorin3A-induced growth cone retraction is correlated with endocytosis. Although its function remains elusive, we showed previously that the cell adhesion molecule of the immunoglobulin super family L1 associates with Neuropilin-1 (NP-1) the Sema3A-binding subunit of the receptor complex and is required for Sema3A to elicit axonal repulsive responses. We report here that upon Sema3A binding to NP-1, L1 and NP-1 are co-internalized through a clathrin-dependent mechanism mediated by L1. We show that in COS7 cells, L1/NP-1 endocytosis is correlated with a cell contraction similar to that observed with the Plexin (Plex)/NP-1 or Plex/NP1/L1 complexes. In neuronal cultures, a L1-mimetic peptide able to switch Sema3A repulsive responses to attraction blocks both endocytosis and growth cone collapse. Similarly, in the COS7 cell model, peptide application prevents both the Sema3-induced L1/NP-1 internalization and cell collapse. These studies demonstrate that the L1/NP-1 complex is able to confer a biological response to Sema3A with L1 mediating receptor internalization following ligand activation. They also reveal that endocytosis controlled by L1/NP-1 cis and trans interactions is pivotal in Sema3A-mediated axon guidance.

Introduction

In the developing nervous system, axon path finding relies on multiple positional signals that specifically orient subpopulations of neuronal projections toward their respective synaptic targets. Time- and region-specific expression of guidance cues and their receptors associated with highly dynamic regulations at protein and mRNA levels are believed to generate the diversity of responses required for the proper wiring of neuronal projections. Thus, growth cones are complex machineries equipped for sensing and integrating coincident guidance signals (Dickson, 2002).

Among these signals, the semaphorins, a large family of membrane-attached and diffusible factors, have been shown to play major roles (reviewed by Raper, 2000). Accumulating evidences indicate that secreted semaphorins activate receptor complexes that contain Neuropilin (NP) as the ligand binding subunit and Plexin (Plex) as the signal propagating subunit He et al., 2002, Tamagnone and Comoglio, 2000.

The immunoglobulin superfamily cell adhesion molecule (IgCAM) L1 has also been implicated in responses to one of the secreted semaphorin, Sema3A Castellani et al., 2000, Castellani et al., 2002. In support, genetic invalidation of L1 in mice generates axon guidance errors (Cohen et al., 1998) that could be due to a lack of response to Sema3A as neurons isolated from these mice are unresponsive to Sema3A-induced chemorepulsion (Castellani et al., 2000). Furthermore, L1 and Neuropilin-1 (NP-1) were found to associate through their extracellular domains, and a pathological mutation in the L1 gene responsible for abnormal development of several axonal tracts specifically disrupts this L1/NP-1 association Castellani et al., 2000, Castellani et al., 2002. Finally, wild type but not L1-deficient axons exposed to soluble L1 switched their response to Sema3A from repulsion to attraction (Castellani et al., 2000). Together, these findings suggested that the IgCAM L1 associates to the Sema3A receptor and is required for this guidance cue to prevent neuronal projections from invading inappropriate territories. Moreover, L1 can be released from the cell surface by an enzymatic cleavage of its ectodomain Gutwein et al., 2000, Nayeem et al., 1999 known to occur in the developing nervous system (Mechtersheimer et al., 2001). This pointed to a possible regulation of the Sema3A signaling by soluble L1. However, many questions remain elusive such as the role of L1 in the Sema3A receptor and how soluble L1 interplays in Sema3A response. In this study, we explored these issues in neuronal and cell culture models. Previous reports showed that Sema3A treatment of dorsal root ganglia (DRG) cultured neurons is accompanied by endocytosis Fournier et al., 2000, Jurney et al., 2002. Other studies reported that L1 is internalized and recycled in the growth cones Kamigushi and Lemmon, 2000, Kamiguchi et al., 1998. We hypothesized that L1 could be responsible for the endocytosis occurring during growth cone response to Sema3A. We report that NP-1 and L1 are co-internalized by Sema3A and that this process accompanies growth cone and cell collapse. Conversely, blockade of receptor internalization correlates with a loss of cell and growth cone collapse. Furthermore, in a COS7 model, we show that both NP-1/Plex-A1 and NP-1/L1 heterocomplexes confer to cells a biological response to Sema3A. This study assigns a possible function of L1 as mediating receptor internalization occurring during growth cone responses to Sema3A. It suggests that the composition of the Sema3A receptor might be modular, with L1 and Plex proteins being recruited either together or independently for association to NP1. It also points to control of endocytosis as a key mechanism underlying the nature of growth cone responses to Sema3A during the development of neuronal projections.