Structural Basis of the Migfilin-Filamin Interaction and Competition with Integrin β Tails

Abstract

A link between sites of cell adhesion and the cytoskeleton is essential for regulation of cell shape, motility, and signaling. Migfilin is a recently identified adaptor protein that localizes at cell-cell and cell-extracellular matrix adhesion sites, where it is thought to provide a link to the cytoskeleton by interacting with the actin cross-linking protein filamin. Here we have used x-ray crystallography, NMR spectroscopy, and protein-protein interaction studies to investigate the molecular basis of migfilin binding to filamin. We report that the N-terminal portion of migfilin can bind all three human filamins (FLNa, -b, or -c) and that there are multiple migfilin-binding sites in FLNa. Human filamins are composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like (IgFLN) domains and we find that migfilin binds preferentially to IgFLNa21 and more weakly to IgFLNa19 and -22. The filamin-binding site in migfilin is localized between Pro 5 and Pro 19 and binds to the CD face of the IgFLNa21 ␤-sandwich. This interaction is similar to the previously characterized ␤7 integrin-IgFLNa21 interaction and migfilin and integrin ␤ tails can compete with one another for binding to IgFLNa21. This suggests that competition between filamin ligands for common binding sites on IgFLN domains may provide a general means of modulating filamin interactions and signaling. In this specific case, displacement of integrin tails from filamin by migfilin may provide a mechanism for switching between different integrin-cytoskeleton linkages.Functional connections between transmembrane adhesion receptors and the intracellular cytoskeleton permit transmission of biochemical signals and mechanical force across the plasma membrane and are essential to the development and functioning of multicellular animals (1). In many cases connections are formed at specialized sites involving assemblies of many adhesion molecules and cytoskeletal and signaling adaptors (2). Although many components of these adhesion complexes have now been identified, a complete understanding of how adhesion complexes function will require detailed information on individual components and how they interact or compete with other elements of this complex machinery. Here we characterize the interaction of a recently identified adaptor protein, migfilin, with the actin-binding protein filamin and examine how this influences interactions of filamin with integrin adhesion receptors.Migfilin, also termed filamin-binding LIM protein-1, is a LIM domain protein that localizes to both cell-extracellular matrix and cell-cell contact sites, where it is thought to provide a link between the actin cytoskeleton and integrin-extracellular matrix contact sites and cadherin-catenin cell-cell junctions, respectively (3-5). In this way, migfilin can modulate cell shape, migration, and cadherin-mediated cell-cell contacts.

Migfilin Interacts with Src and Contributes to Cell-Matrix Adhesion-mediated Survival Signaling

“…Lys-7, Arg-8, and Ser-11) are part of the binding surface for filamin repeat 21 (IgFLN21). The migfilin binding surface in IgFLN21 overlaps with the integrin binding surface (35,36). Thus, the binding of migfilin to Src SH2 domain could potentially be regulated by the interaction between migfilin and filamin, which in turn can be regulated by the interaction between integrins and filamin.…”

“…Our studies suggest that the reduction of the migfilin level induced by loss of cell-ECM adhesion is likely mediated by proteasomes. In this regard it is worth noting that unbound migfilin N-terminal region is unstructured (35,36). The binding of migfilin to Src SH2 domain or actin filament-associated proteins such as filamin may help to stabilize the structure of migfilin N-terminal region and thereby prevent proteasome-mediated degradation of migfilin.…”

“…The C-terminal region is composed of three LIM domains that mediate the interaction with kindlin-2/Mig-2 (27). Migfilin N-terminal region, which does not have obvious sequence homology with any other known proteins, possesses a binding site for filamin (27,35,36). In between the N-terminal region and the C-terminal LIM region lies a proline-rich domain that contains multiple proline-rich sequences.…”

Assembly of a Filamin Four-domain Fragment and the Influence of Splicing Variant-1 on the Structure

“…1A) (7,8). A flexible hinge region (H1) between domains 15 and 16 divides the chain of 24 Ig-like domains into rod 1 (FLN (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)) and rod 2 (FLN(16 -24)) ( Fig. 1A) (9).…”

“…This includes the first ␤-strand of FLNa (20) (Fig. 1B) that masks the integrin and migfilin binding site at the CD face of FLNa (21) (11,(13)(14)(15)(16) (Fig. 1B).…”