The Cell Adhesion Molecule L1 Interacts with Methyl CpG Binding Protein 2 via Its Intracellular Domain

Abstract

Cell adhesion molecule L1 regulates multiple cell functions, and L1 deficiency is linked to several neural diseases. Recently, we have identified methyl CpG binding protein 2 (MeCP2) as a potential binding partner of the intracellular L1 domain. By ELISA we show here that L1’s intracellular domain binds directly to MeCP2 via the sequence motif KDET. Proximity ligation assay with cultured cerebellar and cortical neurons suggests a close association between L1 and MeCP2 in nuclei of neurons. Immunoprecipitation using MeCP2 antibodies and nuclear mouse brain extracts indicates that MeCP2 interacts with an L1 fragment of ~55 kDa (L1−55). Proximity ligation assay indicates that metalloproteases, β-site of amyloid precursor protein cleaving enzyme (BACE1) and ɣ-secretase, are involved in the generation of L1−55. Reduction in MeCP2 expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons as well as the migration of L1-expressing HEK293 cells. Moreover, L1 siRNA, MeCP2 siRNA, or a cell-penetrating KDET-containing L1 peptide leads to reduced levels of myocyte enhancer factor 2C (Mef2c) mRNA and protein in cortical neurons, suggesting that the MeCP2/L1 interaction regulates Mef2c expression. Altogether, the present findings indicate that the interaction of the novel fragment L1−55 with MeCP2 affects L1-dependent functions, such as neurite outgrowth and neuronal migration.

The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions

“…We have shown that the interaction of MeCP2 and HP1 with L1-55 is mediated by the KDET motif in the intracellular domain of L1 (L1-ICD) [ 26 , 27 ]. Besides MeCP2 and HP1α, -β, and -γ, we identified NDUFV2, SFPQ, NonO, PSPC1, ERα, PPARγ, RXRβ, and TOP1 as L1’s binding partners [ 17 , 18 , 28 , 29 ].…”

“…In parallel, we analyzed whether the putative L1 binding partners WDR5, HistH1e, Nup93, Hsc70, SYT1, impβ1, and hnRNP A isoforms [ 28 ], as well as AR and VDR, interact with L1-55. Proximity ligation, which detects interactions between proteins localized at a distance of 40 nm or less, showed that the interactions of L1-55 with its binding partners MeCP2 and HP1 are reduced by the γ-secretase inhibitor DAPT ((2S)-N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1-dimethylethyl ester) [ 26 , 27 ]. On the basis of these findings, cultured cortical neurons were treated with DAPT and subjected to a proximity ligation assay using L1 antibodies and antibodies against NDUFV2, SFPQ, NonO, PSPC1, WDR5, HistH1, TOP1, hnRNP A isoforms, Nup93, Hsc70, SYT1, impβ1, ERα, RXR, PPARγ, AR, and VDR to analyze whether these proteins are associated with L1-55.…”

“…Higher numbers of dots (1–4 dots per cell) ascertained that NDUFV2, SFPQ, NonO, PSPC1, WDR5, TOP1, HistH1e, Nup93, Hsc70, SYT1, impβ1, ERα, RXR, PPARγ, AR, and VDR interact with L1. The numbers of HistH1/L1-positive dots were reduced in neurons treated with the γ-secretase inhibitor DAPT when compared to the numbers in vehicle-treated neurons (approximately 1 dot per cell), as seen for MeCP2 and HP1 ( Figure 1 b,c), indicating that HistH1e interacts with L1-55, like MeCP2 and HP1 [ 26 , 27 ]. The numbers of NDUFV2/L1-, SFPQ/L1-, NonO/L1-, PSPC1/L1-, WDR5/L1-, TOP1/L1-, Nup93/L1-, Hsc70/L1-, SYT1/L1-, impβ1/L1-, ERα/L1-, RXR/L1-, PPARγ/L1-, AR/L1-, and VDR/L1-positive dots were not reduced by DAPT treatment ( Figure 1 c), indicating that NDUFV2, SFPQ, NonO, PSPC1, ERα, RXR, PPARγ, WDR5, TOP1, Nup93, Hsc70, SYT1, impβ1, AR, and VDR are not associated with L1-55.…”

“…In previous studies, we have shown that stimulation of L1 signaling leads to the myelin basic protein-mediated generation of a transmembrane C-terminal fragment of 70 kDa, called L1-70, which plays important roles in neuritogenesis, neuronal migration, and neuronal survival as well as mitochondrial homeostasis [ 5 , 17 , 18 , 19 , 21 , 25 ]. In a more recent study, we have identified a novel C-terminal L1 fragment of 55 kDa, L1-55, which interacts with MeCP2 and the HP1 isoforms α, β, and γ, and is also involved in L1-dependent functions, such as neurite outgrowth and neuronal migration [ 26 , 27 ]. L1-55 binds to the HP1 isoforms α, β, and γ, as well as to MeCP2 via a KDET motif in its intracellular domain [ 26 , 27 ].…”

“…In a more recent study, we have identified a novel C-terminal L1 fragment of 55 kDa, L1-55, which interacts with MeCP2 and the HP1 isoforms α, β, and γ, and is also involved in L1-dependent functions, such as neurite outgrowth and neuronal migration [ 26 , 27 ]. L1-55 binds to the HP1 isoforms α, β, and γ, as well as to MeCP2 via a KDET motif in its intracellular domain [ 26 , 27 ]. Besides MeCP2 and HP1, we had identified NDUFV2 as a mitochondrial binding partner [ 17 ] and, by affinity chromatography using the intracellular domain, as nuclear binding partners, SFPQ (also known as polypyrimidine tract binding associated-splicing factor or PSF), NonO (also known as 54 kDa nuclear RNA- and DNA-binding protein or p54nrb), PSPC1, estrogen receptors α (ERα) and β (ERβ), peroxisome proliferator-activated receptor γ (PPARγ), and retinoid X receptor β (RXRβ) [ 17 , 18 , 28 ] ( Table 1 ).…”