Ligand-of-Numb protein X is an endocytic scaffold for junctional adhesion molecule 4

Abstract

Junctional adhesion molecule 4 (JAM4) is a cell adhesion molecule that interacts with a tight junction protein, membrane-associated guanylate kinase inverted 1 (MAGI-1). Our previous studies suggest that JAM4 is implicated in the regulation of paracellular permeability and the signalings of hepatocyte growth factor. In this study, we performed yeast two-hybrid screening to search for an unidentified JAM4-binding protein and obtained one isoform of Ligand-of-Numb protein X1 (LNX1), LNXp70, that is an interactor of Numb. Ligand-of-Numb protein X1 is expressed in kidney glomeruli and intestinal epithelial cells, where JAM4 is also detected. Immunoprecipitation from kidney lysates supports the in vivo interaction of proteins. Biochemical studies reveal that JAM4 directly binds the second PDZ domain of LNX1 through its carboxyl terminus. Junctional adhesion molecule 4, LNX1 and Numb form a tripartite complex in vitro and are partially colocalized in heterologous cells. Ligand-of-Numb protein X1 facilitates endocytosis of JAM4 and is involved in transforming growth factor b -induced redistribution of JAM4 in mammary epithelial cells. Experiments using dominant-negative constructs and RNA interference insure that Numb is necessary for the LNX1-mediated endocytosis of JAM4. All these findings indicate that LNX1 provides an endocytic scaffold for JAM4 that is implicated in the reorganization of cell junctions.

Ligand of Numb proteins LNX1p80 and LNX2 interact with the human glycoprotein CD8α and promote its ubiquitylation and endocytosis

“…Two splicing variants of LNX1 exist, p70 and p80. The LNX1p70 binds the junctional protein JAM4, and its overexpression facilitates JAM4 endocytosis (Kansaku et al, 2006). On the other hand, LNX1p80 is an interacting partner of claudin-1 and promotes its ubiquitylation, removal from the tight junctions and transport to the lysosomes (Takahashi et al, 2009).…”

“…1C) (Dho et al, 1998;Rice et al, 2001). However, in contrast to LNX2, the function of LNX1p80 as E3 ligase has been partially explored (Kansaku et al, 2006;Takahashi et al, 2009). For these reasons, we decided to test whether LNX1 is also a CD8a binding protein.…”

“…It has been previously shown that LNX1p80 interacts with the junctional proteins JAM-4 via the second PDZ domain (Kansaku et al, 2006), and that the first two PDZ domains of LNX2 are required for binding to the cell surface coxsackievirus and adenovirus receptor (CAR) (Sollerbrant et al, 2003). To identify regions of LNX1p80 and LNX2 important for CD8a recognition, we generated a series of truncation mutants containing separately the distinct domains, and tested their ability to bind the CD8a cytosolic C-terminal tail or its DYV mutant fused to the GST (supplementary material Fig.…”

“…The following antibodies were used: the OKT8 mouse anti-CD8a monoclonal antibody from Ortho (Raritan, NJ); the N1 mouse anti-CD8a monoclonal antibody and rabbit anti-CD8a polyclonal antibody from M. Jackson (Martire et al, 1996); rabbit anti-LNX1 antibody (Kansaku et al, 2006); rabbit and mouse anti-GFP and anti-HA antibodies (Santa Cruz Biotechnology); mouse anti-EEA1 monoclonal antibody (BD Transduction Laboratories, Lexington, KY); mouse anti-CD7107a (LAMP1) monoclonal antibody (Biolegend, San Diego, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich, Milan, Italy); and Texas-Red-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).…”

The E3 ubiquitin ligase LNX1p80 promotes the removal of claudins from tight junctions in MDCK cells

“…Among two isoforms of LNX1, LNX1p80 has a RING-finger domain at the N-terminus and is involved in ubiquitin-dependent degradation of Numb (Nie et al, 2002). One or two PDZ domains of LNX1 isoforms have been reported to interact with several molecules, including CAR, JAM4, Src and cytomatrix at the active-zone-associated structural protein (CAST), whose C-termini have PDZ-domain-binding motifs (Sollerbrant et al, 2003;Kansaku et al, 2006;Weiss et al, 2007;Higa et al, 2007). Similarly to these proteins, the C-termini of claudins appear to bind to PDZ domains of LNX1.…”