Identification Of G3BP1 As A New Filamin A Mechanobinding Protein

Mechanotransduction is the biological process that cells sense internal and external mechanical forces(gravity,shear flow,stretching,compression and so on)and convert these forces into biochemical signals.Mechanotransduction plays a crucial role in tissue repair and regeneration by controlling cell growth,migration,differentiation and apoptosis.It is believed that mechanical forces are sensed by mechanosensors on plasma membrane or in cytoplasm and transmitted to a converter or regulator by transmitter to open a channel or regulate nuclear pore size.Thereby controls localization of trans-acting factors,regulates genes expression,and relays the signal to downstream molecules.Our previous study found that filamin A(FLNA)is a machanosensing molecule,which can mediate mechanotransduction by conformational changes.FLNA is the first actin cross-linked protein found in non-muscle cells and can interact with a variety of proteins,including intracellular signaling molecules,adhesion molecules,and even transcription factors.FLNA can integrate mechanical force and biochemical signals to play an important role in the process of cell proliferation,differentiation,apoptosis and migration.FLNA mutations can cause from mild to severe consequences such as mental retardation,physical malformations,malignant tumors,and heart failure in humans.However,the molecular mechanism of these diseases is still not well known.FLNA consists of two 280 k Da subunits that self-associate to form a 160 nm long semi-flexible strand.Each FLNA subunit consists of a N-terminal spectrin-related actin-binding domain(sr ABD)followed by 24 Ig-like repeats(Ig FLNa or R)with two hinges separating repeats 15 and 16(hinge 1)and R23 and R24(hinge 2).Hinge 1 separates sequence segments,designated rod 1 and rod 2.The most C-terminal repeat,Ig FLNa24,mediates dimerization and bestows a V-shape to the dimeric molecules.FLNA’s rod 2 domain,where FLNA interacts with its many binding partners,has a unique geometry and a compact structure that responds to mechanical force through conformational changes to regulate its partner interactions and thereby converts force to biochemical signals.Previous study demonstrated that force-induced conformational changes in rod 2 of FLNA can regulate partner interactions by exposing a cryptic integrin binding site in R21 CD face.However,our recent research using a mechanosensor probe in living cells found that exposure of the R21 localizes at not only integrin-enriched focal adhesion site but also other regions of the cell,suggesting that there are other mechanobinding partners of FLNA R21.Therefore,this project aims to identify a new mechanobinding protein of FLNA R21 to better understand FLNA mediated mechanotransduction.In order to identify such a new FLNA mechanobinding protein,we used SILAC(stable isotope labeling by amino acids in cell culturing)based quantitative proteomics.FLNA R21-22 was used as affinity ligand and R1-2 was used as a negative control,whose CD face is structurally different from that of R21.The mass spec data suggested several potential binding partners.Using reconstituted system of purified FLNA and the candidate proteins expressed as a GFP-tagged fusion protein,we identified G3BP1,Ras-GTPase-activating protein SH3 domain binding protein 1,as a new binding partner of FLNA.G3BP1 has endonuclease and DNA helicase activity,it relates to RNA metabolism and a variety of cell signaling pathways and plays a crucial role in cell development,tumor cell proliferation,migration and apoptosis.It is required for normal RNA stress granule(SG)assembly,which can protect the transcripts from degradation and makes the cell better adapt to environmental stress.It can prime cyclic GMP-AMP synthase(c GAS)for efficient cytoplasmic DNA binding to increase the expression of type I interferon,playing an important role in innate immunity.Previous study also found that G3BP1 is overexpressed in various tumor and cancer cells,inhibiting apoptosis and promoting tumor cell proliferation and metastasis.We found that G3BP1 binds to FLNA del41 that exposes the cryptic binding site of R21 as expected.Immunofluorescence microscopy demonstrated that FLNA and G3BP1 colocalize in three different cell lines we tested(HEK293A,MEF,hs SKM).Using FLNA fragment proteins,we found that G3BP1 specifically interacts with mechanical exposed R21.We also identified crucial amino acid residues of G3BP1 for FLNA binding and generated point mutant of G3BP1 that does not interact with FLNA,the mutant of G3BP1 can be used to investigate the biological function of FLNA-G3BP1 interaction in living cells.