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

Suresh K. Joseph, Chi Lin, Shawn Pierson, Andrew P. Thomas, Anthony R. Maranto

Research output: Contribution to journalArticle

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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 co-immunoprecipitate 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.

Original languageEnglish (US)
Pages (from-to)23310-23316
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number40
DOIs
StatePublished - Oct 6 1995

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Inositol
Liver
Rats
Protein Isoforms
Epithelial Cells
Immunoprecipitation
Antibodies
Peptides
Hepatocytes
Inositol 1,4,5-Trisphosphate Receptors
Phosphorylation
Cell membranes
Fractionation
Cyclic AMP-Dependent Protein Kinases
Cell Extracts
Fluorescent Antibody Technique
Sucrose
Agglomeration
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry

Cite this

Heteroligomers of type-I and type-III inositol trisphosphate receptors in WB rat liver epithelial cells. / Joseph, Suresh K.; Lin, Chi; Pierson, Shawn; Thomas, Andrew P.; Maranto, Anthony R.

In: Journal of Biological Chemistry, Vol. 270, No. 40, 06.10.1995, p. 23310-23316.

Research output: Contribution to journalArticle

Joseph, Suresh K. ; Lin, Chi ; Pierson, Shawn ; Thomas, Andrew P. ; Maranto, Anthony R. / Heteroligomers of type-I and type-III inositol trisphosphate receptors in WB rat liver epithelial cells. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 40. pp. 23310-23316.
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