Inositol 1,4,5-Trisphosphate Receptor Localization and Stability in Neonatal Cardiomyocytes Requires Interaction with Ankyrin-B

Peter J. Mohler, Jonathan Q. Davis, Lydia H. Davis, Janis A. Hoffman, Peter Michaely, Vann Bennett

Research output: Contribution to journalArticle

69 Scopus citations

Abstract

The molecular mechanisms required for inositol 1,4,5-trisphosphate receptor (InsP3R) targeting to specialized endoplasmic reticulum membrane domains are unknown. We report here a direct, high affinity interaction between InsP3R and ankyrin-B and demonstrate that this association is critical for InsP3R post-translational stability and localization in cultures of neonatal cardiomyocytes. Recombinant ankyrin-B membrane-binding domain directly interacts with purified cerebellar InsP 3R (Kd = 2 nM). 220-kDa ankyrin-B co-immunoprecipitates with InsP3R in tissue extracts from brain, heart, and lung. Alanine-scanning mutagenesis of the ankyrin-B ANK (ankyrin repeat) repeat β-hairpin loop tips revealed that consecutive ANK repeat β-hairpin loop tips (repeats 22-24) are required for InsP3R 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 ∼3-fold reduction in half-life of newly synthesized InsP3R. Furthermore, interactions with ankyrin-B are required for InsP3R stability as abnormal InsP3R 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 InsP3R. These new results provide the first physiological evidence of a molecular partner required for early post-translational stability of InsP3R.

Original languageEnglish (US)
Pages (from-to)12980-12987
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number13
DOIs
StatePublished - Mar 26 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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