The Neural Cell Adhesion Molecule L1 Interacts with the AP-2 Adaptor and Is Endocytosed via the Clathrin-Mediated Pathway

Abstract

Cell-cell interactions mediated via cell adhesion molecules (CAMs) are dynamically regulated during nervous system development. One mechanism to control the amount of cell surface CAMs is to regulate their recycling from the plasma membrane. The L1 subfamily of CAMs has a highly conserved cytoplasmic domain that contains a tyrosine, followed by the alternatively spliced RSLE (Arg-Ser-Leu-Glu) sequence. The resulting sequence of YRSL conforms to a tyrosine-based sorting signal that mediates clathrin-dependent endocytosis of signal-bearing proteins. The present study shows that L1 associates in rat brain with AP-2, a clathrin adaptor that captures plasma membrane proteins with tyrosine-based signals for endocytosis by coated pits. In vitro assays demonstrate that this interaction occurs via the YRSL sequence of L1 and the mu 2 chain of AP-2. In L1-transfected 3T3 cells, L1 endocytosis is blocked by dominant-negative dynamin that specifically disrupts clathrin-mediated internalization. Furthermore, endocytosed L1 colocalizes with the transferrin receptor (TfR), a marker for clathrin-mediated internalization. Mutant forms of L1 that lack the YRSL do not colocalize with TfR, indicating that the YRSL mediates endocytosis of L1. In neurons, L1 is endocytosed preferentially at the rear of axonal growth cones, colocalizing with Eps15, another marker for the clathrin endocytic pathway. These results establish a mechanism by which L1 can be internalized from the cell surface and suggest that an active region of L1 endocytosis at the rear of growth cones is important in L1-dependent axon growth.

Tetanus neurotoxin-mediated cleavage of cellubrevin impairs epithelial cell migration and integrin-dependent cell adhesion

“…Our data are reminiscent of findings on the cell adhesion molecule L1. Indeed, the neuronal form of L1 has an additional exon that encodes an AP-2-binding site, thereby promoting fast recycling of the protein (38) and weaker adhesion (39). Thus, our data are compatible with the concept that optimal migration is obtained with submaximal cell-matrix adhesion.…”

Constitutive GABAA Receptor Endocytosis Is Dynamin-mediated and Dependent on a Dileucine AP2 Adaptin-binding Motif within the β2 Subunit of the Receptor

“…This was surprising given the role of this motif in the endocytosis of integral membrane proteins (22,23). It is possible that the tyrosine motif does not play a role in GABA A receptor endocytosis or that its involvement is dependent on post-translational modification or protein-protein interactions that do not exist under our experimental conditions.…”

Activation of the MAPK Signal Cascade by the Neural Cell Adhesion Molecule L1 Requires L1 Internalization

“…Cell Culture-NIH-3T3 cells (American Type Tissue Culture Collection, Manassas, VA) and dorsal root ganglia from embryonic day 10 chickens were cultured as described previously (20). Briefly, the L1-expressing NIH-3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.) supplemented with 10% fetal calf serum and 600 g/ml G418 (Life Technologies, Inc.) prior to serum starvation.…”

“…In the experiments designed to detect ERK activation, NIH-3T3 cells stably transfected with full-length human L1 (20) were plated at a density of 2 ϫ 10 5 cells/60-mm dish. Prior to stimulation, the cells were maintained in low serum, 0.5% fetal calf serum in DMEM for 48 h followed by 2 h in serum-free DMEM.…”

“…The neuronal form of L1 contains an alternatively spliced exon encoding four amino acids (RSLE) within the L1CD (18), which contributes to a tyrosine based sorting/endocytosis motif (YRSL) (19). This sequence enables the L1CD to directly bind the 2 subunit of the adaptin complex AP-2, linking L1 to the clathrin-mediated endocytotic pathway (20). Adaptin proteins are dynamically regulated by phosphorylation (21)(22)(23), and examples from G-protein-coupled receptors have demonstrated the importance of phosphorylation of both receptors and intracellular machinery in regulating endocytosis (24,25).…”