The native α2β2 tetramer is the only subunit structure of the insulin receptor in intact cells and purified receptor preparations (original) (raw)
Related papers
Insulin Receptor: Covalent Labeling and Identification of Subunits
Proceedings of The National Academy of Sciences, 1979
Two methods were used to label insulin receptors covalently with 125I. In the first, an aryl azide derivative of insulin, 125I-labeled 4-azido-2-nitrophenyl-insulin, was synthesized and used to photolabel the binding region of the insulin receptor in rat liver membranes and human placenta membranes. In the second, insulin receptors were purified from rat liver membranes and labeled with 125I by use of chloramine-T; this method presumably has no specificity for the binding region of the receptor. The proteins labeled by both methods were anal zed by sodium dodecyl sulfate/poyacrylamide gel elec-
Biochemical Journal, 1985
Insulin receptors on RINm5F cell membranes (an insulin-producing rat pancreatic cell line) were studied. To study the insulin receptor alpha-subunit, 125I-labelled photoreactive insulin was covalently bound to the membranes in the absence or presence of unlabelled insulin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions showed specific labelling of an Mr 130 000 protein. The receptor beta-subunit was studied by using a cell-free phosphorylation assay. Analysis under reducing conditions showed a phosphoprotein of Mr 95 000 whose level of phosphorylation was selectively increased by insulin, and which was specifically immunoprecipitated by antibodies to the insulin receptor. Further, covalent hormone-receptor complexes purified with anti-insulin antibodies were able to undergo autophosphorylation, indicating the existence of operational receptor subunit arrangements. RINm5F cell insulin receptors (and, by analogy, possibly those of native B-cells) ...
Oligomeric states of the insulin receptor: binding and autophosphorylation properties
Biochemistry, 1989
Properties of oligomeric states of the insulin receptor were analyzed by polyacrylamide gel electrophoresis in nondenaturing buffer conditions (ND-PAGE). Partially purified insulin receptors resolve in ND-PAGE as three distinct species: (i) the fast electrophoretic mobility, low molecular mass form manifests intense labeling by iodinated insulin and shows basal and insulin-stimulated autophosphorylation; (ii) the middle, intermediate mobility form exhibits strong labeling by iodinated ligand but does not possess the capacity to be autophosphorylated; (iii) the slow mobility, highest molecular mass form necessitates covalent binding with iodinated hormone to withstand electrophoresis and shows autophosphorylation enhanced by insulin. This receptor form is more heavily labeled by phosphorylation than the low form. At 22 "C, binding and autophosphorylation do not appear to be time dependent. At 37 O C , binding and autophosphorylation of low and high species attain a maximum after 15 min and then decrease as time of incubation with insulin is prolonged to 120 min; the middle species exhibits a much slower association rate, and its labeling by iodinated hormone becomes more intense with time. Our data show that in cell-free systems insulin receptors appear in various oligomeric states and that the highest molecular mass oligomer exhibits the most pronounced autophosphorylation. This is compatible with the concept that insulin receptor oligomerization provides a mechanism for transmembrane signaling.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1986
Processing of the insulin receptor by hepatocytes was studied using a t251-1abeiled photoreactive insulin derivative which could be covalently attached to the receptor and facilitate the analysis of receptor structure in isolated subcellular fractions by SDS-polyacrylamide gel electrophoresis. Following binding at the cell surface, the label was rapidly internalised and located in a low-density subcellular fraction ('endosomes'). The intact receptor (350000 molecular weight) and binding (a) subunit (135000), produced by in vitro disulphide reduction of the samples, were found in the plasma membrane fraction but not in endosomes. In endosomes, the label was concentrated in a band at 140000 (non-reduced) which on reduction generated species of 100000 and 68000 predominantly. The insulin receptor therefore undergoes an early structural change during endocytosis. This modification does not involve complete disulphide reduction and may be due to a proteolytic event.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1993
We have studied the structure and the function of a truncated human insulin receptor in which 113 amino acids (aa 1231-1343) at the C-terminus of the fl-subunit were deleted. In this study, wild-type and truncated insulin receptors were expressed by stable transfection in NIH-3T3 cells and CHO cells. The mutation impairs post-translational processing of the insulin receptor; proteolytic cleavage is retarded, and degradation of the truncated receptor is accelerated. Furthermore, insulin-stimulated autophosphorylation of the mutant insulin receptor is impaired. This is associated with a defect in insulin-stimulated endocytosis. Finally, in NIH-3T3 cells, the mutant insulin receptor failed to mediate the mitogenic effects of insulin. In CHO cells, transfection of insulin receptor cDNA (either wild-type or mutant) did not alter mitogenic response to insulin. It has previously been shown that deletion of 43 amino acids at the C-terminus of the fl-subunit did not affect insulin receptor tyrosine kinase activity. Our data suggest that the structural domain located 43-113 amino acids from the C-terminus appears to have several functional roles. First, the domain appears to promote folding of receptor into the optimal conformation for post-translational processing. Second, the presence of this domain appears to promote the stability of the receptor/3-subunit in intact cells. Finally, perhaps as a consequence of the effects upon the stability of the receptor, this domain is required in intact cells for insulin-stimulated autophosphorylation and signal transmission.
Cellular Signalling, 1990
Insulin signal transmission through the plasma membrane was studied in terms of relationship between basal autophosphorylation of the fl-subunit and the ability to bind insulin by the ~t-subunit of the insulin receptor. In a cell free system, receptors phosphorylated on tyrosine residues in the absence of insulin were separated from non-phosphorylated receptors using antiphosphotyrosine antibodies. Insulin binding assays were then performed on basally autophosphorylated and on non-phosphorylated receptors. We found that the tyrosine phosphorylated receptors, which corresponded to 25% of the total number of receptors, were accountable for 60-80% of insulin binding. Scatchard representation of binding data has shown that the plot corresponding to tyrosine phosphorylated receptors was localized above, and was steeper than the plot corresponding to non-phosphorylated receptors. These data make it likely that the conformation of ~-subunit which favours ligand binding is connected to the conformation of fl-subunit which favours phosphate reception on tyrosine residues. Reciprocally, the high-affinity conformation of insulin receptor seems to become stabilized by basal autophosphorylation.
Insulin receptor: Interaction with nonreceptor glycoprotein from liver cell membranes
Proceedings of the National Academy of Sciences, 1978
In crude receptor preparations (either particulate or soluble) of rat liver membranes, the insulin receptor exhibits complicated binding kinetics (two binding plateaus, half-saturated at approximately 60 pM and 700 pM insulin) and an apparent chromatographic heterogeneity, suggested by the presence of two detectable, soluble insulin-binding components with apparent Stokes radii of 72 A and 38 A. In contrast, the insulin receptor isolated by affinity chromatography exhibits a simple binding isotherm (half-maximal saturation of binding at 700 pM insulin) without evidence for negative cooperativity and behaves as a single component (apparent Stokes radius of 38 A) upon chromatography on Sepharose 6B. The apparent discrepancies between the properties of the unpurified insulin receptor and the affinity-purified receptor can be attributed to
High molecular weight forms of the insulin receptor
Biochemistry, 1986
The insulin receptor of liver, adipose, and placental plasma membranes was photoaffinity labeled with radioiodinated NfB29-(monoazidobenzoyl)insulin. Three specifically labeled bands of 450, 360, and 260 kilodaltons (kDa) were identified in each tissue by polyacrylamide gel electrophoresis of the membranes solubilized in sodium dodecyl sulfate (SDS). The 360-and 260-kDa bands corresponded to partially reduced forms of the 450-kDa band. The distribution of radioactivity between the three insulin receptor bands was dependent on the tissue, the purity of the receptor preparation, and the conditions of solubilization in SDS.