Binary and ternary complexes of FLNa-Ig21 with cytosolic tails of αMß2 integrin reveal dual role of filamin mediated regulation.

BACKGROUND: Cytoskeletal protein filamin A is critical for the outside-in signaling of integrins. Although molecular mechanisms of filamin-integrin interactions are not fully understood. Mostly, the membrane distal (MD) part of the cytosolic tail (CT) of ß subunit of integrin is known to interact with filamin A domain 21 (FLNa-Ig2). However, binary and ternary complexes of full-length CTs of leucocyte specific ß2 integrins with FLNa-Ig21 are yet to be elucidated. METHODS: Binding interactions of the CTs of integrin αMß2 with FLNa-Ig21 are extensively investigated by NMR, ITC, cell-based functional assays and computational docking. RESULTS: The αM CT demonstrates interactions with FLNa-Ig21 forming a binary complex. Filamin/αM interface is mediated by sidechain-sidechain interactions among non-polar and aromatic residues involving MP helix of αM and the canonical CD face of FLNa-Ig21. Functional assays delineated an interfacial residue Y1137 of αM CT is critical for in-cell binding to FLNa-Ig2. In addition, full-length ß2 CT occupies two distinct binding sites in complex with FLNa-Ig21. A ternary complex of FLNa-Ig21 with CTs has been characterized. In the ternary complex, αM CT moves away to a distal site of FLNa-Ig21 with fewer interactions. CONCLUSION: Our findings demonstrate a plausible dual role of filamin in integrin regulation. The molecular interactions of the ternary complex are critical for the resting state of integrins whereas stable FLNa-Ig21/αM CT binary complex perhaps be required for the activated state. GENERAL SIGNIFICANCE: Filamin binding to both α and ß CTs of other integrins could be essential in regulating bidirectional signaling mechanisms.

NMR Structure, Dynamics and Interactions of the Integrin ß2 Cytoplasmic Tail with Filamin Domain IgFLNa21.

Integrins are transmembrane proteins that mediate cell adhesion and migration. Each integrin is a heterodimer formed by an α and a ß subunit. A large number of cytoplasmic proteins interact with the cytoplasmic tails (CTs) of integrins. The actin-binding cytoskeletal protein filamin A is a negative regulator of integrin activation. The IgFLNa21 domain of filamin A binds to the C-terminus of ß2 CT that contains a TTT-motif. Based on x-ray crystallography, it has been reported that the integrin ß2 CT forms a ß strand that docks into the ß strands C and D of IgFLNa21. In this study, we performed solution NMR analyses of IgFLNa21 in the presence of integrin ß2 CT peptides, and hybrid IgFLNa21, a construct of covalently linked IgFLNa21 and ß2 CT. The atomic resolution structure of the hybrid IgFLNa21 demonstrated conserved binding mode with ß2 CT. Although, 15N relaxation, model free analyses and H-D exchange studies have uncovered important insights into the conformational dynamics and stability of ß2 CT in complex with IgFLNa21. Such dynamical characteristics are likely to be necessary for the TTT-motif to serve as a phosphorylation switch that regulates filamin A binding to integrin ß2 CT.

Interaction Analyses of the Integrin ß2 Cytoplasmic Tail with the F3 FERM Domain of Talin and 14-3-3ζ Reveal a Ternary Complex with Phosphorylated Tail.

Integrins, which are heterodimeric (α and ß subunits) signal-transducer proteins, are essential for cell adhesion and migration. ß cytosolic tails (ß-CTs) of integrins interact with a number of cytosolic proteins including talin, Dok1, and 14-3-3ζ. The formation of multiprotein complexes with ß-CTs is involved in the activation and regulation of integrins. The leukocyte-specific ß2 integrins are essential for leukocyte trafficking, phagocytosis, antigen presentation, and proliferation. In this study, we examined the binding interactions between integrin ß2-CT and T758-phosphorylated ß2-CT with positive regulators talin and 14-3-3ζ and negative regulator Dok1. Residues of the F3 domain of talin belonging to the C-terminal helix, ß-strand 5, and the adjacent loop were found to be involved in the binding interactions with ß2-CT. The binding affinity between talin F3 and ß2-CT was reduced when ß2 T758 was phosphorylated, but this modification promoted 14-3-3ζ binding. However, we were able to detect stable ternary complex formation of T758-phosphorylated ß2-CT, talin F3, and 14-3-3ζ that involved the repositioning of talin F3 on ß2-CT. We showed that Dok1 binding to ß2-CT was reduced in the presence of 14-3-3ζ and when ß2 T758 was phosphorylated. Based on these data, we propose a sequential model of ß2 integrin activation involving these molecules. Our study provides for the first time insights toward ß2 integrin activation that involves a multiprotein complex.