Force-Dependent Structural Changes of Filamin C Rod Domains Regulated by Filamin C Dimer.

Filamin C (FLNC), a large dimeric actin-binding protein in muscle cells, plays a critical role in transmitting force in the cytoskeleton and that between membrane receptors and the cytoskeleton. It performs crucial mechanosensing and downstream mechanotransduction functions via force-dependent interactions with signaling proteins. Mutations in FLNC have been linked to muscle and heart diseases. The mechanical responses of the force-bearing elements in FLNC have not been determined. This study investigated the mechanical responses of FLNC domains and their dimerization interface using magnetic tweezers. Results showed high stability of the N-terminal domains in the rod-1 segment but significant changes in the rod-2 domains in response to forces of a few piconewtons (pN). The dimerization interface, formed by the R24 domain, has a lifetime of seconds to tens of seconds at pN forces, and it dissociates within 1 s at forces greater than 14 pN. The findings suggest the FLNC dimerization interface provides sufficient mechanical stability that enables force-dependent structural changes in rod-2 domains for signaling protein binding and maintains structural integrity of the rod-1 domains.

Phosphorylation Regulation Mechanism of ß2 Integrin for the Binding of Filamin Revealed by Markov State Model.

Leukocyte adhesion deficiency-1 (LAD-1) disorder is a severe immunodeficiency syndrome caused by deficiency or mutation of ß2 integrin. The phosphorylation on threonine 758 of ß2 integrin acts as a molecular switch inhibiting the binding of filamin. However, the switch mechanism of site-specific phosphorylation at the atom level is still poorly understood. To resolve the regulation mechanism, all-atom molecular dynamics simulation and Markov state model were used to study the dynamic regulation pathway of phosphorylation. Wild type system possessed lower binding free energy and fewer number of states than the phosphorylated system. Both systems underwent local disorder-to-order conformation conversion when achieving steady states. To reach steady states, wild type adopted less number of transition paths/shortest path according to the transition path theory than the phosphorylated system. The underlying phosphorylated regulation pathway was from P1 to P0 and then P4 state, and the main driving force should be hydrogen bond and hydrophobic interaction disturbing the secondary structure of phosphorylated states. These studies will shed light on the pathogenesis of LAD-1 disease and lay a foundation for drug development.

Rate-dependent force-extension models for single-molecule force spectroscopy experiments.

Single-molecule force spectroscopy techniques allow for the measurement of several static and dynamic features of macromolecules of biological origin. In particular, atomic force microscopy, used with a variable pulling rate, provides valuable information on the folding/unfolding dynamics of proteins. We propose here two different models able to describe the out-of-equilibrium statistical mechanics of a chain composed of bistable units. These latter represent the protein domains, which can be either folded or unfolded. Both models are based on the Langevin approach and their implementation allows for investigating the effect of the pulling rate and of the device intrinsic elasticity on the chain unfolding response. The theoretical results (both analytical and numerical) have been compared with experimental data concerning the unfolding of the titin and filamin proteins, eventually obtaining a good agreement over a large range of the pulling rates.

Liquid behavior of cross-linked actin bundles.

The actin cytoskeleton is a critical regulator of cytoplasmic architecture and mechanics, essential in a myriad of physiological processes. Here we demonstrate a liquid phase of actin filaments in the presence of the physiological cross-linker, filamin. Filamin condenses short actin filaments into spindle-shaped droplets, or tactoids, with shape dynamics consistent with a continuum model of anisotropic liquids. We find that cross-linker density controls the droplet shape and deformation timescales, consistent with a variable interfacial tension and viscosity. Near the liquid-solid transition, cross-linked actin bundles show behaviors reminiscent of fluid threads, including capillary instabilities and contraction. These data reveal a liquid droplet phase of actin, demixed from the surrounding solution and dominated by interfacial tension. These results suggest a mechanism to control organization, morphology, and dynamics of the actin cytoskeleton.

Filament rigidity and connectivity tune the deformation modes of active biopolymer networks.

Molecular motors embedded within collections of actin and microtubule filaments underlie the dynamics of cytoskeletal assemblies. Understanding the physics of such motor-filament materials is critical to developing a physical model of the cytoskeleton and designing biomimetic active materials. Here, we demonstrate through experiments and simulations that the rigidity and connectivity of filaments in active biopolymer networks regulates the anisotropy and the length scale of the underlying deformations, yielding materials with variable contractility. We find that semiflexible filaments can be compressed and bent by motor stresses, yielding materials that undergo predominantly biaxial deformations. By contrast, rigid filament bundles slide without bending under motor stress, yielding materials that undergo predominantly uniaxial deformations. Networks dominated by biaxial deformations are robustly contractile over a wide range of connectivities, while networks dominated by uniaxial deformations can be tuned from extensile to contractile through cross-linking. These results identify physical parameters that control the forces generated within motor-filament arrays and provide insight into the self-organization and mechanics of cytoskeletal assemblies.

Structure and Function of Filamin C in the Muscle Z-Disc.

Filamin C (FLNC) is one of three filamin proteins (Filamin A (FLNA), Filamin B (FLNB), and FLNC) that cross-link actin filaments and interact with numerous binding partners. FLNC consists of a N-terminal actin-binding domain followed by 24 immunoglobulin-like repeats with two intervening calpain-sensitive hinges separating R15 and R16 (hinge 1) and R23 and R24 (hinge-2). The FLNC subunit is dimerized through R24 and calpain cleaves off the dimerization domain to regulate mobility of the FLNC subunit. FLNC is localized in the Z-disc due to the unique insertion of 82 amino acid residues in repeat 20 and necessary for normal Z-disc formation that connect sarcomeres. Since phosphorylation of FLNC by PKC diminishes the calpain sensitivity, assembly, and disassembly of the Z-disc may be regulated by phosphorylation of FLNC. Mutations of FLNC result in cardiomyopathy and muscle weakness. Although this review will focus on the current understanding of FLNC structure and functions in muscle, we will also discuss other filamins because they share high sequence similarity and are better characterized. We will also discuss a possible role of FLNC as a mechanosensor during muscle contraction.

Deciphering the Role of Filamin B Calponin-Homology Domain in Causing the Larsen Syndrome, Boomerang Dysplasia, and Atelosteogenesis Type I Spectrum Disorders via a Computational Approach.

Filamins (FLN) are a family of actin-binding proteins involved in regulating the cytoskeleton and signaling phenomenon by developing a network with F-actin and FLN-binding partners. The FLN family comprises three conserved isoforms in mammals: FLNA, FLNB, and FLNC. FLNB is a multidomain monomer protein with domains containing an actin-binding N-terminal domain (ABD 1-242), encompassing two calponin-homology domains (assigned CH1 and CH2). Primary variants in FLNB mostly occur in the domain (CH2) and surrounding the hinge-1 region. The four autosomal dominant disorders that are associated with FLNB variants are Larsen syndrome, atelosteogenesis type I (AOI), atelosteogenesis type III (AOIII), and boomerang dysplasia (BD). Despite the intense clustering of FLNB variants contributing to the LS-AO-BD disorders, the genotype-phenotype correlation is still enigmatic. In silico prediction tools and molecular dynamics simulation (MDS) approaches have offered the potential for variant classification and pathogenicity predictions. We retrieved 285 FLNB missense variants from the UniProt, ClinVar, and HGMD databases in the current study. Of these, five and 39 variants were located in the CH1 and CH2 domains, respectively. These variants were subjected to various pathogenicity and stability prediction tools, evolutionary and conservation analyses, and biophysical and physicochemical properties analyses. Molecular dynamics simulation (MDS) was performed on the three candidate variants in the CH2 domain (W148R, F161C, and L171R) that were predicted to be the most pathogenic. The MDS analysis results showed that these three variants are highly compact compared to the native protein, suggesting that they could affect the protein on the structural and functional levels. The computational approach demonstrates the differences between the FLNB mutants and the wild type in a structural and functional context. Our findings expand our knowledge on the genotype-phenotype correlation in FLNB-related LS-AO-BD disorders on the molecular level, which may pave the way for optimizing drug therapy by integrating precision medicine.

Critical Structural Defects Explain Filamin A Mutations Causing Mitral Valve Dysplasia.

Mitral valve diseases affect ∼3% of the population and are the most common reasons for valvular surgery because no drug-based treatments exist. Inheritable genetic mutations have now been established as the cause of mitral valve insufficiency, and four different missense mutations in the filamin A gene (FLNA) have been found in patients suffering from nonsyndromic mitral valve dysplasia (MVD). The filamin A (FLNA) protein is expressed, in particular, in endocardial endothelia during fetal valve morphogenesis and is key in cardiac development. The FLNA-MVD-causing mutations are clustered in the N-terminal region of FLNA. How the mutations in FLNA modify its structure and function has mostly remained elusive. In this study, using NMR spectroscopy and interaction assays, we investigated FLNA-MVD-causing V711D and H743P mutations. Our results clearly indicated that both mutations almost completely destroyed the folding of the FLNA5 domain, where the mutation is located, and also affect the folding of the neighboring FLNA4 domain. The structure of the neighboring FLNA6 domain was not affected by the mutations. These mutations also completely abolish FLNA's interactions with protein tyrosine phosphatase nonreceptor type 12, which has been suggested to contribute to the pathogenesis of FLNA-MVD. Taken together, our results provide an essential structural and molecular framework for understanding the molecular bases of FLNA-MVD, which is crucial for the development of new therapies to replace surgery.

Novel pathogenic variants in filamin C identified in pediatric restrictive cardiomyopathy.

Restrictive cardiomyopathy (RCM) is a rare and distinct form of cardiomyopathy characterized by normal ventricular chamber dimensions, normal myocardial wall thickness, and preserved systolic function. The abnormal myocardium, however, demonstrates impaired relaxation. To date, dominant variants causing RCM have been reported in a small number of sarcomeric or cytoskeletal genes, but the genetic causes in a majority of cases remain unexplained, especially in early childhood. Here, we describe two RCM families with childhood onset: one in a large family with a history of autosomal dominant RCM and the other a family with affected monozygotic, dichorionic/diamniotic twins. Exome sequencing found a pathogenic filamin C (FLNC) variant in each: p.Pro2298Leu, which segregates with disease in the large autosomal dominant RCM family, and p.Tyr2563Cys in both affected twins. In vitro expression of both mutant proteins yielded aggregates of FLNC containing actin in C2C12 myoblast cells. Recently, a number of variants in FLNC have been described that cause hypertrophic, dilated, and restrictive cardiomyopathies. Our data presented here provide further evidence for the role of FLNC in pediatric RCM, and suggest the need to include FLNC in genetic testing of cardiomyopathy patients including those with early ages of onset.

Differential regulation of two FLNA transcripts explains some of the phenotypic heterogeneity in the loss-of-function filaminopathies.

Loss-of-function mutations in the X-linked gene FLNA can lead to abnormal neuronal migration, vascular and cardiac defects, and congenital intestinal pseudo-obstruction (CIPO), the latter characterized by anomalous intestinal smooth muscle layering. Survival in male hemizygotes for such mutations is dependent on retention of residual FLNA function but it is unclear why a subgroup of males with mutations in the 5' end of the gene can present with CIPO alone. Here, we demonstrate evidence for the presence of two FLNA isoforms differing by 28 residues at the N-terminus initiated at ATG+1 and ATG+82 . A male with CIPO (c.18_19del) exclusively expressed FLNA ATG+82 , implicating the longer protein isoform (ATG+1 ) in smooth muscle development. In contrast, mutations leading to reduction of both isoforms are associated with compound phenotypes affecting the brain, heart, and intestine. RNA-seq data revealed three distinct transcription start sites, two of which produce a protein isoform utilizing ATG+1 while the third utilizes ATG+82 . Transcripts sponsoring translational initiation at ATG+1 predominate in intestinal smooth muscle, and are more abundant compared with the level measured in fibroblasts. Together these observations describe a new mechanism of tissue-specific regulation of FLNA that could reflect the differing mechanical requirements of these cell types during development.

Structural and thermodynamic basis of a frontometaphyseal dysplasia mutation in filamin A.

Filamin-mediated linkages between transmembrane receptors (TR) and the actin cytoskeleton are crucial for regulating many cytoskeleton-dependent cellular processes such as cell shape change and migration. A major TR binding site in the immunoglobulin repeat 21 (Ig21) of filamin is masked by the adjacent repeat Ig20, resulting in autoinhibition. The TR binding to this site triggers the relief of Ig20 and protein kinase A (PKA)-mediated phosphorylation of Ser-2152, thereby dynamically regulating the TR-actin linkages. A P2204L mutation in Ig20 reportedly cause frontometaphyseal dysplasia, a skeletal disorder with unknown pathogenesis. We show here that the P2204L mutation impairs a hydrophobic core of Ig20, generating a conformationally fluctuating molten globule-like state. Consequently, unlike in WT filamin, where PKA-mediated Ser-2152 phosphorylation is ligand-dependent, the P2204L mutant is readily accessible to PKA, promoting ligand-independent phosphorylation on Ser-2152. Strong TR peptide ligands from platelet GP1bα and G-protein-coupled receptor MAS effectively bound Ig21 by displacing Ig20 from autoinhibited WT filamin, but surprisingly, the capacity of these ligands to bind the P2204L mutant was much reduced despite the mutation-induced destabilization of the Ig20 structure that supposedly weakens the autoinhibition. Thermodynamic analysis indicated that compared with WT filamin, the conformationally fluctuating state of the Ig20 mutant makes Ig21 enthalpically favorable to bind ligand but with substantial entropic penalty, resulting in total higher free energy and reduced ligand affinity. Overall, our results reveal an unusual structural and thermodynamic basis for the P2204L-induced dysfunction of filamin and frontometaphyseal dysplasia disease.

Filamin B Loss-of-Function Mutation in Dimerization Domain Causes Autosomal-Recessive Spondylocarpotarsal Synostosis Syndrome with Rib Anomalies.

Spondylocarpotarsal synostosis syndrome (SCT) is a distinct group of disorders characterized by short stature, disrupted vertebral segmentation with vertebral fusion, scoliosis, lordosis, carpal/tarsal synostosis, and lack of rib anomalies. Mutations in filamin B (FLNB) and MYH3 have been reported for autosomal-recessive and autosomal-dominant SCT, respectively. We present a family with two patients suffering from autosomal-recessive SCT with rib anomalies, including malalignment, crowding, and uneven size and shape of ribs. Whole-exome sequencing revealed a novel p.S2542Lfs* 82 (c.7621dup) frameshift mutation in FLNB. This frameshift mutation lies in the C-terminal-most domain involved in FLNB dimerization and resulted in a 20-residue elongation, with complete familial segregation and absence in 376 normal controls. The mutant p.S2542Lfs* 82 FLNB demonstrated a complete loss of ability to form a functional dimer in transiently transfected HEK293T cells. The p.S2542Lfs* 82 mutation also led to significantly reduced protein levels and accumulation of the mutant protein in the Golgi apparatus. This is the first identified mutation in the dimerization domain of FLNB. This loss-of-function frameshift mutation in FLNB causes autosomal-recessive SCT with rarely reported rib anomalies. This report demonstrates the involvement of rib anomaly in SCT and its causative mutation in the dimerization domain of FLNB.

Structural Analysis of G1691S Variant in the Human Filamin B Gene Responsible for Larsen Syndrome: A Comparative Computational Approach.

Larsen syndrome (LRS) is a rare genetic disease associated with variable manifestations including skeletal malformations, dislocations of the large joints, and notable changes in facial and limb features. Genetic variants in the Filamin B (FLNB) gene are associated with the development of LRS. We searched two literature databases (OMIM and PubMed) and three gene variant databases (HGMD, UniProt, & dbSNP) to capture all the possible variants associated with LRS phenotype, which may have an impact on the FLNB function. Our search yielded 77 variants that might impact the FLNB protein function in patients with LRS. We performed rigorous computational analysis such as conservational, biochemical, pathogenicity, and structural computational analyses to understand the deleterious effect of the G1691S variant. Further, the structural changes of the G1691S variant was compared with a null variant (G1691A) and the native protein through a molecular dynamic simulation study of 50 ns. We found that the variant G1691S was highly deleterious and destabilize the protein when compared to the native and variant G1691A. This might be due to the physicochemical changes in the variant G1691S when compared to the native and variant G1691A. The destabilization was further supported by transformation of bend to coil in variant G1691S whereas bend was retained in native and variant G1691A through molecular dynamics analysis. Our study shed light on the importance of computational methods to understand the molecular nature of genetic variants and structural insights on the function of the FLNB protein. J. Cell. Biochem. 118: 1900-1910, 2017. © 2017 Wiley Periodicals, Inc.

Filamin A interaction with the CXCR4 third intracellular loop regulates endocytosis and signaling of WT and WHIM-like receptors.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare congenital immunodeficiency often caused by mutations in the last 10 to 19 C-terminal amino acids of CXCR4. These mutations impair CXCR4 internalization and increase responsiveness to CXCL12. The CXCR4 C-terminal domain (C-tail) also has a binding site for the actin-binding protein filamin A (FLNA); it is not known whether FLNA binds to WHIM CXCR4 mutants or whether this interaction is implicated in the hyperfunction of these receptors. Here we show that, in addition to interacting with the CXCR4 C-tail, FLNA interacted with a region in the receptor third intracellular loop (ICL3) spanning amino acids 238 to 246. This interaction involved specific FLNA repeats and was sensitive to Rho kinase inhibition. Deletion of the 238-246 motif accelerated CXCL12-induced wild-type (WT) receptor endocytosis but enabled CXCL12-mediated endocytosis and normalized signaling by the WHIM-associated receptor CXCR4(R334X). CXCL12 stimulation triggered CXCR4(R334X) internalization in FLNA-deficient M2 cells but not in the FLNA-expressing M2 subclone A7; this suggests a role for FLNA in stabilization of WHIM-like CXCR4 at the cell surface. FLNA increased ß-arrestin2 binding to CXCR4(R334X) in vivo, which provides a molecular basis for FLNA-mediated hyperactivation of WHIM receptor signaling. We propose that FLNA interaction with ICL3 is central for endocytosis and signaling of WT and WHIM-like CXCR4 receptors.

Kinetic Ductility and Force-Spike Resistance of Proteins from Single-Molecule Force Spectroscopy.

Ductile materials can absorb spikes in mechanical force, whereas brittle ones fail catastrophically. Here we develop a theory to quantify the kinetic ductility of single molecules from force spectroscopy experiments, relating force-spike resistance to the differential responses of the intact protein and the unfolding transition state to an applied mechanical force. We introduce a class of unistable one-dimensional potential surfaces that encompass previous models as special cases and continuously cover the entire range from ductile to brittle. Compact analytic expressions for force-dependent rates and rupture-force distributions allow us to analyze force-clamp and force-ramp pulling experiments. We find that the force-transmitting protein domains of filamin and titin are kinetically ductile when pulled from their two termini, making them resistant to force spikes. For the mechanostable muscle protein titin, a highly ductile model reconciles data over 10 orders of magnitude in force loading rate from experiment and simulation.

A mechanism of global shape-dependent recognition and phosphorylation of filamin by protein kinase A.

Protein phosphorylation mediates essentially all aspects of cellular life. In humans, this is achieved by ∼500 kinases, each recognizing a specific consensus motif (CM) in the substrates. The majority of CMs are surface-exposed and are thought to be accessible to kinases for phosphorylation. Here we investigated the archetypical protein kinase A (PKA)-mediated phosphorylation of filamin, a major cytoskeletal protein that can adopt an autoinhibited conformation. Surprisingly, autoinhibited filamin is refractory to phosphorylation by PKA on a known Ser(2152) site despite its CM being exposed and the corresponding isolated peptide being readily phosphorylated. Structural analysis revealed that although the CM fits into the PKA active site its surrounding regions sterically clash with the kinase. However, upon ligand binding, filamin undergoes a conformational adjustment, allowing rapid phosphorylation on Ser(2152). These data uncover a novel ligand-induced conformational switch to trigger filamin phosphorylation. They further suggest a substrate shape-dependent filtering mechanism that channels specific exposed CM/kinase recognition in diverse signaling responses.

An adventitious interaction of filamin A with RhoGDI2(Tyr153Glu).

Filamin A (FLNA) is an actin filament crosslinking protein with multiple intracellular binding partners. Mechanical force exposes cryptic FLNA binding sites for some of these ligands. To identify new force-dependent binding interactions, we used a fusion construct composed of two FLNA domains, one of which was previously identified as containing a force-dependent binding site as a bait in a yeast two-hybrid system and identified the Rho dissociation inhibitor 2 (RhoGDI2) as a potential interacting partner. A RhoGDI2 truncate with 81 N-terminal amino acid residues and a phosphomimetic mutant, RhoGDI(Tyr153Glu) interacted with the FLNA construct. However, neither wild-type or full-length RhoGDI2 phosphorylated at Y153 interacted with FLNA. Our interpretation of these contradictions is that truncation and/or mutation of RhoGDI2 perturbs its conformation to expose a site that adventitiously binds FLNA and is not a bona-fide interaction. Therefore, previous studies reporting that a RhoGDI(Y153E) mutant suppresses the metastasis of human bladder cancer cells must be reinvestigated in light of artificial interaction of this point mutant with FLNA.

Actin crosslinking protein filamin A during early pregnancy in the rat uterus.

During early pregnancy the endometrium undergoes a major transformation in order for it to become receptive to blastocyst implantation. The actin cytoskeleton and plasma membrane of luminal uterine epithelial cells (UECs) and the underlying stromal cells undergo dramatic remodelling to facilitate these changes. Filamin A (FLNA), a protein that crosslinks actin filaments and also mediates the anchorage of membrane proteins to the actin cytoskeleton, was investigated in the rat uterus at fertilisation (Day 1) and implantation (Day 6) to determine the role of FLNA in actin cytoskeletal remodelling of UECs and decidua during early pregnancy. Localisation of FLNA in UECs at the time of fertilisation was cytoplasmic, whilst at implantation it was distributed apically; its localisation is under the influence of progesterone. FLNA was also concentrated to the first two to three stromal cell layers at the time of fertilisation and shifted to the primary decidualisation zone at the time of implantation. This shift in localisation was found to be dependent on the decidualisation reaction. Protein abundance of the FLNA 280-kDa monomer and calpain-cleaved fragment (240kDa) did not change during early pregnancy in UECs. Since major actin cytoskeletal remodelling occurs during early pregnancy in UECs and in decidual cells, the changing localisation of FLNA suggests that it may be an important regulator of cytoskeletal remodelling of these cells to allow uterine receptivity and decidualisation necessary for implantation in the rat.

Familial periventricular nodular heterotopia, epilepsy and Melnick-Needles Syndrome caused by a single FLNA mutation with combined gain-of-function and loss-of-function effects.

BACKGROUND: Loss-of-function mutations of the FLNA gene cause a neuronal migration disorder defined as X-linked periventricular nodular heterotopia (PNH); gain-of-function mutations are associated with a group of X-linked skeletal dysplasias designed as otopalatodigital (OPD) spectrum. We describe a family in which a woman and her three daughters exhibited a complex phenotype combining PNH, epilepsy and Melnick-Needles syndrome (MNS), a skeletal disorder assigned to the OPD spectrum. All four individuals harboured a novel non-conservative missense mutation in FLNA exon 3. METHODS: In all affected family members, we performed mutation analysis of the FLNA gene, RT-PCR, ultradeep sequencing analysis in FLNA cDNAs and western blot in lymphocyte cells to further characterise the mutation. We also assessed the effects on RT-PCR products of treatment of patients' lymphocytes with cycloheximide, a nonsense mediated mRNA decay (NMD) inhibitor. RESULTS: We identified a novel c.622G>C change in FLNA exon 3, leading to the substitution of a highly conserved aminoacid (p.Gly208Arg). Gel electrophoresis and ultradeep sequencing revealed the missense mutation as well as retention of intron 3. Cycloheximide treatment demonstrated that the aberrant mRNA transcript-retaining intron 3 is subjected to NMD. Western blot analysis confirmed reduced FLNA levels in lymphocyte cells. CONCLUSIONS: The novel c.622G>C substitution leads to two aberrant FLNA transcripts, one of which carries the missense mutation, plus a longer transcript resulting from intron 3 retention. We propose that the exceptional co-occurrence of PNH and MNS, two otherwise mutually exclusive allelic phenotypes, is the consequence of a single mutational event resulting in co-occurring gain-of-function and loss-of-function effects.

G protein-coupled receptors directly bind filamin A with high affinity and promote filamin phosphorylation.

Although interaction of a few G protein-coupled receptors (GPCRs) with Filamin A, a key actin cross-linking and biomechanical signal transducer protein, has been observed, a comprehensive structure-function analysis of this interaction is lacking. Through a systematic sequence-based analysis, we found that a conserved filamin binding motif is present in the cytoplasmic domains of >20% of the 824 GPCRs encoded in the human genome. Direct high-affinity interaction of filamin binding motif peptides of select GPCRs with the Ig domain of Filamin A was confirmed by nuclear magnetic resonance spectroscopy and isothermal titration calorimetric experiments. Engagement of the filamin binding motif with the Filamin A Ig domain induced the phosphorylation of filamin by protein kinase A in vitro. In transfected cells, agonist activation as well as constitutive activation of representative GPCRs dramatically elicited recruitment and phosphorylation of cellular Filamin A, a phenomenon long known to be crucial for regulating the structure and dynamics of the cytoskeleton. Our data suggest a molecular mechanism for direct GPCR-cytoskeleton coupling via filamin. Until now, GPCR signaling to the cytoskeleton was predominantly thought to be indirect, through canonical G protein-mediated signaling cascades involving GTPases, adenylyl cyclases, phospholipases, ion channels, and protein kinases. We propose that the GPCR-induced filamin phosphorylation pathway is a conserved, novel biochemical signaling paradigm.