The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.

Human filamins are large actin-crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 Å resolution structure of a three-domain fragment of human filamin A (IgFLNa19-21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N-terminus of IgFLNa20 forms a b-strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20-IgFLNa21 interaction enhances filamin binding to integrin b-tails. Structural and functional analysis of other IgFLN domains suggests that auto-inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin-ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure.

SummaryThe integrin family of heterodimeric cell-surface receptors are fundamental in cell-cell and cell-matrix adhesion. Changes to either integrin-ligand affinity or integrin gene expression are central to a variety of disease processes, including inflammation, cardiovascular disease and cancer. In screening for novel activators of integrin-ligand affinity we identified the previously uncharacterised multitransmembrane domain protein Fam38A, located at the endoplasmic reticulum (ER). siRNA knockdown of Fam38A in epithelial cells inactivates endogenous b1 integrin, reducing cell adhesion. Fam38A mediates integrin activation by recruiting the small GTPase RRas to the ER, which activates the calcium-activated protease calpain by increasing Ca 2+ release from cytoplasmic stores. Fam38A-induced integrin activation is blocked by inhibition of either R-Ras or calpain activity, or by siRNA knockdown of talin, a welldescribed calpain substrate. This highlights a novel mechanism for integrin activation by Fam38A, utilising calpain and R-Ras signalling from the ER. These data represent the first description of a novel spatial regulator of R-Ras, of an alternative integrin activationsuppression pathway based on direct relocalisation of R-Ras to the ER, and of a mechanism linking R-Ras and calpain signalling from the ER with modulation of integrin-ligand affinity.

The activation of integrin adhesion receptors from low to high affinity in response to intracellular cues controls cell adhesion and signaling. Binding of the cytoskeletal protein talin to the ␤3 integrin cytoplasmic tail is required for ␤3 activation, and the integrin-binding PTB-like F3 domain of talin is sufficient to activate ␤3 integrins. Here we report that, whereas the conserved talin-integrin interaction is also required for ␤1 activation, and talin F3 binds ␤1 and ␤3 integrins with comparable affinity, expression of the talin F3 domain is not sufficient to activate ␤1 integrins. ␤1 integrin activation could, however, be detected following expression of larger talin fragments that included the N-terminal and F1 domains, and mutagenesis indicates that these domains cooperate with talin F3 to mediate ␤1 activation. This effect is not due to increased affinity for the integrin ␤ tail and we hypothesize that the N-terminal domains function by targeting or orienting talin in such a way as to optimize the interaction with the integrin tail. Analysis of ␤3 integrin activation indicates that inclusion of the N-terminal and F1 domains also enhances F3-mediated ␤3 activation. Our results therefore reveal a role for the N-terminal and F1 domains of talin during integrin activation and highlight differences in talin-mediated activation of ␤1 and ␤3 integrins.Integrins are a family of ␣␤ heterodimeric transmembrane receptors that mediate cell adhesion to extracellular matrix, cell surface, or soluble protein ligands and modulate a variety of intracellular signaling cascades. Cells regulate integrin function through tight temporal and spatial control of integrin affinity for extracellular ligands. This is achieved by rapid, reversible changes in the conformation of the integrin extracellular domains; integrin activation (1-3). Activation of the platelet integrin ␣IIb␤3 is a pivotal event in thrombus formation (4), and ␣IIb␤3 has served as a prototype in studies on integrin activation. However, activation of other integrins, including the widely expressed ␤1 family, is essential for normal development because it controls cell adhesion, migration, and assembly of an extracellular matrix (5-10), and deregulated ␤1 integrin activation contributes to neoplasia (11) and impairs cardiac function (12) and the immune response (13).A large body of evidence points to regulation of integrin activation through interactions of the ␤ subunit tail (3, 14), although ␣ tail-binding proteins also have a role (15, 16). Using ␣IIb␤3 as a model system we and others have shown that binding of talin to the ␤3 cytoplasmic tail is necessary and sufficient for integrin activation (17-23). Talin, a cytoskeletal actin-binding protein, consists of an N-terminal ϳ50-kDa globular head and an ϳ220-kDa C-terminal rod (3,24,25). The talin head is composed of an N-terminal 85-amino acid region followed by a FERM (4.1, ezrin, radixin, moesin) domain and a 33-amino acid stretch (18, 24 -26). FERM domains are made up of three subdomains, F1, F2, and F3 (27...

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.