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

A prominent labelled polypeptide having the same mobility as type-I inosito l 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 radioac tive polypeptides released from calnexin-antibody immunoprecipitates with t ype-I IP3R antibody. The interaction of calnexin with newly synthesized typ e-I IP3R was transient and inhibited by treatment of the cells with dithiot hreitol or the glucosidase inhibitor N-methyldeoxynojirimicin. In similar e xperiments, there was no evidence for the binding of type-I IP3R to calreti culin, an ER luminal chaperone. Calnexin (but not calreticulin) associated with newly synthesized FLAG (DYKDDDDK epitope)-tagged type-III IP3R express ed in COS-7 cells. Tn order to further define the mechanism of interaction of nascent IP3R with chaperones, we have utilized an in vitro rabbit reticu locyte translation assay programmed with RNA templates encoding the six put ative transmembrane (TM) domains of IP(3)Rs. In accordance with the known p reference of calnexin for monoglucosylated oligosaccharide chains, calnexin antibody preferentially immunoprecipitated a proportion of glycosylated ty pe-I translation product. Addition of glucosidase inhibitors prevented the association of calnexin with in I vitro translated type-I TM construct. Usi ng truncated RNA templates we found that calnexin did not associate with th e first four TM domains but retained affinity for the construct encoding TM domains 5 and 6, which contains the glycosylation sites. We propose that c alnexin is a key chaperone involved in the folding, assembly and oligomeriz ation of newly synthesized IP3 receptors in the ER.