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

• Published in: The Journal of biological chemistry 2008-07, Vol.283 (30), p.20959
• Main Authors: Richard J. Chi, Alanna R. Simon, Ewa A. Bienkiewicz, Augustine Felix, Thomas C. S. Keller III
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M709621200

Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells contain the actin filament-cross-linking protein α-actinin. In striated muscle Z-disks, α-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 α-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 α-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 α-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 α-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 α-helix formation decreased apparent Zq domain dimer formation and decreased Zq interaction with the α-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 α-actinin central rod.