title: Secretion of soluble functional insulin receptors by transfected NIH3T3 cells.
abstract: In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.
authors.affiliation: Howard Hughes Medical Institute, University of Chicago, Illinois 60637.
authors.forename: J
authors.initials: J
authors.lastname: Whittaker
authors.forename: A
authors.initials: A
authors.lastname: Okamoto
mesh_terms: D000818:Animals; D052688:Bovine papillomavirus 1 / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D007163:Immunosorbent Techniques; D007328:Insulin / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D009154:Mutation; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D007328:Insulin; D046911:Macromolecular Substances; D004247:DNA; D011972:Receptor, Insulin; D006026:Glycoside Hydrolases; C020668:glycanase