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The characterization of a novel 59-kD cytoskeletal protein is described. It is exclusively observed in smooth muscle cells by Northern blotting and immunohistochemical analysis and therefore designated "smoothelin." A human smooth muscle cDNA library was screened with the monoclonal antibody R4A, and a full-size cDNA of the protein was selected. The cDNA was sequenced and appeared to contain a 1,113-bp open reading frame. Based on the cDNA sequence, the calculated molecular weight of the polypeptide was 40 kD and it was demonstrated to contain two N-glycosylation sites. Computer assisted analysis at the protein level revealed a 56-amino acid domain with homologies of approximately 40% with a sequence bordering the actin-binding domains of dystrophin, utrophin, beta-spectrin and alpha-actinin. In situ hybridization demonstrated that human smoothelin is encoded by a single copy gene which is located on chromosome 22. Immunohistochemistry and Western blotting revealed synthesis of smoothelin in smooth muscle of species evolutionarily as far apart as human and teleost. Northern blotting indicated that sequence as well as size of the mRNA (approximately 1,500 bases) are conserved among vertebrates. Cell fractionation studies and differential centrifugation showed that the protein cannot be extracted with Triton X-100, which indicates that it is a part of the cytoskeleton. Transfection of the human cDNA into smooth muscle cells and COS7 cells produced a protein of 59 kD, which assembled into a filamentous network. However, in rat heart-derived myoblasts association with stress fibers was most prominent. Smoothelin was not detected in primary or long term smooth muscle cell cultures. Also, transcription of smoothelin mRNA was almost instantly halted in smooth muscle tissue explants. We conclude that smoothelin is a new cytoskeletal protein that is only found in contractile smooth muscle cells and does not belong to one of the classes of structural proteins presently known.