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J. Biol. Chem., Vol. 283, Issue 30, 20959-20967, July 25, 2008

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Smooth Muscle Titin Zq Domain Interaction with the Smooth Muscle -Actinin Central Rod^*

Richard J. Chi¹, Alanna R. Simon, Ewa A. Bienkiewicz², Augustine Felix^¶, and Thomas C. S. Keller, III¹

From the Department of Biological Science, College of Medicine, and ^¶Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306

Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells contain the actin filament-cross-linking protein -actinin. In striated muscle Z-disks, -actinin interacts with N-terminal domains of titin to provide a structural linkage crucial for the integrity of the sarcomere. We previously discovered a long titin isoform, originally smitin, hereafter sm-titin, in smooth muscle and demonstrated that native sm-titin interacts with C-terminal EF hand region and central rod R2-R3 spectrin-like repeat region sites in -actinin. Reverse transcription-PCR analysis of RNA from human adult smooth muscles and cultured rat smooth muscle cells and Western blot analysis with a domain-specific antibody presented here revealed that sm-titin contains the titin gene-encoded Zq domain that may bind to the -actinin R2-R3 central rod domain as well as Z-repeat domains that bind to the EF hand region. We investigated whether the sm-titin Zq domain binds to -actinin R2 and R3 spectrin repeat-like domain loops that lie in proximity with two-fold symmetry on the surface of the central rod. Mutations in -actinin R2 and R3 domain loop residues decreased interaction with expressed sm-titin Zq domain in glutathione S-transferase pull-down and solid phase binding assays. Alanine mutation of a region of the Zq domain with high propensity for -helix formation decreased apparent Zq domain dimer formation and decreased Zq interaction with the -actinin R2-R3 region in surface plasmon resonance assays. We present a model in which two sm-titin Zq domains interact with each other and with the two R2-R3 sites in the -actinin central rod.

Received for publication, November 26, 2007 , and in revised form, May 8, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant R01EB006158 (to J. Schlenoff (PI) and T. K. (co-PI)). This work was also funded by Grant 017297 from the Florida State University Cornerstone Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by an American Heart Association Predoctoral Fellowship.

² To whom correspondence should be addressed: 303 BIO, Florida State University, Tallahassee, FL 32306-4370. Fax: 850-644-1406; E-mail: tkeller{at}bio.fsu.edu.

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