Biosynthesis of the inositol trisphosphate receptor in WB rat liver epithelial cells.
We have studied the biosynthesis and turnover of the inositol 1,4,5-trisphosphate receptor (IP3R) in WB cells, a rat liver epithelial cell line. In detergent extracts of 35S-labeled WB cells, an affinity-purified antibody directed at the C terminus of the IP3R immunoprecipitated two labeled polypeptides, a major band at 233 kDa and a minor band at 222 kDa. The major band was shown to correspond to the IP3R by immunoblotting. The minor band was not a proteolytic clip of the IP3R because, unlike the IP3R, the 222-kDa band was not a glycoprotein or a substrate for protein kinase A and was not recognized by three different IP3R antibodies on immunoblots. The identity and function of this co-immunoprecipitating protein is unknown. The IP3R in WB cells was 5 kDa smaller than the rat cerebellar IP3R. Significant changes in the molecular weight of the IP3R were not observed in pulse-chase experiments, indicating that extensive proteolytic or carbohydrate processing events do not occur during biosynthesis of the receptor. From the analysis of such experiments in confluent WB cells, it was determined that the half-life of the IP3R protein was 11 h. Chromatography of the 35S-labeled extracts on Sephacryl S-400 columns revealed rapid oligomerization of newly synthesized protein with > 95% of newly synthesized protein forming tetramers during a short (10 min) pulse labeling period. However, significant amounts of mature IP3R were found as incompletely oligomerized forms. Mature IP3R was bound by concanavalin A-Sepharose beads, and binding was greatly reduced by endoglycosidase H treatment of the receptor. Endoglycosidase H sensitivity, absence of binding to wheat germ agglutinin-Sepharose, and insensitivity of biosynthesis or oligomerization to brefeldin A suggest that processing of the WB IP3R does not involve transit through the Golgi apparatus.
Joseph
S K
SK
Department of Pathology and Cell Biology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107.
D000818:Animals; D015220:Calcium Channels / Q000096:biosynthesis* / Q000378:metabolism; D002478:Cells, Cultured; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis* / Q000378:metabolism
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate