Baculovirus-directed expression of the human insulin receptor and an insulin-binding ectodomain.
In this report we describe the use of the baculovirus expression system to overproduce the human insulin holoreceptor (HIR) and a truncated, secretory version of the HIR cDNA (HIRsec) consisting of the alpha subunit and the extracellular portion of the beta subunit (beta'). Sf9 cells infected with the full-length HIR viruses synthesize recombinant HIR (rHIR) with an insulin-binding alpha subunit of apparent Mr = 110,000 and a beta subunit of apparent Mr = 80,000. Uncleaved alpha beta proreceptor accumulates in infected cells. Both of these forms assemble into higher order disulfide-linked dimers or heterotetramers of apparent Mr greater than 350,000. Insulin-binding activity in cells infected with rHIR viruses is present predominantly on the extracellular aspect of the plasma membrane (greater than 80%). Insulin binding to the full-length rHIR occurs with typical complex kinetics with Kd1 = 0.5-1 x 10(-9) M and Kd2 = 10(-7) M and receptors are present in large amounts in infected cells (1 x 10(6) receptors/cell; 1-2 mg HIR/10(9) cells). The full-length rHIR undergoes insulin-dependent autophosphorylation; half-maximal activation of beta subunit autophosphorylation occurs at 1-2 x 10(-8) M. The alpha beta proreceptor also becomes phosphorylated in vitro. Analysis of tryptic phosphopeptides derived from in vitro autophosphorylated beta subunit and alpha beta proreceptor reveals a pattern of phosphorylation that is indistinguishable from that of authentic placental HIR. Sf9 cells infected with rHIRsec viruses synthesize and secrete an (alpha beta')2 heterotetrameric complex having an insulin-binding alpha subunit of apparent Mr = 110,000 and a truncated beta' subunit of apparent Mr = 45,000 that lacks kinase activity. The rHIRsec complex purified from the conditioned medium of infected cells binds insulin with high affinity (Kd = 10(-9) M).
Howard Hughes Medical Institute, University of Chicago, Illinois 60637.
J I
JI
Paul
J
J
Tavaré
R M
RM
Denton
D F
DF
Steiner
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D004279:DNA, Viral; D007328:Insulin; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D011972:Receptor, Insulin