Insulin receptor monoclonal antibodies that mimic insulin action without activating tyrosine kinase

D M Hawley,B A Maddux,R G Patel,K Y Wong,P W Mamula,G L Firestone,A Brunetti,E Verspohl,I D Goldfine

doi:10.1016/s0021-9258(19)81632-0

Abstract

HTC rat hepatoma cells were transfected with human insulin receptor cDNA to a level of 40,000 receptors/cell. In these cells, as well as in nontransfected cells, insulin stimulated the uptake of alpha-aminoisobutyric acid. Two monoclonal antibodies directed against the human insulin receptor alpha subunit, like insulin, stimulated amino acid uptake in transfected HTC cells, but not in nontransfected HTC cells. The antibodies, in contrast to insulin, failed to stimulate insulin receptor tyrosine kinase activity, both in intact transfected cells and in cell free extracts prepared from them. These data suggest, therefore, that activation of insulin receptor tyrosine kinase may not be an obligatory step in all of the transmembrane signaling mechanisms of the insulin receptor.

Highlights

HTC rat hepatoma cells were transfected with human insulin receptor cDNA to a level of 40,000 receptors/cell
The insulinreceptor is a tetrameric disulfide-linked glycoprotein consisting of two identical extracellular a subunits that bind the hormone and two identical transmembrane /3 subunits that containa typical tyrosine kinase in their cytoplasmic domains [1].The receptor is synthesized asa precursor polypeptide, and is subsequently cleaved into one a and one /3 subunit [1].After insulin bindtso its receptor, p subunit tyrosine kinase activity increases, followed by /3 subunit autophosphorylation on specific tyrosine residues
Several lines of evidence suggest that insulin receptor tyrosine kinaseactivity may be involvedin hormoneaction

Summary

EXPERIMENTAL PROCEDURES

Transfection of Cultured Cells-HTC rat hepatoma cells were grown in Dulbecco’s modified Eagle’s(DME)’ medium supplemented with 10%fetal bovine serum (FBS)as previously described [9].These cells were transfected with an expression plasmid containing the human insulin receptor cDNA [10] and elements of the first exon of the insulin receptor genomic DNA(ll),under the control of the Rous sarcoma virus long terminal repeat and theSV40 late gene termination and polyadenylation sites [12].Stably transfected cell lines were constructed using the calcium phosphate/glycerol shock method and coselection for the neomycin resistance gene in pSV2neo [13].After transfection, resistantcolonies were subcloned and tested for binding of 1251-insulinas previously described [9]. The cell lysate was purified over a wheat germ agglutinin column [17], and the pooled, partially purified receptors were precipitated with rabbit polyclonal antireceptor antiserum [16] These immunoprecipitates were reduced with dithiothreitol, denaturedwith sodium dodecyl sulfate (SDS),andthen subjected to electrophoresis on. The abbreviations used are: DME, Dulbecco’smodifiedEagle’s medium; AIB,a-aminoisobutyric acid FBS, fetal bovine serum; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonicacid; PBS, phosphate buffered saline; EGTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid. Tibody MA-5 enhance AIB uptake, the initial rate of AIB uptake was the solubilized cells were centrifuged at 100,000 X g for 1 h and measured as a function of AIB concentration, in the presence of purified on a 2-ml column containing wheat germ agglutinin coupled buffer, 100 nM insulin, and 100 nM MA-5. Cells were grown to confluence on 100-mmtissue culture dishes. 18h prior to labeling, the media was changed to DME-H16 containing

RESULTS

Monoclonal Antibodies

Insulin Receptor Monoclonal Antibodies abcdefghi

DISCUSSION