High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells.
In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10(6) receptors per cell. The cell line with the highest insulin binding (NIH 3T3 HIR3.5) had 6 X 10(6) receptors with a Kd of 10(-9) M. This level was not dependent on exposure to metals but could be increased further to 2 X 10(7) receptors per cell by addition of sodium butyrate to the culture medium. The alpha and beta subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the alpha and beta subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis.
Whittaker
J
J
Okamoto
A K
AK
Thys
R
R
Bell
G I
GI
Steiner
D F
DF
Hofmann
C A
CA
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D005786:Gene Expression Regulation; D006003:Glycogen / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007668:Kidney / Q000032:analysis; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D009439:Neuraminidase / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics*
D007328:Insulin; D006003:Glycogen; D004247:DNA; D008715:Methionine; D011972:Receptor, Insulin; D009439:Neuraminidase