Molecular cloning and characterization of human trabeculin-alpha a giant protein defining a new family of actin-binding proteins

We describe the molecular cloning and characterization of a novel giant hum an cytoplasmic protein, trabeculin-alpha (M-r = 614,000), Analysis of the d educed amino acid sequence reveals homologies with several putative functio nal domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 c opies of a spectrin-like motif in the central region of the protein; two po tential Ca2+-binding EF-hand motifs; and a Ser-rich region containing a rep eated GSRX motif, With similarities to both plakins and spectrins, trabecul in-alpha appears to have evolved as a hybrid of these two families of prote ins. The functionality of the actin binding domains located near the N term inus was confirmed with an F-actin binding assay using glutathione S-transf erase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that t rabeculin is ubiquitously expressed and is distributed throughout the cytop lasm, though the protein was found to be greatly up-regulated upon differen tiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests th at trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.