Abstract
To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells. Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195. When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding. The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189. Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding. Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin. Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells. Furthermore, an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro.
Highlights
888 localized to actin-containing structures when expressed in Cos cells
An E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro
Chem. 268 :1298912993) have shown that antibodies to the 27 amino acids implicated in binding of ABP120 to actin can immune precipitate ABP120 from cell lysates, suggesting that these residues are on the surface of the protein as expected for an actin-binding site
Summary
888 localized to actin-containing structures when expressed in Cos cells. an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro. A-ACTININ is a rod-shaped F-actin cross-linking protein found in both muscle and nonmuscle cells at sites where actin is attached to a variety of intracellular structures. It is a homodimer with subunits of molecular mass 94-103 kD arranged in an antiparallel orientation. Analysis of the deduced sequence of the chick smooth muscle isoform of the protein shows that a-actinin can be divided into three distinct domains, an NH2-terminal actin-binding domain spanning. 268 :1298912993) have shown that antibodies to the 27 amino acids implicated in binding of ABP120 to actin can immune precipitate ABP120 from cell lysates, suggesting that these residues are on the surface of the protein as expected for an actin-binding site. At present there is no direct evidence that dystrophin can bind actin
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