title: The ankyrin-B C-terminal domain determines activity of ankyrin-B/G chimeras in rescue of abnormal inositol 1,4,5-trisphosphate and ryanodine receptor distribution in ankyrin-B (-/-) neonatal cardiomyocytes.
abstract: Ankyrins are a closely related family of membrane adaptor proteins that are believed to participate in targeting diverse membrane proteins to specialized domains in the plasma membrane and endoplasmic reticulum. This study addresses the question of how individual ankyrin isoforms achieve functional specificity when co-expressed in the same cell. Cardiomyocytes from ankyrin-B (-/-) mice display mis-localization of inositol 1,4,5-trisphosphate receptors and ryanodine receptors along with reduced contraction rates that can be rescued by expression of green fluorescent protein (GFP)-ankyrin-B but not GFP-ankyrin-G. We developed chimeric GFP expression constructs containing all combinations of the three major domains of ankyrin-B and ankyrin-G to determine which domain(s) of ankyrin-B are required for ankyrin-B-specific functions. The death/C-terminal domain of ankyrin-B determined activity of ankyrin-B/G chimeras in localization in a striated pattern in cardiomyocytes and in restoration of a normal striated distribution of both ryanodine and inositol 1,4,5-trisphosphate receptors as well as normal beat frequency of contracting cardiomyocytes. Further deletions within the death/C-terminal domain demonstrated that the C-terminal domain determines ankyrin-B activity, whereas deletion of the death domain had no effect. C-terminal domains are the most divergent between ankyrin isoforms and are candidates to encode the signal(s) that enable ankyrins to selectively target proteins to diverse cellular sites.
authors.affiliation: Howard Hughes Medical Institute and Department of Cell Biology, 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: Anthony O
authors.initials: AO
authors.lastname: Gramolini
authors.forename: Vann
authors.initials: V
authors.lastname: Bennett
mesh_terms: D000185:Actinin / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D017487:Ankyrins / Q000737:chemistry*; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: C498994:ANK2 protein, human; C490613:ANK3 protein, human; C498995:Ank2 protein, mouse; C094681:Ank3 protein, mouse; C490614:Ank3 protein, rat; D017487:Ankyrins; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D000185:Actinin; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate