Smooth muscle titin Zq domain interaction with the smooth muscle alpha-actinin central rod.
Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells contain the actin filament-cross-linking protein alpha-actinin. In striated muscle Z-disks, alpha-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 alpha-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 alpha-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 alpha-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 alpha-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 alpha-helix formation decreased apparent Zq domain dimer formation and decreased Zq interaction with the alpha-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 alpha-actinin central rod.
Department of Biological Science, College of Medicine, Florida State University, Tallahassee, FL 32306, USA.
Richard J
RJ
Chi
Alanna R
AR
Simon
Ewa A
EA
Bienkiewicz
Augustine
A
Felix
Thomas C S
TC
Keller
D000185:Actinin / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000378:metabolism; D001665:Binding Sites; D002645:Chickens; D064211:Connectin; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014599:Uterus / Q000378:metabolism
D064211:Connectin; D009124:Muscle Proteins; C577285:TTN protein, human; D000185:Actinin; D011494:Protein Kinases