Abstract
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.
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