Insulin-receptor Complex Research Articles

Selective degradation of insulin within rat liver endosomes.

To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor. Dissociated insulin is then degraded by an insulin specific protease(s) leading to further dissociation and degradation.

Assembly of insulin/insulin-like growth factor-1 hybrid receptors in vitro

Insulin and Mn/MgATP treatment of immunoaffinity-purified alpha beta heterodimeric insulin receptors induced the formation of an alpha 2 beta 2 heterotetrameric insulin receptor complex. In contrast, insulin-like growth factor-1 (IGF-1) treatment was completely ineffective in inducing the association of alpha beta heterodimeric insulin receptors. Similarly, IGF-1 or Mn/MgATP, but not insulin, treatment of immunoaffinity-purified alpha beta heterodimeric IGF-1 receptors induced the formation of an alpha 2 beta 2 heterotetrameric IGF-1 receptor complex. A monoclonal antibody specific for the insulin receptor (MA5) completely immunoprecipitated all the insulin binding activity from both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes but did not immunoprecipitate IGF-1 receptors. Conversely, the IGF-1 receptor-specific monoclonal antibody (alpha IR-3) immunoprecipitated all the IGF-1 binding activity, but not insulin receptors. The simultaneous treatment of pooled equal amounts of alpha beta heterodimeric insulin and IGF-1 receptors with a combination of insulin and IGF-1 resulted in the formation of alpha 2 beta 2 heterotetrameric insulin and IGF-1 receptor complexes. However, in the mixed alpha 2 beta 2 heterotetrameric receptor fraction MA5 immunoprecipitated 94% of the insulin binding in addition to 27% of the IGF-1 binding activity whereas alpha IR-3 immunoprecipitated 97% of the IGF-1 binding in addition to 38% of the insulin binding activity. Treatment of the mixed alpha beta heterodimeric insulin and IGF-1 receptors with Mn/MgATP also resulted in the formation of cross-immunoreactive (42-46%) alpha 2 beta 2 heterotetrameric receptors. These data directly demonstrate the formation of insulin/IGF-1 hybrid receptors by both a combination of insulin plus IGF-1 or Mn/MgATP treatment of purified human placenta alpha beta heterodimeric insulin and IGF-1 half-receptors in vitro.

IGF-1-dependent subunit communication of the IGF-1 holoreceptor: interactions between alpha beta heterodimeric receptor halves.

Examination of 125I-IGF-1 affinity cross-linking and beta-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated alpha beta heterodimeric IGF-1 receptors into an alpha 2 beta 2 heterotetrameric state, in a similar manner to that observed for the insulin receptor [Morrison, B.D., Swanson, M.L., Sweet, L.J., & Pessin, J.E. (1988) J. Biol. Chem. 263, 7806-7813]. The formation of the alpha 2 beta 2 heterotetrameric IGF-1 receptor complex from the partially purified alpha beta heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulate the protein kinase activity of the purified alpha beta heterodimeric insulin receptor complex. Incubation of the alpha 2 beta 2 heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter 125I-IGF-1 binding of IGF-1 stimulation of protein kinase activity. In addition, IAN did not affect the Mn/MgATP-dependent noncovalent association of IGF-1 receptor alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state. However, IAN treatment of the alpha beta heterodimeric IGF-1 receptors inhibited the IGF-1-dependent covalent formation of the disulfide-linked alpha 2 beta 2 heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated alpha beta heterodimeric IGF-1 receptor complexes into a disulfide-linked alpha 2 beta 2 heterotetrameric state whereas Mn/MgATP induces a noncovalent association.(ABSTRACT TRUNCATED AT 250 WORDS)

Defects in insulin-receptor internalization and processing in monocytes of obese subjects and obese NIDDM patients.

We investigated intracellular processing of the insulin-receptor complex in monocytes from 12 healthy control subjects, 11 obese nondiabetic subjects, and 13 obese patients with non-insulin-dependent diabetes mellitus (NIDDM) by measuring receptor internalization, recovery of cell-surface insulin binding after receptor internalization, and the release of intracellular intact insulin (insulin retroendocytosis). When monocytes from the three groups of subjects were exposed to 100 nM unlabeled insulin for 30 min at 37 degrees C, the subsequent cell-surface 125I-labeled insulin binding was reduced, but the total number of insulin receptors, measured by radioimmunoassay, was not changed. These findings indicate a redistribution of insulin receptors from the surface to the cell interior. Insulin-receptor internalization was significantly lower in monocytes of obese NIDDM patients (mean +/- SE 17.8 +/- 4.7%) than in obese subjects and healthy control subjects (33.5 +/- 4.5%, P less than .05, and 34.4 +/- 3.7%, P less than .02, respectively). Moreover, in downregulated cells, a complete recovery of the initial insulin binding was observed in control subjects but not in obese NIDDM patients or obese nondiabetic subjects. The release of internalized insulin was also reduced in obese NIDDM patients and obese subjects (t 1/2 = 49.0 +/- 2.4 min, P less than .02; 47.4 +/- 5.7 min, P less than .05; and 32.9 +/- 3.8 in NIDDM patients, obese subjects, and control subjects, respectively). In the radioactivity released from monocytes of obese subjects and obese NIDDM patients, the percentage of intact insulin was higher (P less than .05) than in control subjects, suggesting reduced intracellular insulin degradation in obese subjects and obese NIDDM patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Lilly lecture 1989. Toward physiological understanding of glucose tolerance. Minimal-model approach.

Glucose tolerance depends on a complex interaction among insulin secretion from the beta-cells, clearance of the hormone, and the actions of insulin to accelerate glucose disappearance and inhibit endogenous glucose production. An additional factor, less well recognized, is the ability of glucose per se, independent of changes in insulin, to increase glucose uptake and suppress endogenous output (glucose effectiveness). These factors can be measured in the intact organism with physiologically based minimal models of glucose utilization and insulin kinetics. With the glucose minimal model, insulin sensitivity (SI) and glucose effectiveness (SG) are measured by computer analysis of the frequently sampled intravenous glucose tolerance test. The test involves intravenous injection of glucose followed by tolbutamide or insulin and frequent blood sampling. SI varied from a high of 7.6 x 10(-4) min-1.microU-1.ml-1 in young Whites to 2.3 x 10(-4) min-1.microU-1.ml-1 in obese nondiabetic subjects; in all of the nondiabetic subjects, SG was normal. In subjects with non-insulin-dependent diabetes mellitus (NIDDM), not only was SI reduced 90% below normal (0.61 +/- 0.16 x 10(-4) min-1.microU-1.ml-1), but in this group alone, SG was reduced (from 0.026 +/- 0.008 to 0.014 +/- 0.002 min-1); thus, defects in SI and SG are synergistic in causing glucose intolerance in NIDDM. One assumption of the minimal model is that the time delay in insulin action on glucose utilization in vivo is due to sluggish insulin transport across the capillary endothelium. This was tested by comparing insulin concentrations in plasma with those in lymph (representing interstitial fluid) during euglycemic-hyperinsulinemic glucose clamps. Lymph insulin was lower than plasma insulin at basal (12 vs. 18 microU/ml) and at steady state, indicating significant loss of insulin from the interstitial space, presumably due to cellular uptake of the insulin-receptor complex. Additionally, during clamps, lymph insulin changed more slowly than plasma insulin, but the rate of glucose utilization followed a time course identical with that of lymph (r = .96) rather than plasma (r = .71). Thus, lymph insulin, which may be reflective of interstitial fluid, is the signal to which insulin-sensitive tissues are responding. These studies support the concept that, at physiological insulin levels, the time for insulin to cross the capillary endothelium is the process that determines the rate of insulin action in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of modified insulin B21-B26 fragments on glycogenesis and on insulin-receptor complex fate in cultured fetal hepatocytes

Biological activity and interference with insulin receptor complex fate of two modified sequences of insulin B21–B26, β-Ala-Arg-Gly-Phe-Phe-Tyr-NH 2 (DP-432) and β-Ala-Arg-Pro-Phe-Phe-Tyr-NH 2 (DP-640), were studied in cultured 18-day-old fetal rat hepatocytes known to respond to insulin by an acute stimulation of glycogenesis. The two derivatives stimulated [ 14C]glucose incorporation into glycogen in the absence of insulin independently of the deprivation of serum in the medium. The maximal effect of 3 mM DP-640 after 2 h, more pronounced than with 3 mM DP-432, was of the same order as that obtained with 10 nM insulin alone (stimulation index: 4.7 ± 0.7 2.5 ± 0.2 and 3.6 ± 0.9, n = 4, with DP-640, DP-432 and insulin, respectively) whereas insulin B-chain decreased glycogen labeling. Simultaneous addition of derivatives and insulin at maximal concentrations produced nearly additive effects. DP-640, as well as DP-432, increased the amount of [ 125I](A14) or (B26) human insulin associated with cells at 37°C and inhibited intracellular insulin degradation with differences depending on the kind of insulin isomer and derivative, while the rapid insulin receptor cycle was not affected. Thus, the two derivatives specifically modified the cellular processing of insulin in cultured fetal hepatocytes, and exerted an insulin-like effect on glycogenesis clearly enhanced through modification of DP-432 by substitution of glycine for proline (DP-640).

The cell biology of the insulin receptor

Following initial binding to specific cell surface receptors insulin is internalized in target cells. The fate of the internalized insulin-receptor complexes and how the processes involved are regulated is reviewed. The implications of these events in the effects of insulin on its target cells and in the physiopathology of diabetes and insulin resistance states are also considered.

High affinity insulin binding in the human placenta insulin receptor requires alpha beta heterodimeric subunit interactions.

Insulin binding to human placenta membranes treated at pH 7.6 or 8.5 in the presence or absence of 2.0 mM DTT for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, displayed curvilinear (heterogeneous) insulin binding plots when analyzed by the method of Scatchard. However, Triton X-100 solubilization followed by Bio-Gel A-1.5m gel filtration chromatography of the placenta membranes previously treated with DTT at pH 8.5 generated a nearly straight line (homogeneous) Scatchard plot. 125I-insulin affinity crosslinking studies coupled with Bio-Gel A-1.5m gel filtration chromatography demonstrated that the alkaline pH and DTT treatment of placenta membranes followed by detergent solubilization generated an alpha beta heterodimeric insulin receptor complex from the alpha 2 beta 2 heterotetrameric disulfide-linked state. The ability of alkaline pH and DTT to produce a functional alpha beta heterodimeric insulin receptor complex was found to be time dependent with maximal formation and preservation of tracer insulin binding occurring at 5 min. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of placenta membranes can result in the formation of a functional alpha beta heterodimeric insulin receptor complex. (ii) the alpha beta heterodimeric complex displays homogeneous insulin binding. (iii) the insulin receptor membrane environment maintains the alpha 2 beta 2 association state, which displays heterogeneous insulin binding, despite reduction of the critical domains that are responsible for the covalent interaction between the alpha beta heterodimers.

Wheat Germ Agglutinin Stimulation ofαβHeterodimeric Insulin Receptor β-Subunit Autophosphorylation by Noncovalent Association into an α2/β2Heterotetrameric State*

The purified human placental alpha 2 beta 2 heterotetrameric insulin-receptor complex was reduced and dissociated into functional alpha beta heterodimers by a combination of alkaline pH and dithiothreitol treatment. Wheat germ agglutinin (WGA) was observed to stimulate the beta-subunit autophosphorylation of both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes in the absence of insulin. However, WGA was without effect on the insulin stimulation of beta-subunit autophosphorylation in these insulin-receptor complexes. In contrast, monomeric WGA was unable to stimulate the basal exogenous substrate protein kinase activity in either the alpha 2 beta 2 heterotetrameric or alpha beta heterodimeric insulin receptor complexes. The stimulatory effect of WGA was biphasic, increasing the basal insulin receptor beta-subunit autophosphorylation at low concentrations (250 ng/ml to 2.5 micrograms/ml) and inhibiting at high concentrations (greater than 25 micrograms/ml). Similarly, equilibrium tracer insulin binding was not significantly altered by low concentrations of WGA (less than 1 microgram/ml) but was dramatically reduced at high WGA concentrations (greater than 2.5 micrograms/ml). In contrast to the insulin-induced covalent association of the alpha beta heterodimeric insulin receptors to form a disulfide-linked alpha 2 beta 2 heterotetrameric complex, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the WGA stimulation of beta-subunit autophosphorylation in the alpha beta heterodimer preparation occurred in the absence of covalent association. Nondenaturing Bio-Gel A-1.5m gel filtration chromatography and [125I]insulin affinity cross-linking demonstrated that WGA treatment of the alpha beta heterodimeric insulin receptors induced a noncovalent association into an alpha 2 beta 2 heterotetrameric state. These data support the hypothesis that the insulin receptor protein kinase activity, although dependent upon alpha beta heterodimeric subunit interactions, does not necessarily require covalent disulfide bond formation between the individual alpha beta heterodimeric species.

Relationship between insulin receptor subunit association and protein kinase activation: insulin-dependent covalent and manganese/magnesium ATP dependent noncovalent association of .alpha..beta. heterodimeric insulin receptors into an .alpha.2.beta.2 heterotetrameric state

The purified human placenta alpha 2 beta 2 heterotetrameric insulin receptor was reduced and dissociated into a functional alpha beta heterodimeric complex by a combination of alkaline pH and dithiothreitol treatment. In the presence of Mn/MgATP, insulin binding to the isolated alpha beta heterodimeric insulin receptor was found to induce the formation of a covalent disulfide-linked alpha 2 beta 2 heterotetrameric complex. In the absence of insulin, a noncovalent association of the alpha beta heterodimeric insulin receptor complex into an alpha 2 beta 2 heterotetrameric state required the continuous presence of both a divalent metal ion (Mn or Mg) and an adenine nucleotide (ATP, ADP, or AMPPCP). Thus, Mn/MgATP binding and not insulin receptor autophosphorylation was responsible for the noncovalent association into the alpha 2 beta 2 heterotetrameric state. However, the divalent metal ions or NaATP separately was ineffective in inducing the noncovalent association between the alpha beta heterodimers. The specific sulfhydryl agent iodoacetamide (IAN) was observed to inhibit the insulin-dependent covalent association of the alpha beta heterodimers without affecting the Mn/MgATP-induced noncovalent association into the alpha 2 beta 2 heterotetrameric state. Insulin treatment of the isolated alpha beta heterodimeric complex in the presence of IAN demonstrated that the Mn/MgATP-induce noncovalent association into the alpha 2 beta 2 heterotetrameric state was sufficient for insulin stimulation of beta-subunit autophosphorylation and exogenous substrate protein kinase activity. These data indicate that although interaction between the individual insulin receptor alpha beta heterodimers is necessary for insulin stimulation of protein kinase activity it does not require covalent disulfide bond formation.

Hormone Binding Site of the Insulin Receptor: Analysis Using Photoaffinity-Mediated Avidin Complexing

A trifunctional reagent was designed which allows derivatization of ligands, particularly peptides and proteins, for subsequent photoaffinity labelling of receptors and specific isolation of the covalent complex or its fragments. B29-(2-nitro-4-azidophenyl)-biocytinyl-insulin (NB-insulin) was synthesized, radioiodinated, and the B26-mono-iodo derivative isolated by HPLC. It was used to photoaffinity label human placental membranes and the purified insulin receptor. Extensive digestion of the covalent insulin-receptor complex with trypsin (EC 3.4.21.4) led to the generation of a fragment of Mr 14,000. Specific complexing with avidin, derivatized avidin or streptavidin could be demonstrated for the photoaffinity labelled alpha-subunit and the 14,000 core fragment. The latter was isolated (approx. 100 pmol from 3-4 placentae) by streptavidin affinity chromatography and HPLC. According to microsequencing based on the known primary structure of the insulin receptor, the N-terminus of the core peptide appears to be Leu20-His21-Glu22-Leu23. We thus conclude: a part of the insulin-binding region of the receptor is located close to the N-terminus of its alpha-subunit in a remarkably stable domain of the sequence 20--(approx.) 120.

Receptor-mediated endocytosis and degradation of insulin.

Receptor-mediated endocytosis and degradation of insulin.O SonneO SonneInstitute of Physiology, University of Aarhus, Denmark.Published Online:01 Oct 1988https://doi.org/10.1152/physrev.1988.68.4.1129MoreSectionsPDF (11 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByInsulin Therapy Improves Adeno-Associated Virus Transduction of Liver and Skeletal Muscle in Mice and Cultured CellsHuman Gene Therapy, Vol. 27, No. 11Transport studies of insulin across rat jejunum in the presence of chicken and duck ovomucoids18 February 2010 | Journal of Pharmacy and Pharmacology, Vol. 53, No. 8The journey of the insulin receptor into the cell: from cellular biology to pathophysiologyHistochemistry, Vol. 100, No. 3Characterization of insulin degradation products generated in liver endosomes: In vivo and in vitro studiesMolecular and Cellular Endocrinology, Vol. 72, No. 3Evidence for targeted gene transfer by receptor-mediated endocytosisBiochemical Pharmacology, Vol. 40, No. 2Regulation of insulin receptor functions by a peptide derived from a major histocompatibility complex class I antigenCell, Vol. 62, No. 2Effects of modified insulin B21-B26 fragments on glycogenesis and on insulin-receptor complex fate in cultured fetal hepatocytesMolecular and Cellular Endocrinology, Vol. 66, No. 2 More from this issue > Volume 68Issue 4October 1988Pages 1129-96 Copyright & PermissionsCopyright © 1988 by American Physiological Societyhttps://doi.org/10.1152/physrev.1988.68.4.1129PubMed3054947History Published online 1 October 1988 Published in print 1 October 1988 Metrics

The structure of 2Zn pig insulin crystals at 1.5 A resolution.

The paper describes the arrangement of the atoms within rhombohedral crystals of 2Zn pig insulin as seen in electron density maps calculated from X-ray data extending to 1.5 A (1 A = 10(-10) m = 10(-1) nm) at room temperature and refined to R = 0.153. The unit cell contains 2 zinc ions, 6 insulin molecules and about 3 x 283 water molecules. The atoms in the protein molecules appear well defined, 7 of the 102 side chains in the asymmetric unit have been assigned alternative disordered positions. The electron density over the water molecules has been interpreted in terms of 349 sites, 217 weighted 1.0, 126 weighted 0.5, 5 at 0.33 and 1 at 0.25 giving ca. 282 molecules. The positions and contacts of all the residues belonging to the two A and B chains of the asymmetric unit are shown first and then details of their arrangement in the two insulin molecules, 1 and 2, which are different. The formation from these molecules of a compact dimer and the further aggregation of three dimers to form a hexamer around two zinc ions, follows. It appears that in the packing of the hexamers in the crystal there are conflicting influences; too-close contacts between histidine B5 residues in neighbouring hexamers are probably responsible for movements of atoms at the beginning of the A chain of one of the two molecules of the dimer that initiate movements in other parts, particularly near the end of the B chain. At every stage of the building of the protein structure, residues to chains of definite conformation, molecules, dimers, hexamers and crystals, we can trace the effect of the packing of like groups to like, aliphatic groups together, aromatic groups together, hydrogen-bonded structures, positive and negative ions. Between the protein molecules, the water is distributed in cavities and channels that are continuous throughout the crystals. More than half the water molecules appear directly hydrogen bonded to protein atoms. These are generally in contact with other water molecules in chains and rings of increasing disorder, corresponding with their movement through the crystals. Within the established crystal structure we survey next the distribution of hydrogen bonds within the protein molecules and between water and protein and water and water; all but eight of the active atoms in the protein form at least one hydrogen bond.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-dependent covalent reassociation of isolated alpha beta heterodimeric insulin receptors into an alpha 2 beta 2 heterotetrameric disulfide-linked complex.

The purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex was reduced and dissociated into functional alpha beta heterodimers by a combination of alkaline pH and dithiothreitol treatment. Insulin treatment of the isolated alpha beta heterodimeric complex was observed to induce the complete reassociation to an alpha 2 beta 2 heterotetrameric state when analyzed by nondenaturing Bio-Gel A-1.5m gel filtration chromatography. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-insulin affinity cross-linked and 32P-autophosphorylated alpha beta heterodimers demonstrated that the insulin-dependent reassociation to the alpha 2 beta 2 heterotetrameric state occurred both covalently and noncovalently under these conditions. Comparison by reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the insulin-dependent covalent reassociation to an alpha 2 beta 2 heterotetrameric complex was due to the formation of a disulfide linkage(s) between the alpha beta heterodimers. beta subunit autophosphorylation of the control alpha 2 beta 2 heterotetrameric insulin receptor preparation was maximally stimulated within 5 min of insulin preincubation and occurred exclusively in the Mr = 400,000 alpha 2 beta 2 complex. Similarly, maximal insulin-stimulated beta subunit autophosphorylation of the alpha beta heterodimeric preparation occurred within 5 min of insulin pretreatment in the Mr = 210,000 alpha beta complex. However, 4 h of insulin pretreatment of the alpha beta heterodimer preparation induced the formation (6-fold) of a covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Maximal stimulation of substrate phosphorylation for the alpha 2 beta 2 heterotetrameric complex was also observed to occur within 5 min of insulin treatment, whereas maximal insulin-stimulated substrate phosphorylation of the alpha beta heterodimeric complex required greater than 4 h. These data demonstrate that (i) insulin treatment can induce the reassociation of the alpha beta heterodimeric complex into a covalent alpha 2 beta 2 heterotetrameric state, and (ii) insulin-dependent protein kinase activation of the alpha beta heterodimeric insulin receptor correlates with the covalent reassociation into a disulfide-linked alpha 2 beta 2 heterotetrameric complex.

Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene.

The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblast insulin receptors. These cells bind and internalize insulin normally. Biochemical assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 degrees C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 degrees C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The total number of complexes reached a maximum by 5 min and decreased rapidly thereafter with a t 1/2 of approximately 10 min. There was a distinct delay in the appearance, rate of rise, and peak of intracellular free and degraded insulin. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored, based on the ability of dissociated insulin to rebind to receptor upon neutralization of acidic intracellular vesicles with monensin. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.

Ligand-dependent intersubunit association within the insulin receptor complex activates its intrinsic kinase activity.

Insulin receptor halves (alpha beta) were obtained upon selective reduction of the holoreceptor (alpha 2 beta 2) and were isolated in concentrated form. Autophosphorylation of concentrated alpha beta receptor halves can be stimulated by insulin an average of 4.0-fold, whereas nonreduced holoreceptor can be stimulated 5.4-fold. If alpha beta half-receptors are immobilized on wheat germ agglutinin-agarose, no insulin-stimulated autophosphorylation is observed, whereas immobilized holoreceptor retains insulin responsiveness. Treatment of alpha beta half-receptors with glutathione in the presence of insulin results in reoxidation to the holoreceptor form (alpha 2 beta 2) with an efficiency of 60-70% as visualized by immunoblotting, thus providing evidence that two alpha beta halves are in close physical proximity. This reoxidation reaction, which is evident prior to autophosphorylation, is rapid and strictly dependent on the presence of insulin, consistent with the hypothesis that insulin promotes the association of two alpha beta halves. Furthermore, the insulin-induced reoxidation reaction and the insulin-induced autophosphorylation show the same dose dependence ED50 3-4 x 10(-8) M insulin), suggesting that the noncovalent association of alpha beta half-receptors upon insulin binding is a prerequisite for insulin-stimulated autophosphorylation in concentrated alpha beta half-receptor preparations. If the alpha beta half-receptor forms are phosphorylated in the presence of an anti-phosphotyrosine antibody and separated from nonphosphorylated alpha beta receptors, we observed that the phosphorylated alpha beta receptor halves contain bound insulin. This excludes the possibility that alpha beta half-receptors that bind insulin, preferentially phosphorylate alpha beta halves that have no insulin bound.

Internalized insulin-receptor complexes are unidirectionally translocated to chloroquine-sensitive degradative sites. Dependence on metabolic energy.

Insulin receptors on the surface of isolated rat adipocytes were photoaffinity labeled at 12 degrees C with the iodinated photoreactive insulin analogue, 125I-B2 (2-nitro-4-azidophenylacetyl)-des-PheB1-insulin, and the pathways in the intracellular processing of the labeled receptors were studied at 37 degrees C. During 37 degrees C incubations, the labeled 440-kDa insulin receptors were continuously internalized (as assessed by trypsin inaccessibility) and degraded such that up to 50% of the initially labeled receptors were lost by 120 min. Metabolic poisons (0.125-0.75 mM 2,4-dinitrophenol (DNP) and 1-10 mM NaF), which led to dose-dependent depletion of adipocyte ATP pools, inhibited receptor loss, and caused up to 3-fold increase in intracellular receptor accumulation. This effect was due to inhibition of intracellular receptor degradation, and there was no apparent effect of the metabolic poisons on initial internalization of the receptors. Following maximal intracellular accumulation of labeled insulin receptors in the presence of NaF or DNP, removal of these agents resulted in a subsequent, time-dependent degradation of the accumulated receptors. However, when the lysosomotropic agent, chloroquine (0.2 mM), was added immediately following removal of the metabolic poisons, further degradation of the intracellularly accumulated receptors was prevented, suggesting that the chloroquine-sensitive degradation of insulin receptors occurs distal to the site of inhibition by NaF or DNP. To confirm this, maximal intracellular accumulation of labeled receptors was first allowed to occur in the presence of chloroquine and the cells were then washed and reincubated in chloroquine-free media in the absence or presence of NaF or DNP. Under these conditions, degradation of the intracellularly accumulated receptors continued to occur, and NaF or DNP failed to block the degradation. In summary, these results indicate that the loss of cell surface insulin receptors in adipocytes involves: 1) initial internalization of the receptors to a nondegradative intracellular compartment by a process that is relatively insensitive to ATP depletion, followed by 2) a highly energy-dependent unidirectional translocation of the receptors from this compartment to chloroquine-sensitive site(s) of degradation.

Kinetic studies of insulin degrading enzyme in cultured human lymphocytes

After insulin binds to its receptors, insulin-receptor complexes are internalized by absorptive endocytosis, and then insulin seems to be degraded in the intracellular sites. Although the degradation of insulin has been extensively studied, the sites and enzymes of intracellular degradation have still not been appeared. In order to clarify this problem, following experiments were performed. The effects on insulin degradation of the various inhibitors and anti-IDE serum were investigated in isolated rat hepatocytes and Bri-7 human cultured lymphocytes. N-ethylmaleimide and bacitracin, inhibitors which inhibit the activity of insulin-degrading enzyme (IDE), and anti-IDE serum were decreased insulin degradation in Bri-7 cells which does not contain lysosomal pathway. IDE mainly exists in the cytosol fraction, but also on the surface of various cell types. The kinetic changes of insulin receptors and cell surface IDE was determined in Bri-7 cells after preincubation with or without insulin. The concentration of cell surface IDE was determined by immunoenzymatic labeling method using anti-IDE serum. Bri-7 cells were preincubated with 10(-6) M insulin for 30 min to 18 h. Fifty percent of the receptors were lost at 6 h after the preincubation, and level of the receptors achieved a steady state at 18 h. Although the loss of surface IDE was slightly slower than that of receptors, the curves were essentially parallel to each other. Thus, the loss of cell surface receptors and IDE was directly related to the preincubation time. Furthermore, the recovery of decreased surface receptors and IDE was needed for 36 and 72 h, respectively. The alpha-subunit of insulin receptor (135 K) and IDE (110 K) were assessed by cross-linking of 125I-insulin to the plasma membrane and the cytosol of Bri-7 cells, respectively. Cell surface insulin receptor was decreased, whereas cytosolic IDE was increased in Bri-7 cells incubated with insulin. Thus, it is likely that both cell surface and cytosolic IDE, acting either individually or in concert, constitute a physiological mechanism by which the cellular response to insulin is terminated. These results suggest that IDE may play a major role in insulin degradation in the intact cell. Moreover, one possible mechanism of intracellular insulin degradation is that cell surface IDE may be internalized with the insulin receptor complex and may degrade insulin during the intracellular process.

Insulin-degrading enzyme is capable of degrading receptor-bound insulin

In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ aggulutinin column and monoclonal anti-IDE antibody affinity column, respectively, [ 125I]insulin-receptor complex was incubated with various amounts of IDE at 0°C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135kd band (insulin receptor α-subunit) decreased, whereas that of 110kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37°C, about half of the [ 125I]insulin was dissociated from the complex. However, the time course of [ 125I]insulin degradation in this incubation was essentially identical to that of free [ 125I]insulin degradation. Cross-linked, non-dissociable receptor-bound [ 125I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [ 125I]insulin from insulin-receptor complex incubated with IDE was significantly (p<0.001) decreased as compared with that without the enzyme. These results, therefore, show that IDE could recognize and degrade receptor-bound insulin, and suggest that IDE may be involved in insulin metabolism during receptor-mediated endocytosis through the degradation of receptor-bound insulin in early neutral vesicles before their internal pH is acidified.

Reversal of insulin-induced negative cooperativity by monoclonal antibodies that stabilize the slowly dissociating (“K super”) state of the insulin receptor

Two monoclonal antibodies to the insulin receptor, MA-5 and MA-20, unlike other monoclonal antibodies, do not mimick the accelerating effect of insulin on the dissociation of 125I-insulin from the receptors (negative cooperativity). On the contrary, MA-5 and MA-20 markedly slow down the dissociation rate. We show now that MA-5 and MA-20 are potent antagonists of the negative cooperativity induced by insulin, and reverse the insulin-induced acceleration whether added simultaneously with insulin or after insulin. The reversal of the insulin-induced acceleration is almost immediate. These data strengthen the concept therefore that the insulin-receptor complex has access to alternative conformational states that can be stabilized by ligand-induced site-site interactions.