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The striated muscle Z-line is a complex network of proteins in which N-terminal domains of titin interact with the actin filament cross-linker alpha-actinin, and other proteins to establish and maintain the structural integrity of the sarcomere. Actin-myosin II filament-based contractile structures in smooth muscle and nonmuscle cells also contain the actin filament-crosslinking protein alpha-actinin. We previously discovered a titin isoform - originally named smitin, hereafter called sm-titin - in smooth muscle cells suggesting that similar interactions may exist in the alpha-actinin rich dense bodies and dense plaques that act as the smooth muscle equivalent of the Z-line. I have found that purified native smooth muscle alpha-actinin binds with nanomolar affinity to sm-titin in sm-titin-myosin coassemblies in vitro. Smooth muscle alpha-actinin also interacts with striated muscle titin. In contrast to striated muscle alpha-actinin interaction with titin and sm-titin, which is significantly enhanced by PIP2, smooth muscle alpha-actinin interacts with sm-titin and titin equally well in the presence and absence of PIP2. Using expressed regions of smooth muscle alpha-actinin, I have demonstrated sm-titin-binding sites in the smooth muscle alpha-actinin R2-R3 spectrin-like repeat rod domain and a C-terminal domain formed by cryptic EF hand structures. These sm-titin-binding sites are highly homologous to the titin-binding sites of striated muscle alpha-actinin. This suggests that sm-titin contains at least some of the domains found in titin that are known to bind to the alpha-actinin sites. In striated muscle Z-disks, titin N-terminal Z-repeat domains interact with the alpha-actinin EF hand region and the titin Zq domain interacts with the alpha-actinin central rod. RT-PCR analysis of RNA from various smooth muscle sources and western blot analysis with an N-terminal region-specific antibody presented here reveals that sm-titin contains the alpha-actinin-binding Z repeats Zr1, Zr2, Zr3, and Zr7 and the Zq domain encoded by the titin gene. I investigated whether the sm-titin Zq domain interacts with R2 and R3 spectrin repeat-like domain loops that lie in proximity on the surface of the smooth muscle alpha-actinin central rod. I found alanine and phosphomimetic mutations of alpha-actinin R2 and R3 loop residues decreased binding to expressed sm-titin Zq domain in GST-pull-down and solid phase binding assays. Likewise, surface plasmon resonance experiments revealed that alanine mutagenesis of a Zq domain region with high propensity to form alpha-helix decreased binding to the alpha-actinin R2-R3 region. Additionally, the Zq peptide migrates on FPLC size exclusion chromatography as an apparent dimer. I present a model of how the sm-titin Zq domain, which may form an anti-parallel homodimer, could interact with mirror image sites formed by R2 and R3 loops in the smooth muscle alpha-actinin central rod domain. Taken together, our results suggest that direct interaction between alpha-actinin and titin or titin isoforms is a common feature of actin-myosin II contractile structures in striated muscle and smooth muscle cells and that the molecular bases for alpha-actinin interaction with these proteins are similar, although regulation of these interactions may differ according to tissue.
A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
Thomas C. S. Keller, III, Professor Directing Dissertation; Michael Blaber, Outside Committee Member; Kenneth A. Taylor, Committee Member; Piotr G. Fajer, Committee Member; P. Bryant Chase, Committee Member.
Florida State University
Chi, R. J. -H. (2007). Smooth Muscle Titin Interactions with Alpha-Actinin. Retrieved from http://purl.flvc.org/fsu/fd/FSU_migr_etd-3821