Biosynthesis and processing of the human insulin receptor.
We investigated the biosynthesis of the human insulin receptor in IM-9 lymphocytes and HEP-G2 hepatoma cells. Cells were first pulse labeled for 15 min with [35S]methionine and then chased for up to 4 h. At each time, the cells were solubilized in 1% Triton X-100; the insulin receptor was immunoprecipitated and then analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6%) and fluorography. At 15 min, a major precursor protein of 190,000 Mr was precipitated. During the chase period, two smaller proteins became apparent, which evolved into two major species of 130,000 and 95,000 Mr, the mature alpha- and beta-subunits, respectively. When IM-9 cells were trypsinized after pulse chase, the alpha- and beta-subunits were completely digested, whereas the 190,000-Mr precursor was unaffected. 125I-surface labeling of cells, followed by immunoprecipitation, revealed the presence of only the alpha- and beta-subunits, indicating that only these two species were on the cell surface. To study this biosynthetic pathway, several inhibitors were used (tunicamycin, monensin, and swainsonine). These inhibitors revealed the following. The receptor is first synthesized as a 170,000-Mr protein that is cotranslationally N-glycosylated to yield a high-mannose 190,000-Mr precursor. This precursor is rapidly transported from the endoplasmic reticulum to the Golgi apparatus where it is cleaved into two subunits of 120,000 Mr (alpha) and 90,000 Mr (beta). These subunits then increase in molecular weight by processing of the high-mannose oligosaccharides to the low-mannose complex type. The two subunits then migrate to the cell surface where they function to transmit the insulin signal.
D002460:Cell Line; D011232:Chemical Precipitation; D006023:Glycoproteins / Q000096:biosynthesis; D006026:Glycoside Hydrolases; D006801:Humans; D007120:Immunochemistry; D011498:Protein Precursors / Q000096:biosynthesis; D011499:Protein Processing, Post-Translational; D011869:Radioligand Assay; D011972:Receptor, Insulin / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism; D012995:Solubility; D014357:Trypsin
D006023:Glycoproteins; D011498:Protein Precursors; D011972:Receptor, Insulin; D006026:Glycoside Hydrolases; D014357:Trypsin