Heteroligomers of type-I and type-III inositol trisphosphate receptors in WB rat liver epithelial cells

J Biol Chem. 1995 Oct 6;270(40):23310-6. doi: 10.1074/jbc.270.40.23310.

Abstract

We have previously shown that a 222-kDa polypeptide co-immunoprecipitates together with the type-I myoinositol 1,4,5-trisphosphate receptor (IP3R) in WB rat liver epithelial cell extracts, when the immunoprecipitation is carried out with a type-I isoform specific antibody (Joseph, S. K. (1994) J. Biol. Chem. 269, 5673-5679). Utilizing isoform-specific antibodies raised to unique sequences within the COOH-terminal region of IP3 receptors, we now report that the co-immunoprecipitating 222-kDa polypeptide is the type-III IP3R isoform and that type-III IP3R antibodies (Abs) can co-immunoprecipitate the type-I IP3R isoform. Co-immunoprecipitation of IP3R isoforms was not due to cross-reactivity of the antibodies for the following reasons: (a) on immunoblots the type-III antibodies did not cross-react with type-I IP3R and vice versa; (b) inclusion of the COOH-terminal type-III peptide had no effect on the ability of type-I IP3R Ab to co-immunoprecipitate the type-III IP3R but blocked the ability of type-III IP3R Ab to coimmunoprecipitate the type-I isoform; and (c) crude hepatocyte lysates contain undetectable amounts of type-III IP3R, and immunoprecipitation with type-III IP3R Ab does not co-immunoprecipitate any other isoforms. However, type-I and type-II IP3R isoforms were co-immunoprecipitated by their respective antibodies in hepatocyte lysates. Sucrose density gradient analysis of WB cell lysates indicated that the co-immunoprecipitating fraction is exclusively located at the density expected for tetrameric receptors, suggesting that co-immunoprecipitation was not a reflection of the nonspecific aggregation of IP3R isoforms. Phosphorylation of either type-I or type-III immunoprecipitates by protein kinase A indicated that only the type-I IP3R could be phosphorylated in vitro. Fractionation of WB cell membranes and immunofluorescence studies showed that the type-I and type-III isoforms have very similar sub-cellular localizations. We conclude that the WB cell contains both type-I and type-III IP3R isoforms and that a proportion of these receptors exist as heterotetramers.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Calcium Channels / chemistry*
  • Calcium Channels / genetics
  • Calcium Channels / isolation & purification
  • Cell Line
  • Epithelium / metabolism
  • Immunochemistry
  • In Vitro Techniques
  • Inositol 1,4,5-Trisphosphate / metabolism*
  • Inositol 1,4,5-Trisphosphate Receptors
  • Liver / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • Phosphorylation
  • Precipitin Tests
  • Protein Conformation
  • Rats
  • Receptors, Cytoplasmic and Nuclear / chemistry*
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / isolation & purification
  • Subcellular Fractions / metabolism

Substances

  • Calcium Channels
  • Inositol 1,4,5-Trisphosphate Receptors
  • Receptors, Cytoplasmic and Nuclear
  • Inositol 1,4,5-Trisphosphate