Abstract
We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.
Highlights
The cellular cytoskeleton is a highly organized structure composed of three types of filaments: microfilaments, whose principal polymeric component is actin; intermediate filaments, which come in vimentin, keratin, and other “flavors”; and microtubules made of polymeric tubulin
Plakins are a family of large cytoskeletal proteins (Ͼ200 kDa) that serve as cross-linkers between cytoskeletal filaments and in some cases as filament attachment points with the plasma membrane [12]
The region of homology to the actin binding domain (ABD) of ␣-actinin suggested that trabeculin-␣ may bind F-actin [37], which we have confirmed
Summary
Cell Culture—The MRC-5 (human lung fibroblast), CCL-105 (human adrenal cortex carcinoma), and BSC-1 (African green monkey kidney epithelium) cell lines were purchased from ATCC, and the FS-2 (human foreskin fibroblasts) and JMN (human mesothelioma) cells were kindly provided by the late Dr Ruth Sager (Dana-Farber Cancer Institute) and Dr Jim Rheinwald (Brigham and Women’s Hospital), respectively. Nuclei and cell debris were removed by centrifugation, and either total supernatant proteins were precipitated using acetone or immunoprecipitation was performed. Cells were fixed in 2% paraformaldehyde at room temperature for 20 min, followed by washing in PBS and 10 min incubation in sucrose buffer (10 mM Hepes, 3 mM MgCl2, 50 mM NaCl, 300 mM sucrose, 0.5% Triton X-100). The anti-␣-tubulin monoclonal antibody (Sigma) was applied at 37 °C for 1 h, followed by incubation with secondary rhodamine-conjugated anti-mouse antibody (Jackson ImmunoResearch), 100 ng/ml for 30 min at 37 °C, and washing and mounting in glycerol/gelatin (Sigma). Cells were washed, fixed, and labeled as described before
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