Using normalized protein standards for FlnA and FlnB, the FlnA protein expression is usually 1

Using normalized protein standards for FlnA and FlnB, the FlnA protein expression is usually 1.35 fold that of FlnB in 293 cells. diminished expression of 1-integrin, whereas FlnB loss promotes integrin expression. Finally, fibronectin mediated integrin activation has been shown to activate RhoA and activated RhoA leads to stress fiber formation and cell spreading. 7-Methylguanine Fibronectin stimulation in null FlnA cells impairs enhanced spreading whereas FlnB inhibited cells show enhanced spreading. While filamins serve a primary static function in stabilization of 7-Methylguanine the actin cytoskeleton, these studies are the first to demonstrate a dynamic and antagonistic relationship between different filamin isoforms in the dynamic Cd248 regulation of integrin expression, RhoGTPase activity and actin stress fiber remodeling. Introduction Filamins comprise a family of actin-binding proteins responsible for diverse biological functions. In general, they are comprised of an N-terminal actin-binding domain name, followed by immunoglobulin-like repeat domains that form a receptor binding region at the C-terminus. This structure allows for receptor activation and transduction of signals onto the actin cytoskeleton, thereby directing various cell functions including membrane stability, protrusion, and motility (1,2).The three members of the filamin family of proteins (Filamin A, B, and C) share a high degree of homology between the conserved exon/intron structure (3). 7-Methylguanine Moreover, previous studies showed that Filamin A (FlnA) and Filamin B (FlnB) actually interact and heterodimerize, potentially suggesting a shared mechanism with which to regulate each others function (4). Disruption of these proteins has been shown to give rise to multiple human developmental disorders. Humans harboring mutations in the Filamin A are known to develop a wide variety of disorders, including periventricular heterotopia (malformation of brain development), otopalatodigital syndrome and Melnick-Needles syndrome. However, more recent work has also exhibited problems in skeletal, cardiac, pulmonary, dermal, and gastrointestinal development (5,6). Recessively and dominantly inherited mutations in FLNB can result in dwarfism and skeletal dysplasia with joint dislocations, respectively (7). Filamin C (FlnC) defects lead to an underlying myopathy (8). These varied phenotypes reiterate the broad and important role that filamins and actin play in both development and maintenance of numerous cell types. A fundamental question exists as to whether the different filamins play comparable roles in different organ systems or whether each filamin gene subserves specific functions in 7-Methylguanine a shared pathway. In the current studies, we focus on FlnA and FlnB because loss of either protein results in skeletal defects, with both shared as well as distinct bone phenotypes (7,9). We find that FlnA and FlnB are broadly expressed in multiple organ systems although FlnA is usually more highly expressed in certain tissues, the two proteins actually interact to form heterodimers, and they share overlapping expression with cytoplasmic RhoA in chondrocytes. Both filamin proteins share overlapping static functions by stabilizing the actin cytoskeleton in unstimulated chondrocytes. Loss of expression of one filamin isoform leads to upregulation of the other, consistent with compensation. Actin assembly can be regulated by RhoGTPases and we find that FlnA more strongly binds RhoA GTPase than FlnB. While total RhoA levels are unchanged following FlnA/B inhibition, activated RhoA levels are increased with prolonged loss of FlnB and decreased with loss of FlnA. Moreover, loss of FlnA inhibits integrin expression and decreases stress fiber formation whereas FlnB knockdown promotes these processes. Finally, cell spreading (an indicator of RhoA activation and stress fiber formation) is usually impaired with loss of FlnA and promoted by loss of FlnB, after RhoA activation through fibronectin-integrin stimulation. Collectively, these findings suggest that FlnA actually binds to RhoA and upregulate its activity to affect downstream changes. While FlnB binds to RhoA to a lesser extent, it antagonizes RhoA activation though the formation of homo-dimers and hetero-dimers with FlnA. As a result, the dynamic interactions between FlnA and.