Monoclonal antibodies distinctively recognizing the subtypes of inositol 1,4,5-trisphosphate receptor: Application to the studies on inflammatory cells

Abstract

Monoclonal antibodies were raised that specifically recognize the COOH-terminal sequences and the loop sequences between the fifth and the sixth transmembrane spanning regions of human inositol 1,4,5-trisphosphate receptor (IP₃R) type 1, 2 and 3. Western blot analysis using Jurkat cells, mouse cerebellum, COS-7 expressing IP₃R type 3 cDNA showed that those monoclonal antibodies reacted specifically with each of these three IP₃R subtypes and that they do not cross-react. These antibodies could be used for the specific immunoprecipitation of IP₃Rs. Using these monoclonal antibodies, the expression profiles of IP₃R-subtype proteins were found to be different among inflammatory cells such as macrophages, polymorphonuclear cells, mast cells, eosinophils, splenocytes, thymocytes and megakaryocytic cells. Usually, more than one type of IP₃R were expressed in a cell simultaneously. The observation of CMK cells under immunofluorescence confocal microscopy revealed that IP₃R type 1 and type 2 are located at different subcellular fractions.