Inositol 1,4,5-trisphosphate receptor localization and stability in neonatal cardiomyocytes requires interaction with ankyrin-B.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Mohler, PJ; Davis, JQ; Davis, LH; Hoffman, JA; Michaely, P; Bennett, V

Published Date

  • March 26, 2004

Published In

Volume / Issue

  • 279 / 13

Start / End Page

  • 12980 - 12987

PubMed ID

  • 14722080

Pubmed Central ID

  • 14722080

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M313979200

Language

  • eng

Conference Location

  • United States