title: Ankyrin-B targets beta2-spectrin to an intracellular compartment in neonatal cardiomyocytes.
abstract: Ankyrin-B is a spectrin-binding protein that is required for localization of inositol 1,4,5-trisphosphate receptor and ryanodine receptor in neonatal cardiomyocytes. This work addresses the interaction between ankyrin-B and beta(2)-spectrin in these cells. Ankyrin-B and beta(2)-spectrin are colocalized in an intracellular striated compartment overlying the M-line and distinct from T-tubules, sarcoplasmic reticulum, Golgi, endoplasmic reticulum, lysosomes, and endosomes. Beta(2)-Spectrin is absent in ankyrin-B-null cardiomyocytes and is restored to a normal striated pattern by rescue with green fluorescent protein-220-kDa ankyrin-B. We identified two mutants (A1000P and DAR976AAA) located in the ZU5 domain which eliminate spectrin binding activity of ankyrin-B. Ankyrin-B mutants lacking spectrin binding activity are normally targeted but do not reestablish beta(2)-spectrin in ankyrin-B(+/-) cardiomyocytes. However, both mutant forms of ankyrin-B are still capable of restoring inositol 1,4,5-trisphosphate receptor localization and normal contraction frequency of cardiomyocytes. Therefore, direct binding of beta(2)-spectrin to ankyrin-B is required for the normal targeting of beta(2)-spectrin in neonatal cardiomyocytes. In contrast, ankyrin-B localization and function are independent of beta(2)-spectrin. In summary, this work demonstrates that interaction between members of the ankyrin and beta-spectrin families previously established in erythrocytes and axon initial segments also occurs in neonatal cardiomyocytes with ankyrin-B and beta(2)-spectrin. This work also establishes a functional hierarchy in which ankyrin-B determines the localization of beta(2)-spectrin and operates independently of beta(2)-spectrin in its role in organizing membrane-spanning proteins.
authors.affiliation: Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA. p.mohler@cellbio.duke.edu
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Mohler
authors.forename: Woohyun
authors.initials: W
authors.lastname: Yoon
authors.forename: Vann
authors.initials: V
authors.lastname: Bennett
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D017487:Ankyrins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D015220:Calcium Channels / Q000378:metabolism; D002451:Cell Compartmentation / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009200:Myocardial Contraction / Q000502:physiology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D013049:Spectrin / Q000378:metabolism*
chemical_list: C498994:ANK2 protein, human; C498995:Ank2 protein, mouse; D017487:Ankyrins; D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C452439:SPTBN1 protein, human; D013049:Spectrin