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/sup 6/ receptors per cell. The cell line with the highest /sup 125/I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10/sup 6/ receptors with a K/sub d/ of 10/sup -9/ M. This level was not dependent on exposure to metals but could be increased further to 2 x 10/sup 7/ receptors per cell by addition of sodium butyrate to the culture medium. The ..cap 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 ..cap 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
- Research Organization:
- Univ. of Chicago, IL (USA)
- OSTI ID:
- 5172521
- Journal Information:
- Proc. Natl. Acad. Sci. U.S.A.; (United States), Vol. 84:15
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
CARBON 14 COMPOUNDS
BIOSYNTHESIS
INSULIN
RADIORECEPTOR ASSAY
OLIGOSACCHARIDES
RECEPTORS
MOLECULAR STRUCTURE
FIBROBLASTS
IODINE 125
KIDNEYS
MAN
METHIONINE
MICE
MUTAGENESIS
PHOSPHORUS 32
RECOMBINANT DNA
SULFUR 35
AMINO ACIDS
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARBOHYDRATES
CARBOXYLIC ACIDS
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
DNA
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
EVEN-ODD NUCLEI
HORMONES
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPOTROPIC FACTORS
MAMMALS
MEMBRANE PROTEINS
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
PEPTIDE HORMONES
PHOSPHORUS ISOTOPES
PRIMATES
PROTEINS
RADIOISOTOPES
RODENTS
SACCHARIDES
SOMATIC CELLS
SULFUR ISOTOPES
SYNTHESIS
TRACER TECHNIQUES
VERTEBRATES
550601* - Medicine- Unsealed Radionuclides in Diagnostics
550201 - Biochemistry- Tracer Techniques