Identification Of LARP4 As A New Filamin A Mechanobinding Protein

Mechanotransduction is the ability of a cell to sense mechanical cues from its microenvironment and convert them into biochemical signals to induce cellular responses and gene expression.Increasing evidence suggests that mechanotransduction is involved in many physiological and disease conditions,ranging from early embryonic development,organogenesis,to a variety of human diseases including cancer.Mechanotransduction is mediated through several classes of senor proteins on the cell surface,intracellular signaling mediators,and core transcriptional regulation networks.Previous studies found that filamin A(FLNA)is a mechanosensor and mechanotransducer that senses and converts mechanical forces into biochemical signals and thus mediates the mechnotransduction.FLNA is an actin-crosslinking protein,which also anchors a variety of proteins in the cytoskeleton and regulates cell adhesion and migration.It is involved in signal transduction to regulate cell proliferation and differentiation as well.Loss or overexpression of FLNA results in aberrant development of the brain,heart,bone,gastrointestinal tract and megakaryocyte.Many studies have shown that FLNA plays a crucial role in the occurrence of genetic diseases and the development of tumors.FLNA is a homodimer protein which consists of an N-terminal actin-binding domain followed by 24 immunoglobulin(Ig)-like repeats(R).Two hinges separate the subunit into two rods.Repeat 1–15 is referred to as the rod 1 and 16–23 as the rod 2,and the C-terminal repeat 24 is responsible for the dimerization.The rod 2,where interactions of FLNA with its over 150 binding partners take place,has a unique geometry and a compact structure that responds to mechanical force through conformational changes to regulate its partner interactions.Force-induced conformational changes in FLNA rod 2 can regulate partner interactions by exposing a cryptic binding site of R21 CD face.In fact,it was experimentally demonstrated that the CD face of R21 is covered by the strand A of R20,but the mechanical force opens the CD face,allowing the integrin to bind.A probe to monitor R20/R21 domain unfolding of FLNA in living cells demonstrated that this domain unfolds predominately in protruding cytoplasm at the cell edges but not at integrin-rich focal adhesions.This unexpected result suggesting that other molecules recruit the exposed R21 binding site.Thus,this project aims to identify such molecule,a mechanobinding protein of FLNA R21.To discover a new FLNA mechanobinding protein,we have established a method to identify a binding partner that specifically interacts with the mechanosensitive binding site by means of isotope labeling amino acids in cell culturing(SILAC)-based proteomics using R21-22 as affinity ligand.As a negative control,R1-2 was used because CD faces of R1-2 are structurally different from that of R21.The mass spectrum data indicated several potential binding partners.Here,we identified LARP4,La-related protein-4,as a new mechanobinding partner of FLNA.LARP4 is an RNA binding protein that binds to the poly-A sequence of m RNA.Overexpression of LARP4 resulted in increased m RNA stability whereas knockdown of LARP4 caused a 15-20% reduction in translation,indicating that LARP4 promotes m RNA stability.LARP4 also regulate cell morphology through translational regulation of m RNAs encoding cytoskeletal regulators.Furthermore,the interaction of LARP4 with Receptor for Activated C Kinase(RACK1)may be particularly relevant in morphological regulation,as RACK1 has been reported to play a role in cell adhesion and migration.Importantly catalogue of somatic mutations in cancer(COSMIC)reports more than 224 LARP4 mutations in various cancer types,but their genotype-phenotype relationships are not known.We validated the interaction between LARP4 and FLNA R21 both in vivo and in vitro.Immunofluorescence microscopy clearly demonstrated the co-localization of FLNA and LARP4 in human embryonic kidney(HEK)293A and homo sapience skeletal muscle(hs SKM)line,especially localized in the cell-cell junction.Using the in vitro site-directed mutagenesis,we determined the critical amino acid that is responsible for the interaction and generated the non-FLNA-binding mutant LARP4 that disrupts the interaction.Fluorescence recovery after photobleaching(FRAP)of GFP-labeled LARP4 in living cells demonstrated that non-FLNA-binding mutant LARP4 diffuses faster than wild-type LARP4.Taken together,our finding reveals that LARP4 is a new FLNA mechanobinding partner which specifically interacts with cryptic FLNA R21 binding site that is exposed by mechanical forces.