Adipocyte insulin-binding species: the 40 Å Stoke's radius protein

1984 ◽  
Vol 62 (7) ◽  
pp. 566-570
Author(s):  
C. Elliott ◽  
H. Joseph Goren
Keyword(s):  
Insulin Receptor ◽  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Relative Mass ◽  
Single Polypeptide Chain ◽  
Dodecyl Sulfate ◽  
Single Polypeptide ◽  
Adipocyte Plasma Membranes

Several laboratories have demonstrated the presence of large (70 Å) (1 Å = 0.1 nm) and small (40 Å) insulin receptors. This report provides evidence that the 40 Å insulin receptor migrates on dodecyl sulfate – acrylamide gel electrophoresis as a 90 000 dalton protein and that this protein is a single polypeptide chain. 125I-labeled insulin was bound to plasma membranes from isolated rat adipocytes. Following removal of unbound 125I-labeled insulin, the mixture was exposed to disuccinimidyl suberate. Proteins tagged with 125I-labeled insulin were separated by dodecyl sulfate gel electrophoresis or Sepharose 6B chromatography. Autoradiography of the gels demonstrated several large (relative mass (Mr) > 300 000) and one small (Mr ~ 90 000) labeled protein in nonreduced membrane proteins. Dithiothreitol reduction decreased the large insulin-binding species to its known subunits, but the 90 000 dalton protein did not decrease in size. Triton X-100 solubilized plasma membranes were separated by Sepharose 6B chromatography. One labeled protein, with Kav = 0.57 elution position, on dodecyl sulfate gel electrophoresis migrated as a 90 000 dalton protein. Thus, rat adipocyte plasma membranes contain both an oligomeric insulin-binding species and a monomeric insulin-binding species. The relationship of the monomeric to the oligomeric insulin receptor is discussed.

Desensitization of beta-adrenergic receptors in adipocytes causes increased insulin sensitivity of glucose transport

1996 ◽  
Vol 271 (2) ◽  
pp. E271-E276 ◽  
Author(s):  
A. Green ◽  
R. M. Carroll ◽  
S. B. Dobias
Keyword(s):  
Insulin Sensitivity ◽  
Glucose Transport ◽  
Adrenergic Receptors ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
High Dose ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Adipocyte Plasma Membranes

To determine the effect of desensitization of adipocyte beta-adrenergic receptors on insulin sensitivity, rats were continuously infused with isoproterenol (50 or 100 micrograms.kg-1.h-1) for 3 days by osmotic minipumps. Epididymal adipocytes were isolated. The cells from treated animals were desensitized to isoproterenol, as determined by response of lipolysis (glycerol release). Binding of [125I]iodocyanopindolol was decreased by approximately 80% in adipocyte plasma membranes isolated from treated rats, indicating that beta-adrenergic receptors were downregulated. Cellular concentrations of Gn alpha and Gi alpha were not altered. Insulin sensitivity was determined by measuring the effect of insulin on glucose transport (2-deoxy-[3H]glucose uptake). Cells from the isoproterenol-infused rats were markedly more sensitive to insulin than those from control rats. This was evidenced by an approximately 50% increase in maximal glucose transport rate in cells from the high-dose isoproterenol-treated rats and by an approximately 40% decrease in the half-maximal effective concentration of insulin in both groups. 125I-labeled insulin binding to adipocytes was not altered by the isoproterenol infusions, indicating that desensitization of beta-adrenergic receptors results in tighter coupling between insulin receptors and stimulation of glucose transport.

scholarly journals Insulin receptor function is inhibited by guanosine 5′-[γ-thio]triphosphate (GTP[S])

1990 ◽  
Vol 270 (2) ◽  
pp. 401-407 ◽  
Author(s):  
H W Davis ◽  
J M McDonald
Keyword(s):  
Insulin Receptor ◽  
G Proteins ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Peptide Sequence ◽  
Receptor Kinase ◽  
Receptor Function ◽  
Gtp Binding ◽  
Insulin Receptor Kinase

The regulatory role of GTP-binding proteins (G-proteins) in insulin receptor function was investigated using isolated insulin receptors and plasma membranes from rat adipocytes. Treatment of isolated insulin receptors with 1 mM-guanosine 5′-[gamma-thio]triphosphate (GTP[S]) inhibited insulin-stimulated phosphorylation of the beta-subunit, histone Hf2b and poly(GluNa4,Tyr1) by 22%, 65% and 65% respectively. Phosphorylation of calmodulin by the insulin receptor kinase was also inhibited by 1 mM-GTP[S] both in the absence (by 88%) and in the presence (by 81%) of insulin. In the absence of insulin, 1 mM-GTP had the same effect on calmodulin phosphorylation as 1 mM-GTP[S]. However, when insulin was present, GTP was less effective than GTP[S] (41% versus 81% inhibition). Concentrations of GTP[S] greater than 250 microM are necessary to inhibit phosphorylation. Although these concentrations are relatively high, the effect of GTP[S] is not due to competition with [32P]ATP for the insulin receptor kinase since (1) other nucleotide triphosphates did not inhibit phosphorylation as much as did GTP[S] (or GTP) and (2) the Vmax of the ATP-dependent kinase reaction was decreased in the presence of GTP[S]. GTP[S] (1 mM) also inhibited insulin binding to isolated receptors and plasma membranes, by 80% and 50% respectively. Finally, an antibody raised to a peptide sequence common to the alpha-subunits of G-proteins Gs, Gi, Go and transducin detected G-proteins in plasma membranes but failed to detect them in the insulin receptor preparation. These results indicate that GTP inhibits insulin receptor function, but does so through a mechanism that does not require a conventional GTP-binding protein.

scholarly journals Guanosine nucleotides regulate hormone binding of insulin receptors

1992 ◽  
Vol 281 (3) ◽  
pp. 735-743 ◽  
Author(s):  
E R Mortensen ◽  
J Drachman ◽  
G Guidotti
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Scatchard Analysis ◽  
Hormone Binding ◽  
Temperature Dependent ◽  
High Affinity ◽  
Mild Reduction ◽  
Adipocyte Plasma Membranes

Insulin receptors in turkey erythrocyte and rat adipocyte plasma membranes display non-linear hormone binding by Scatchard analysis. This result is consistent with evidence that the insulin-binding sites are heterogeneous and have at least two affinities for the hormone. Mild reduction of plasma membranes with dithiothreitol, before insulin binding, increased the fraction of hormone binding with high affinity without significantly changing the total number of receptor-binding sites. In the presence of guanosine 5′-[gamma-thio]triphosphate, the amount of receptor with high affinity for insulin in the reduced membranes decreased to that present in the absence of reduction; the effect of the nucleotide was concentration- and temperature-dependent. This decrease in insulin binding was specific for guanine nucleotides.

scholarly journals The structure of the hepatic insulin receptor and insulin binding

1986 ◽  
Vol 239 (1) ◽  
pp. 127-133 ◽  
Author(s):  
F J Haynes ◽  
E Helmerhorst ◽  
C C Yip
Keyword(s):  
Insulin Receptor ◽  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Insulin Binding ◽  
Intact Cells ◽  
Collagenase Treatment ◽  
Receptor Component ◽  
Predominant Band ◽  
Cytoplasmic Side

Hepatocytes or hepatic plasma membranes were photoaffinity-labelled with radioiodinated N epsilon B29-monoazidobenzoyl-insulin. Analysis of the samples by SDS/polyacrylamide-gel electrophoresis and autoradiography revealed the insulin receptor as a predominant band of 450 kDa. When hepatic plasma membranes were first treated with clostridial collagenase and then photolabelled, the insulin receptor appeared as a predominant band of 360 kDa. This effect of collagenase treatment on the insulin receptor was due to Ca2+-dependent heat-labile proteinases contaminating the preparation of collagenase, and it could be mimicked by elastase. The decrease in size of the insulin receptor to 360 kDa resulted from the loss of a receptor component that was inaccessible to photolabelling. In contrast, the size of the insulin receptor of intact cells was not affected by collagenase treatment. This suggests that the site sensitive to proteolysis was located on the cytoplasmic side of the plasma membrane. In hepatic plasma membranes that were treated with collagenase or elastase, and contained the 360 kDa form of the insulin receptor, the binding affinity for insulin was increased by up to 2-fold. These findings support the concept that a component which is either a part of, or closely associated with, the insulin receptor may regulate its affinity for insulin.

scholarly journals A thiol-sensitive degradative process of liver uncouples autophosphorylation of the insulin receptor from insulin binding

1986 ◽  
Vol 236 (2) ◽  
pp. 535-542 ◽  
Author(s):  
K M Lerea ◽  
J N Livingston
Keyword(s):  
Plasma Membrane ◽  
Insulin Receptor ◽  
Plasma Membranes ◽  
Proteinase Inhibitors ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Binding Activity ◽  
Binding Characteristics ◽  
Golgi Membranes ◽  
Plasma Membrane Receptor

Insulin receptors derived from highly purified rat liver plasma membranes and Golgi membranes showed differences in insulin-mediated receptor autophosphorylation, even though their insulin-binding characteristics were similar. This difference was related to the generation of a Mr-84,000 fragment of the Mr-90,000 beta subunit of the plasma-membrane receptor, a fragment that was not present in the receptor from Golgi membranes, in the absence of a change in the insulin-binding alpha subunit. When autophosphorylation activity was based on insulin binding, the activity of the plasma-membrane-derived insulin receptor was decreased to 25-30% that of the Golgi-derived receptor. Endoglycosidase F digestion produced changes in the Mr values for both species, but they were not converted into a single subunit, thereby suggesting differences in the protein component of the two subunits. Although the proteinase inhibitors phenylmethanesulphonyl fluoride, ovomucoid and aprotinin failed to block the formation of the Mr-84,000 fragment, the presence of iodoacetamide or EDTA during liver homogenization markedly inhibited fragment generation and allowed the plasma-membrane insulin receptor to retain an autophosphorylation activity comparable with that present in insulin receptors from Golgi membranes. Thus a thiol-sensitive, cation-dependent, degrading activity has been identified that can uncouple the insulin-binding activity of the plasma-membrane insulin receptor from its tyrosine kinase activity.

scholarly journals Reversible reduction of insulin receptor affinity by ATP depletion in rat adipocytes

1983 ◽  
Vol 214 (1) ◽  
pp. 203-207 ◽  
Author(s):  
H J Steinfelder ◽  
H G Joost
Keyword(s):  
Insulin Receptor ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Dissociation Rate ◽  
Atp Depletion ◽  
Metabolic Inhibitor ◽  
Receptor Affinity ◽  
Rat Fat Cells ◽  
Reversible Reduction

The effects of the metabolic inhibitor NaN3 on insulin receptors in isolated rat fat-cells were investigated. The agent reduced insulin binding in parallel to a decrease of the ATP content of cells. Both effects were observed in the same concentration range of NaN3, and were fully reversible. According to the binding curves the affinity rather than the number of receptors was reduced. Kinetic experiments revealed an increased dissociation rate of the insulin-receptor complex. The effects outlasted cell disruption, since the receptor affinity was still lowered in plasma membranes obtained from NaN3-treated cells. Thus an inhibition of insulin internalization could not account for the observed effects. It is suggested that the observed ATP-dependence of insulin receptor affinity reflects a reversible structural alteration of the receptor, or of some closely related membrane protein.

scholarly journals Tyrosine kinase activity of liver insulin receptor is inhibited in rats at term gestation

1989 ◽  
Vol 263 (1) ◽  
pp. 267-272 ◽  
Author(s):  
C Martínez ◽  
P Ruiz ◽  
A Andrés ◽  
J Satrústegui ◽  
J M Carrascosa
Keyword(s):  
Insulin Receptor ◽  
Kinase Activity ◽  
Insulin Signalling ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Late Gestation ◽  
Receptor Kinase ◽  
Pregnant Rats ◽  
Insulin Resistant ◽  
32P Incorporation

Late gestation is associated with insulin resistance in rats and humans. It has been reported that rats at term gestation show active hepatic gluconeogenesis and glycogenolysis, and diminished lipogenesis, despite normal or mildly elevated plasma insulin concentrations, indicating a state of resistance to the hormone action. Since autophosphorylation of the insulin receptor has been reported to play a key role in the hormone signal transduction, we have partially purified plasma-membrane liver insulin receptors from virgin and 22-day-pregnant rats and studied their binding and kinase activities. (1) Insulin binding to partially purified receptors does not appear to be influenced by gestation, as indicated by the observed KD and Bmax. values. (2) The rate of autophosphorylation and the maximal 32P incorporation into the receptor beta-subunit from pregnant rats at saturating concentrations of insulin are markedly decreased with respect to the corresponding values for virgin rats. (3) The diminished autophosphorylation rate was due to a decreased responsiveness of the kinase activity to the action of insulin. (4) Phosphorylation of the exogenous substrates casein and poly(Glu80Tyr20) by insulin-receptor kinase was also less when receptors from pregnant rats were used. These results show the existence of an impairment at the receptor kinase level of the insulin signalling mechanism that might be related to the insulin-resistant state characteristic of term gestation in rats.

scholarly journals The αβ monomer of the insulin receptor has hormone-responsive tyrosine kinase activity

1991 ◽  
Vol 273 (1) ◽  
pp. 49-56 ◽  
Author(s):  
E R Mortensen ◽  
J G Drachman ◽  
G Guidotti
Keyword(s):  
Plasma Membranes ◽  
Kinase Activity ◽  
Gradient Centrifugation ◽  
Insulin Receptors ◽  
Sucrose Density Gradient ◽  
Erythrocyte Membranes ◽  
Protein Kinase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Adipocyte Plasma Membranes ◽  
Receptor Dimers

Insulin receptors from turkey erythrocyte membranes exist as monomers and dimers when membranes are solubilized with detergent. We examined the ability of monomers and dimers to act as protein kinases to effect both autophosphorylation of the receptor and phosphorylation of an exogenous substrate. After separation by sucrose-density-gradient centrifugation, only receptor dimers show significant basal and insulin-stimulated kinase activity, whereas material at the position of receptor monomers is not active. Partial reduction of the membrane-bound receptors with dithiothreitol, however, produces a receptor monomer containing an alpha and a beta chain which has protein kinase activity similar to that of the original dimers. With rat adipocyte plasma membranes, which in the absence of reducing agents only contain receptor dimers, reduction with dithiothreitol also produces monomers with receptor kinase activity. Receptor monomer hormone-dependent kinase activity is insensitive to receptor concentration and shows stimulation after immobilization on an affinity support.

Effect of exercise on insulin receptor binding and kinase activity in skeletal muscle

1989 ◽  
Vol 256 (1) ◽  
pp. E138-E144 ◽  
Author(s):  
J. L. Treadway ◽  
D. E. James ◽  
E. Burcel ◽  
N. B. Ruderman
Keyword(s):  
Skeletal Muscle ◽  
Insulin Receptor ◽  
Insulin Action ◽  
White Muscle ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Similar Data

Insulin action in skeletal muscle is markedly enhanced for several hours after an acute bout of exercise. The purpose of this study was to examine the possible involvement of the intrinsic tyrosine kinase activity of the insulin receptor in mediating these effects. Red and white muscles were removed from rats either at rest or following a treadmill run (45 min at 18 m/min), and insulin receptors were isolated in partially purified form. Basal and insulin-stimulated receptor kinase activity was higher in red than in white muscle, in agreement with previous studies (J. Biol. Chem. 261: 14939-14944, 1986). There was no effect of exercise on insulin binding, basal and insulin-stimulated receptor autophosphorylation, or basal and insulin-stimulated exogenous kinase activity, in either red or white muscle. Similar data were obtained when phosphatase inhibitors were used during receptor isolation. The structure of insulin receptors isolated from the muscle of exercised and control rats was similar as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity cross-linked insulin receptors. We conclude that enhanced insulin action in muscle during the postexercise state is not related to increased kinase activity of the insulin receptor.

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