Purification and characterization of an alpha-actinin-binding PDZ-LIM protein that is up-regulated during muscle differentiation

alpha-Actinin is required for the organization and function of the contract ile machinery of muscle. In order to understand more precisely the molecula r mechanisms by which alpha-actinin might contribute to the formation and m aintenance of the contractile apparatus within muscle cells, we performed a screen to identify novel alpha-actinin binding partners present in chicken smooth muscle cells. In this paper, we report the identification, purifica tion, and characterization of a 36-kDa smooth muscle protein (p36) that int eracts with alpha-actinin. Using a variety of in vitro binding assays, we d emonstrate that the association between alpha-actinin and p36 is direct, sp ecific, and saturable and exhibits a moderate affinity. Furthermore, native co-immunoprecipitation reveals that the two proteins are complexed in vice . p36 is expressed in cardiac muscle and tissues enriched in smooth muscle. Interestingly, in skeletal muscle, a closely related protein of 40 kDa (p4 0) is detected. The expression of p36 and p40 is dramatically up-regulated during smooth and skeletal muscle differentiation, respectively, and p40 co localizes with alpha-actinin at the Z-lines of differentiated myotubes. me have established the relationship between p36 and p40 by molecular cloning of cDNAs that encode both proteins and have determined that they are the pr oducts of a single gene. Both proteins display an identical N-terminal PDZ domain and an identical C-terminal LIM domain; an internal 63-amino acid se quence present in p36 is replaced by a unique 111-amino acid sequence in p4 0. Analysis of the sequences of p36 and p40 suggest that they are the avian forms of the actinin-associated LIM proteins (ALPs) recently described in rat (Xia, H., Winokur, S. T., Kuo, W.-L., Altherr, M. R., and Bredt, D. S. (1997) J. Cell Biol. 139, 507-515). The expression of the human ALP gene ha s been postulated to be affected by mutations that cause facioscapulohumera l muscular dystrophy; thus, the characterization of ALP function may ultima tely provide insight into the mechanism of this disease.