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

Abstract

Filamins are large actin-binding proteins that stabilize threedimensional F-actin networks and link them to the cell membrane by binding to transmembrane receptors, e.g. integrins or ion channels. In addition, filamins bind to various other proteins with diverse function, including signaling and adaptor proteins, such as migfilin (1-6). Accordingly, filamins are considered as scaffolding proteins that integrate multiple cellular functions. Filamins are also associated with various human genetic diseases including malformations of the skeleton, brain, and heart (5).The filamin family has three members; filamins (FLNs) 4 A, B and C. The FLN genes are highly conserved, and the encoded proteins share about 70% overall sequence identity (6). FLNA is located on the X chromosome, whereas FLNB and FLNC are on autosomal chromosomes 3 and 7 (6). All three FLN genes are widely expressed during development, but in adults the most abundant isoform is FLNA. FLNC expression is predominantly restricted to skeletal and cardiac muscle cells (3). FLN proteins are homodimers of two 280-kDa polypeptide chains consisting of an N-terminal actin binding domain followed by 24 immunoglobulin (Ig)-like domains (FLN(1-24)) (6). The last domain, FLN(24), mediates dimerization (Fig. 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(1-15)) and rod 2 (FLN(16 -24)) (Fig. 1A) Fig. 1) (10, 11). A similar intertwined interaction is, however, not seen with the domain pair FLNa(16 -17) (10).The diversity of the filamin family is increased by alternative splicing of FLN mRNA. These changes in amino acid sequence can affect the binding of other interacting partners. FLNa and FLNb splice variants (var-1), which are widely expressed at low levels, lack 41 amino acids including the C-terminal part of FLNa(19) and the N-terminal part of FLNa(20) (residues 2127-2167) (6,12). 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). FLN var-1 binding to various integrins is increased compared with nonspliced filamins (11, 12), implying that alternative splicing could be a possible regulatory mechanism for binding to integrin and other interaction partners. Other possible mechanisms include mechanical force-induced exposure of the cryptic binding site on the CD face (17, 18) and/or the binding of other binding partners.Other splice variants are also found in FLNb and FLNc. The hinge region H1, which is responsible for the flexibility of FLN dimers, is missing in some FLNb and FLNc splice variants (12). Its absence might therefore affect the orthogonal cross-linking patterns made by FLN. The expression of different FLN splice variants appears to differ according to tissue type. The predominant isoforms in thyroid are FLNb containing H1 and FLNc lacking H1 (⌬H1) (19,20

An adventitious interaction of filamin A with RhoGDI2(Tyr153Glu)

“…We co-expressed wild-type or mutant Myc-RhoGDI2-HA with Flag-tagged FLNA or a FLNA del41 variant in Hela cells. FLNA del41 removes the auto-inhibitory domain (strand A) of repeat 20 from repeat 21 constitutively revealing its cryptic binding site [31–33] (Figure 2A). Wild-type RhoGDI2 failed to co-immunoprecipitate either form of FLNA, whereas mutant RhoGDI2(Y153E) collected FLNA even though the expression level of mutant RhoGDI2(Y153E) was lower than that of wild-type.…”

Exon skipping causes atypical phenotypes associated with a loss-of-function mutation in FLNA by restoring its protein function

“…Several reports revealed that the N-terminus of Ig20 forms a β-strand that interacts with, and occupies, the C and D β-strands (known as the CD face) of the adjacent Ig21. [26][27][28] When mechanical stresses stretch the FLNA protein, the masked CD face is exposed, increasing the affinity of Ig21 toward its binding partners, including integrins and migfilin, and leading to downstream signaling. 29 In this study, the exon-skipped FLNA protein had stronger binding to β7 and a similar capacity to induce focal adhesions, compared with that of wild-type FLNA.…”

Dynamic force sensing of filamin revealed in single-molecule experiments

“…4D. This ensures a precise and well-controlled gating curve even though the involved binding energies lie below 4 k B T. It has been argued that an autoinhibition of filamin seems unlikely in in vitro experiments because it could be shown that an excess of ligand alone is able to convert the filamin domain pairs from a closed to an open conformation (33,34). However, this stood in apparent contrast to earlier reports that the isolated domain 21 binds stronger to ligands than the domain pairs (14) as well as to binding studies on strained filamin-cross-linked actin networks (17).…”