HETEROLIGOMERS OF TYPE-I AND TYPE-III INOSITOL TRISPHOSPHATE RECEPTORS IN WB RAT-LIVER EPITHELIAL-CELLS

We have previously shown that a 222-kDa polypeptide co-immunoprecipitates together with the type-I myoinositol 1,4,5-trisphosphate receptor (IP(3)R) 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 IP(3)R isoform and that type-III IP(3)R antibodies (Abs) can co-immunoprecipitate the type-I IP(3)R isoform. Co-immunoprecipitation of IP(3)R 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 IP(3)R and vice versa; (b) inclusion of the COOH-terminal type-III peptide had no effect on the ability of type-I IP(3)R Ab to co-immunoprecipitate the type III IP(3)R but blocked the ability of type-III IP(3)R Ab to coimmunoprecipitate the type-I isoform; and (c) crude hepatocyte lysates contain undetectable amounts of type III IP(3)R, and immunoprecipitation with type-III IP(3)R Ab does not co-immunoprecipitate any other isoforms, However, type-I and type-II IP(3)R 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 IP(3)R isoforms. Phosphorylation of either type-I or type-III immunoprecipitates by protein kinase A indicated that only the type-I IP(3)R 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 WE cell contains both type-I and type III IP(3)R isoforms and that a proportion of these receptors exist as heterotetramers.