The insulin receptor functions normally in Chinese hamster ovary cells after truncation of the C terminus.
We studied the structure and function of the human insulin receptor (IR) and a mutant which lacked the last 43 amino acids of the beta-subunit (IR delta ct). This deletion removed tyrosine (Tyr1322, Tyr1316) and threonine (Thr1336) phosphorylation sites. In Chinese hamster ovary (CHO) cells, insulin binding to the mutant receptor was normal, and [35S]methionine labeling indicated that both the IR and IR delta ct were processed normally; however, the beta-subunit of IR delta ct was 5 kDa smaller than that of the IR. The time course of insulin-stimulated autophosphorylation of the partially purified IR delta ct was normal, but the maximum autophosphorylation was reduced 20-30%. Tryptic phosphopeptide mapping confirmed the absence of the C-terminal phosphorylation sites and indicated that phosphorylation of the regulatory region (Tyr1146, Tyr1150, Tyr1151) occurred normally; kinase activity of the IR and IR delta ct was activated normally by insulin-stimulated autophosphorylation. In the intact CHO cells, insulin-stimulated serine and threonine phosphorylation of the IR delta ct was reduced 20%, suggesting that most Ser/Thr phosphorylation sites are located outside of the C terminus. During insulin stimulation, the wild-type and mutant insulin receptor activated the phosphatidylinositol 3-kinase. Moreover, insulin itself or human-specific anti-insulin receptor antibodies stimulated glycogen and DNA synthesis equally in both CHO/IR and CHO/IR delta ct cells. These data suggest that the C terminus plays a minimal role in IR function and signal transmission in CHO cells.
Myers
M G
MG
Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215.
Backer
J M
JM
Siddle
K
K
White
M F
MF
D019870:1-Phosphatidylinositol 4-Kinase; D000818:Animals; D001345:Autoradiography; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006003:Glycogen / Q000096:biosynthesis; D006801:Humans; D009154:Mutation; D010053:Ovary / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000502:physiology*; D013936:Thymidine / Q000378:metabolism; D014357:Trypsin; D014443:Tyrosine / Q000378:metabolism
D014443:Tyrosine; D006003:Glycogen; D010770:Phosphotransferases; D019870:1-Phosphatidylinositol 4-Kinase; D011972:Receptor, Insulin; D014357:Trypsin; D013936:Thymidine