Biosynthesis of inositol trisphosphate receptors: selective association with the molecular chaperone calnexin

Abstract

A prominent labelled polypeptide having the same mobility as type-I inositol trisphosphate receptor (IP₃R) was immunoprecipitated from WB-cell lysates by antibodies to calnexin, an ER integral membrane chaperone. The identity of this polypeptide was confirmed by re-immunoprecipitation of the radioactive polypeptides released from calnexin-antibody immunoprecipitates with type-I IP₃R antibody. The interaction of calnexin with newly synthesized type-I IP₃R was transient and inhibited by treatment of the cells with dithiothreitol or the glucosidase inhibitor N-methyldeoxynojirimicin. In similar experiments, there was no evidence for the binding of type-I IP₃R to calreticulin, an ER luminal chaperone. Calnexin (but not calreticulin) associated with newly synthesized FLAG (DYKDDDDK epitope)-tagged type-III IP₃R expressed in COS-7 cells. In order to further define the mechanism of interaction of nascent IP₃R with chaperones, we have utilized an in vitro rabbit reticulocyte translation assay programmed with RNA templates encoding the six putative transmembrane (TM) domains of IP₃Rs. In accordance with the known preference of calnexin for monoglucosylated oligosaccharide chains, calnexin antibody preferentially immunoprecipitated a proportion of glycosylated type-I translation product. Addition of glucosidase inhibitors prevented the association of calnexin with in vitro translated type-I TM construct. Using truncated RNA templates we found that calnexin did not associate with the first four TM domains but retained affinity for the construct encoding TM domains 5 and 6, which contains the glycosylation sites. We propose that calnexin is a key chaperone involved in the folding, assembly and oligomerization of newly synthesized IP₃ receptors in the ER.