Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles.
Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of approximately 800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils. In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.
Bagnato
Paola
P
Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, Italy.
Barone
Virigina
V
Giacomello
Emiliana
E
Rossi
Daniela
D
Sorrentino
Vincenzo
V
D016475:3T3 Cells; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D009119:Muscle Contraction / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D009210:Myofibrils / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D012519:Sarcoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D020798:Two-Hybrid System Techniques
C110202:ANK1 protein, human; C491707:Ank1 protein, mouse; D017487:Ankyrins; D020662:Guanine Nucleotide Exchange Factors; D009124:Muscle Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; C578224:OBSCN protein, human; D017346:Protein Serine-Threonine Kinases