Actin-binding protein filamin A is displayed on the surface of human neuroblastoma cells

Abstract

We previously reported the identification of natural human IgM antibodies, which recognize a M(r) 260 000 surface protein (NB-p260) and induce both complement-mediated cytotoxicity and apoptosis of human neuroblastoma cells. NB-p260 was shown to belong to the family of filamin proteins. Filamin A is a high molecular weight actin-binding protein, previously thought to be only located intracellularly. Here we show that NB cells as well as three NB-unrelated human cell lines express filamin A also on the cell surface. Our findings suggest new biological functions for filamins, including a role as mediators in anti-NB IgM-induced apoptosis, and they add to the growing body of evidence of the interaction of cytoskeletal proteins with the extracellular matrix.

Figure 1

Structure of human filamin A (FLNa) and filamin B (FLNb). Filamins are cytoskeleton‐associated proteins and consist of an N‐terminal actin‐binding domain (ABD), 24 tandem repeats, two flexible hinge regions (H1 and H2) and a C–terminal self association domain (repeat 24).

Figure 2

Detection of endogenous filamin A (FLNa) by confocal laser microscopy (A, C, E, G) and corresponding differential interference contrast images (B, D, F, H) of human NB cell lines LAN‐1 (A, B) and LAN‐5 (C, D), and human melanoma cell lines A7 (E, F) and FLNa‐deficient M2 (G, H).

Figure 3

Detection of surface‐expressed filamin A (FLNa) in LAN‐1 cells. Subconfluent cells were surface‐biotinylated, lyzed, and immunoprecipitated with the anti‐FLNa antibody MAB1678. Cell lysates (right panel) or precipitated proteins (left panel) were resolved by 7.5% SDS‐PAGE under reducing conditions, electroblotted onto a PVDF membrane, and probed with the MAB1678 antibody or streptavidin.

Figure 4

Detection of endogenous filamin A (FLNa) in the integral membrane protein fraction of NMB‐7 NB cells. Integral membrane proteins of NMB‐7 cells were extracted into a solubilized membrane protein fraction. Both the integral membrane protein fraction (lane 2) and the hydrophilic protein fraction containing cytoplasmic and peripheral membrane proteins (lane 1) were subjected to 7.5% SDS‐PAGE and western blotting using (A) the anti‐FLNa antibody MAB1678 or (B) an anti‐β‐catenin antibody (as a cytoplasmic control protein).

Figure 5

Detection of filamin A (FLNa) on the cell surface of NB cells by flow cytometry. The binding analysis was performed with LAN‐1 cells using five different mouse monoclonal antibodies (A, B, D, E, F) and one goat polyclonal antiserum (C) against FLNa. The red line represents the FLNa‐specific antibody. Controls include secondary antibody alone (blue line), and cells incubated with PBS in the absence of antibodies (black line). The two monoclonal antibodies (MAB1680 and MAB1678) and the goat polyclonal antiserum (F2762) showed strong binding to the NB cells. No binding was detected with the monoclonal antibodies ABP‐280, AB‐1, and AB‐2.

Figure 6

Detection of filamin A (FLNa) on the cell surface of human cell lines by flow cytometry. The binding analysis was performed with HeLa, SKOV‐3, and HEK293 cells using the FLNa‐recognizing mouse monoclonal antibody MAB1680 (red line). Controls include secondary antibody alone (blue line), and cells incubated with PBS in the absence of primary and secondary antibodies (black line).

Figure 7

Computational analysis of the filamin A (FLNa) structure. (A) Using the Dense Alignment Surface transmembrane prediction program, a transmembrane domain was predicted in the N‐terminal part of FLNa. (B) A detailed analysis of residues 77–217 of FLNa localized the predicted transmembrane domain between residues 137 and 155. TM, transmembrane domain; aa, amino acids.

Figure 8

Hypothetical structure model for filamin A (FLNa). The actin binding domain‐containing N‐terminus of FLNa is in the intracellular space and continues to interact with the actin cytoskeleton, while the C‐terminus is exposed to the extracellular environment. Possible integrin binding sites (RGD) are indicated (residues 437–439 and 1312–1314).