Filamin and its interactions in cell migration

Human filamin is a large dimeric, modular protein made up of two calponin homology domains and twenty-four immunoglobulin-like (Ig_FLMN) domains. Filamin cross-links actin filaments and is deeply involved in cell migration processes, largely through interactions with plasma membrane proteins, especially integrins. In the work presented here, the structural properties of filamin fragments involved in integrin binding have been studied. NMR investigations of individual dissected Ig_FLMN modules from human . filamin A (called FLNa here) revealed that, although homologous at the sequence level, they have variable folding properties. A series of NMR titrations located the binding interface between the cytoplasmic tails of integrins and filamin modules, showing that binding of FLNa19 and FLNa21 to the tails was very similar. Further analysis of NMR experimental results quantitatively characterized a dimerization of the integrin-filamin complex in solution. Inter-module interactions in a triple module (FLNa19-21) were also observed and studied. NMRstudies on module pairs of filamin (FLNaI9-20 and FLNa20-21) disclosed that part ofFLNa20 'zips' along FLNa21 in a similar pattern as integrin tails. NMR studies show that this structure is, however, readily disrupted by addition of integrin tails. The observed affinities, dimerization behaviour and the structure of FLNa19-21 led to an 'integrin clip' hypothesis. This model was supported by evidence that suggested a module rearrangement of FLNa19-21 was induced by integrin binding. Several potential regulation mechanisms for filamin-integrin binding, including selective expression of isoforrns, splicing variation and phosphorylation, were also studied. Based on previous knowledge of Ig_FLMN complexes, a strategy to predict filamin binding affinity was developed and applied to the binding site detennination for two newly identified filamin binding proteins,' migfilin and FILIP; this was successfully confinned by NMR titration experiments.EThOS - Electronic Theses Online ServiceGBUnited Kingdo