Inositol 1,4,5-trisphosphate receptor localization and stability in neonatal cardiomyocytes requires interaction with ankyrin-B.
The molecular mechanisms required for inositol 1,4,5-trisphosphate receptor (InsP(3)R) targeting to specialized endoplasmic reticulum membrane domains are unknown. We report here a direct, high affinity interaction between InsP(3)R and ankyrin-B and demonstrate that this association is critical for InsP(3)R post-translational stability and localization in cultures of neonatal cardiomyocytes. Recombinant ankyrin-B membrane-binding domain directly interacts with purified cerebellar InsP(3)R (K(d) = 2 nm). 220-kDa ankyrin-B co-immunoprecipitates with InsP(3)R in tissue extracts from brain, heart, and lung. Alanine-scanning mutagenesis of the ankyrin-B ANK (ankyrin repeat) repeat beta-hairpin loop tips revealed that consecutive ANK repeat beta-hairpin loop tips (repeats 22-24) are required for InsP(3)R interaction, thus providing the first detailed evidence of how ankyrin polypeptides associate with membrane proteins. Pulse-chase biosynthesis experiments demonstrate that reduction or loss of ankyrin-B in ankyrin-B (+/-) or ankyrin-B (-/-) neonatal cardiomyocytes leads to approximately 3-fold reduction in half-life of newly synthesized InsP(3)R. Furthermore, interactions with ankyrin-B are required for InsP(3)R stability as abnormal InsP(3)R phenotypes, including mis-localization, and reduced half-life in ankyrin-B (+/-) cardiomyocytes can be rescued by green fluorescent protein (GFP)-220-kDa ankyrin-B but not by GFP-220-kDa ankyrin-B mutants, which do not associate with InsP(3)R. These new results provide the first physiological evidence of a molecular partner required for early post-translational stability of InsP(3)R.
Mohler
Peter J
PJ
Howard Hughes Medical Institute and Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA. p.mohler@cellbio.duke.edu
Davis
Jonathan Q
JQ
Davis
Lydia H
LH
Hoffman
Janis A
JA
Michaely
Peter
P
Bennett
Vann
V
D000818:Animals; D000831:Animals, Newborn; D017487:Ankyrins / Q000378:metabolism*; D001921:Brain / Q000196:embryology; D015220:Calcium Channels / Q000096:biosynthesis*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D032383:Myocytes, Cardiac / Q000166:cytology*; D010641:Phenotype; D011233:Precipitin Tests; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014018:Tissue Distribution
C498994:ANK2 protein, human; C498995:Ank2 protein, mouse; D017487:Ankyrins; D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008164:Luminescent Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins