Thiol-specific biotinylation of the insulin receptor in permeabilized cells enhances receptor function.
We examined the reactivity of insulin receptor sulfhydryls to biotinylation in Chinese hamster ovary cells that express high levels of human insulin receptors (CHO/HIRc cells). Following the biotinylation reaction, the insulin receptor was purified by immunoprecipitation, and resolved by SDS-polyacrylamide gel electrophoresis before electrotransfer to membranes. The use of enzyme-linked streptavidin in conjunction with a chemiluminescent technique allowed the detection of thiol-biotinylated receptor beta-subunit, with no modification of the alpha-subunit. In cells expressing large numbers of IGF-1 receptors, the same technique enabled the detection of thiol-biotinylated IGF-1 receptors as well. Thiol-alkylation of intact CHO/HIRc cells with an impermeant reagent did not impair the ability of maleimidodibutyrylbiocytin (MBB) to biotinylate sulfhydryls on the receptor beta-subunit after cell permeabilization with digitonin. In contrast, thiol-alkylation of digitonin-permeabilized cells prevented MBB-induced receptor biotinylation. The basal and insulin-activated insulin receptors exhibited a comparable reactivity to MBB. Furthermore, the use of affinity purification on monomeric avidin-agarose enabled us to learn that the biotinylation reaction was near-quantitative. MBB had no effect on insulin binding nor on receptor autophosphorylation and insulin-dependent receptor kinase activity. However, basal levels of receptor kinase activity were significantly elevated by thiol-biotinylation. Further, in the presence of vanadate, MBB retained the ability to enhance receptor kinase activity in permeabilized cells, consistent with the notion that this increased exogenous substrate phosphorylation was not accounted for by inactivation of protein tyrosine phosphatases. The dephosphorylation of thiol-biotinylated, 32P-labeled insulin receptors by particulate protein tyrosine phosphatases was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)
Bernier
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M
Diabetes Unit, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
Nadiv
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Kole
H K
HK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives / Q000494:pharmacology; D016466:CHO Cells; D002463:Cell Membrane Permeability; D002846:Chromatography, Affinity; D006224:Cricetinae; D004072:Digitonin; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008163:Luminescent Measurements; D008239:Lysine / Q000031:analogs & derivatives* / Q000494:pharmacology; D046911:Macromolecular Substances; D008301:Maleimides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D011972:Receptor, Insulin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology
D007328:Insulin; D046911:Macromolecular Substances; D008301:Maleimides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D014638:Vanadates; D001710:Biotin; D000255:Adenosine Triphosphate; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin; D008239:Lysine; D004072:Digitonin