Drosophila insulin receptor: lectin-binding properties and a role for oxidation-reduction of receptor thiols in activation.
The glycosylation of the Drosophila insulin receptor (DIR) has been compared to that of the rat insulin receptor by examining the binding of receptors to the lectins wheat germ agglutinin, Concanavalin-A, and lentil lectin. Although rat insulin receptors bound and were specifically eluted from all three lectins, only a small fraction of the DIR (< 5%) was retained on wheat germ agglutinin. In contrast, the DIR bound strongly to Concanavalin-A and lentil lectin and was recovered from lentil lectin columns after elution with alpha-methyl-mannoside. The pattern of lectin binding indicates that glycosylation of the DIR and rat insulin receptors differs, with the DIR containing primarily high mannose-type oligosaccharides. After lectin chromatography, the DIR exhibited an elevated level of basal autophosphorylation and kinase activity, which could be restored to a low level by incubation with 0.5 mM dithiothreitol (DTT). DTT did not, however, affect ligand-stimulated kinase activity. The ability of low concentrations of DTT to deactivate the DIR kinase suggests that, like the mammalian receptor, beta-subunit thiols may be involved in regulation of conformational changes between activated and unactivated receptor states. Interestingly, DTT-induced deactivation of the DIR was blocked by preincubation with an antipeptide antibody against the carboxy-terminal domain of the DIR. This suggests that the DIR carboxyl terminus undergoes a conformational change during the activation-inactivation cycle of the kinase, which can be sterically hindered by the antibody. Conformational changes in this region of the mammalian receptor have been observed, and these data suggest that features of the insulin receptor activation mechanism have been substantially conserved during evolution.
Marin-Hincapie
M
M
Department of Anatomy and Cell Biology, State University of New York, Brooklyn 11203, USA.
Garofalo
R S
RS
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D004229:Dithiothreitol / Q000494:pharmacology; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D037102:Lectins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D010766:Phosphorylation; D051381:Rats; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000906:Antibodies; D037102:Lectins; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D011972:Receptor, Insulin; D004229:Dithiothreitol