Chicken gizzard filamin, retina filamin and cgABP260 are respectively, smooth muscle-, non-muscle- and pan-muscle-type isoforms: distribution and localization in muscles

Abstract

We determined the full cDNA sequences of chicken gizzard filamin and cgABP260 (chicken gizzard actin-binding protein 260). The primary and secondary structures predicted by these sequences were similar to those of chicken retina filamin and human filamins. Like mammals, chickens have 3 filamin isoforms. Comparison of their amino acid sequences indicated that gizzard filamin, retina filamin, and cgABP260 were the counterparts of human FLNa (filamin a), b, and c, respectively. Antibodies against the actin-binding domain (ABD) of these 3 filamin isoforms were raised in rabbits. Using immunoabsorption and affinity chromatography, we prepared the monospecific antibody against the ABD of each filamin. In immunoblotting, the antibody against the gizzard filamin ABD detected a single band in gizzard, but not in striated muscles or brain. In brain, only the antibody against the retina filamin ABD produced a strong single band. The antibody against the cgABP260 ABD detected a single peptide band in smooth, skeletal, and cardiac muscle. In immunofluorescence microscopy of muscular tissues using these antibodies, the antibody against the gizzard filamin ABD only stained smooth muscle cells, and the antibody against the retina filamin ABD strongly stained endothelial cells of blood vessels and weakly stained cells in connective tissue. The antibody against the cgABP260 ABD stained the Z-lines and myotendinous junctions of breast muscle, the Z-lines and intercalated disks of cardiac muscle, and dense plaques of smooth muscle. These findings indicate that chicken gizzard filamin, retina filamin, and cgABP260 are, respectively, smooth muscle-type, non-muscle-type, and pan-muscle-type filamin isoforms.