scholarly journals An Mr 180000 protein is an endogenous substrate for the insulin-receptor-associated tyrosine kinase in human placenta

1987 ◽  
Vol 243 (3) ◽  
pp. 797-801 ◽  
Author(s):  
F Machicao ◽  
H Häring ◽  
M F White ◽  
J M Carrascosa ◽  
B Obermaier ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Human Placenta ◽  
Insulin Binding ◽  
Specific Protein ◽  
Beta Subunit ◽  
Phosphorylated Proteins ◽  
Endogenous Substrate ◽  
32P Incorporation ◽  
Insulin Receptor Kinase

The beta-subunit of the insulin receptor contains a tyrosine-specific protein kinase. Insulin binding activates this kinase and causes phosphorylation of the beta-subunit of the insulin receptor. It is believed that phosphorylation of other proteins might transmit the insulin signal from the receptor to the cell. In the present study we used a polyclonal anti-phosphotyrosine antibody to detect other proteins that become tyrosine phosphorylated upon insulin stimulation. Glycoproteins from human placenta membranes were enriched by wheat germ agglutinin chromatography and phosphorylation was studied with [gamma-32P]ATP and insulin in vitro. Phosphorylated proteins were immunoprecipitated by antibodies against the insulin receptor and by serum containing the anti-phosphotyrosine antibody. Beside the insulin-stimulated phosphorylation of the 95 kDa beta-subunit of the insulin receptor, an insulin-stimulated phosphorylation of a 180 kDa protein was found. The phosphorylation of both proteins occurred only on tyrosine residues. Insulin increased 32P incorporation into the 180 kDa band 2.7-fold (S.E.M. +/- 0.3, n = 5). The 180 kDa protein was not precipitated by antibodies against the insulin receptor. H.p.l.c. chromatograms of tryptic fragments of the phosphorylated 180 kDa protein and of the beta-subunit of the insulin receptor revealed different patterns for both proteins. Insulin-stimulated phosphorylation of the 180 kDa protein was also detectable in unfractionated detergent-solubilized membranes. The phosphorylation of the 180 kDa protein was stimulated by insulin with the same dose-response curve as the phosphorylation of the beta-subunit, suggesting that this protein might be another endogenous substrate of the insulin receptor kinase.

scholarly journals Tyrosine kinase activity of insulin receptors from human placenta. Effects of autophosphorylation and cyclic AMP-dependent protein kinase

1986 ◽  
Vol 233 (3) ◽  
pp. 677-681 ◽  
Author(s):  
H G Joost ◽  
H J Steinfelder ◽  
C Schmitz-Salue
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Insulin Receptor ◽  
Human Placenta ◽  
Kinase Activity ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The kinase activity of partially purified insulin receptor obtained from human placenta was studied. When autophosphorylation of the beta-subunit of the receptor was initiated by ATP prior to the addition of the exogenous substrate, both basal and insulin-stimulated kinase activity was increased. However, half-maximum effective insulin concentrations were unchanged. Insulin receptor autophosphorylation as stimulated by ATP and insulin failed to affect significantly 125I-insulin binding to partially purified insulin receptor from human placenta. It is concluded that autophosphorylation of the insulin receptors regulates its kinase activity but not its affinity for insulin. The catalytic subunit of cyclic AMP-dependent protein kinase failed to phosphorylate either subunit of the insulin receptor, and each kinase failed to affect the affinity of the other one. Thus no functional interaction between cyclic AMP-dependent protein kinase and insulin receptors was observed in the in vitro system.

Insulin resistance in uremia: insulin receptor kinase activity in liver and muscle from chronic uremic rats

1988 ◽  
Vol 254 (4) ◽  
pp. E394-E401 ◽  
Author(s):  
F. Cecchin ◽  
O. Ittoop ◽  
M. K. Sinha ◽  
J. F. Caro
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Insulin Receptor ◽  
Kinase Activity ◽  
Insulin Binding ◽  
Beta Subunit ◽  
Receptor Kinase ◽  
Ad Libitum ◽  
Insulin Receptor Kinase ◽  
Insulin Receptor Kinase Activity

We have studied the structure and function of the partially purified insulin receptors from liver and skeletal muscle in a rat model of severe chronic uremia. 125I-insulin binding was higher in the liver from uremic rats when compared with ad libitum- and pair-fed controls. Furthermore, the ability of insulin to stimulate the autophosphorylation of the beta-subunit and insulin receptor kinase activity using Glu80, Tyr20 as exogenous phosphoacceptor was increased in the liver of the uremic animals. The structural characteristic of the receptors, as determined by electrophoretic mobilities of affinity labeled alpha-subunit and the phosphorylated beta-subunit, were normal in uremia. 125I-insulin binding and insulin receptor kinase activity were similar in the skeletal muscle from uremic and pair- and ad libitum-fed animals. Thus our data are supportive of the hypothesis that in liver and muscle of chronic uremic rats, insulin resistance is due to a defect(s) distal to the insulin receptor kinase.

Biological activity of immunoreactive insulin-like activity extracted from rat submandibular gland

1995 ◽  
Vol 269 (2) ◽  
pp. E277-E282 ◽  
Author(s):  
M. Taouis ◽  
D. Deville de Periere ◽  
D. Hillaire-Buys ◽  
M. Derouet ◽  
R. Gross ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Amino Acid Uptake ◽  
Biologically Active ◽  
Beta Subunit ◽  
Immunoreactive Insulin ◽  
Acid Uptake ◽  
Intact Cells

Earlier studies indicate the presence of an insulin-like immunoreactivity (ILI) in rat submandibular salivary glands (SSG). Previous observations also showed that streptozotocin (STZ)-induced diabetes was accompanied by an increase in SSG ILI concentrations. In the present work we studied the effect of SSG ILI from normal and STZ diabetic rats (ILI-N and ILI-D, respectively) on insulin receptor binding and function in LMH cell line. ILI-N and ILI-D inhibited 125I-insulin binding to intact cells and wheat germ agglutinin (WGA)-purified insulin receptors with a high affinity. Furthermore, ILI-N and ILI-D activated, although weakly, the beta-subunit autophosphorylation of solubilized and WGA-purified insulin receptors. An ATP hydrolytic activity was present in ILI-N and, to a greater extent, in ILI-D extracts, which can at least in part explain their low potency for activating autophosphorylation and kinase activity of insulin receptors in vitro. However, after ILI treatment of intact cells and immunoprecipitation of insulin receptors, ILI induced a dose-dependent tyrosine phosphorylation of the insulin receptor beta-subunit. Finally, ILI-N and ILI-D stimulated amino acid uptake and lipogenesis in LMH cells. These findings suggest that SSG ILI is biologically active and can participate in metabolic regulations.

scholarly journals Decreased tyrosine kinase activity of insulin receptor isolated from rat adipocytes rendered insulin-resistant by catecholamine treatment in vitro

1986 ◽  
Vol 234 (1) ◽  
pp. 59-66 ◽  
Author(s):  
H Häring ◽  
D Kirsch ◽  
B Obermaier ◽  
B Ermel ◽  
F Machicao
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Kinase Activity ◽  
Insulin Binding ◽  
Receptor Kinase ◽  
Rat Adipocytes ◽  
Insulin Resistant ◽  
32P Incorporation ◽  
Insulin Responsiveness

Catecholamine treatment of isolated rat adipocytes decreases insulin binding and inhibits insulin stimulation of the glucose-transport system. There is increasing evidence that the insulin signal is transmitted after insulin is bound to the receptor via a tyrosine kinase, which is an intrinsic part of the receptor. To find whether the receptor kinase is modified by catecholamines, we solubilized and partially purified the insulin receptor of isoprenaline-treated adipocytes and studied the effect of insulin on its kinase activity. (1) Insulin increased the tyrosine autophosphorylation of the insulin receptor kinase from catecholamine-treated cells only 4-fold, compared with a 12-fold stimulation in control cells. (2) The rate of insulin-stimulated 32P incorporation into the receptor of isoprenaline-treated cells at non-saturating [32P]ATP concentrations (5 muM) was decreased to 5-8% of the values for receptor from control cells. (3) 125I-insulin binding to the partially purified receptor from catecholamine-treated cells was also markedly decreased. The insulin receptor from catecholamine treated cells bound 25-50% of the amount of insulin bound by the receptor from control cells at insulin concentrations of 10 pM-0.1 muM. Part of the impaired insulin-responsiveness of the receptor kinase of catecholamine-treated cells is therefore explained by impaired binding properties; however, an additional inhibition of the kinase activity of the insulin receptor from catecholamine-treated cells is evident. (4) This inhibition of kinase activity decreased when the concentration of [gamma-32P]ATP in the phosphorylation assay was increased. A Lineweaver-Burk analysis revealed that the Km for ATP of the receptor kinase from isoprenaline-treated cells was increased to approx. 100 muM, compared with approx. 25 muM for receptor of control cells. (5) We conclude from the data that catecholamine treatment of rat adipocytes modulates the kinase activity of the insulin receptor by increasing its Km for ATP and that this is part of the mechanism leading to insulin-resistance in these cells.

scholarly journals Effect of basic polycations and proteins on purified insulin receptor. Insulin-independent activation of the receptor tyrosine-specific protein kinase by poly(l-lysine)

1989 ◽  
Vol 263 (3) ◽  
pp. 813-822 ◽  
Author(s):  
Y Fujita-Yamaguchi ◽  
D B Sacks ◽  
J M McDonald ◽  
D Sahal ◽  
S Kathuria
Keyword(s):  
Protein Kinase ◽  
Insulin Receptor ◽  
Kinase Activity ◽  
Specific Protein ◽  
Beta Subunit ◽  
Protein Kinase Activity ◽  
Receptor Kinase ◽  
Kinase Activation ◽  
Insulin Receptor Kinase ◽  
Specific Protein Kinase

Since the studies on tyrosine phosphorylation of calmodulin by the insulin receptor kinase in vitro suggested that protamine and poly(L-lysine) may activate phosphorylation of the receptor beta subunit [Sacks & McDonald (1988) J. Biol. Chem. 263, 2377-2383], we examined the effects of a variety of basic polycations/proteins and polyamines on insulin receptor kinase activity. The insulin receptor purified from human placental membranes was incubated with each basic polycation/protein or polyamine and assayed for tyrosine-specific protein kinase activity by measuring 32P incorporation into the src-related peptide. At a concentration of 1 microM, poly(L-lysine) and poly(L-ornithine) markedly stimulated kinase activity, whereas poly(L-arginine) and histones H1 and H2B inhibited insulin receptor kinase. In contrast, at a concentration of 1 mM, three polyamines (spermine, spermidine and putrescine) did not alter kinase activity. Poly(L-lysine) and poly(L-ornithine) stimulated the insulin receptor kinase by 5-10-fold at concentrations of 0.1-1 microM. Protamine sulphate also showed a significant stimulatory effect at a concentration of 100 microM. Preincubation of the receptor with poly(L-lysine) or poly(L-ornithine) for 20-60 min resulted in maximal kinase activation. Poly(L-lysine), the most effective activator of the receptor kinase, was used to characterize further the mechanisms of the kinase activation. Poly(L-lysine) activates the insulin receptor kinase by increasing the Vmax. without changing the Km. Poly(L-lysine) markedly stimulates the kinase activity of insulin receptor preparations that have lost both basal kinase activity and the ability to be stimulated by insulin. Insulin and poly(L-lysine) also differed in their ability to stimulate the kinase activity of prephosphorylated receptors. Prephosphorylation of the receptors did not affect the stimulation of the kinase by insulin. In contrast, prephosphorylation of receptors resulted in a markedly enhanced ability of poly(L-lysine) to stimulate kinase activity. These studies suggest that the mechanisms by which poly(L-lysine) and insulin activate the kinase are different. In conjunction with other additional evidence, it is suggested that poly(L-lysine) interacts directly with the beta-subunit of the receptor, thereby activating the receptor kinase.

scholarly journals Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping

1988 ◽  
Vol 252 (2) ◽  
pp. 607-615 ◽  
Author(s):  
J M Tavaré ◽  
R M Denton
Keyword(s):  
Thin Layer ◽  
Insulin Receptor ◽  
Peptide Mapping ◽  
Beta Subunit ◽  
Two Dimensional ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Domain 3 ◽  
Receptor Beta

1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells.

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 juxtamembrane but not the carboxyl-terminal domain of the insulin receptor mediates insulin's metabolic functions in primary adipocytes and cultured hepatoma cells

2000 ◽  
Vol 24 (3) ◽  
pp. 419-432 ◽  
Author(s):  
K Paz ◽  
S Boura-Halfon ◽  
LS Wyatt ◽  
D LeRoith ◽  
Y Zick
Keyword(s):  
Insulin Receptor ◽  
Alternative Pathway ◽  
Insulin Binding ◽  
Hepatoma Cells ◽  
Metabolic Effects ◽  
Quantitative Expression ◽  
Metabolic Functions ◽  
Downstream Effectors ◽  
Carboxyl Terminal

Insulin-stimulated signaling pathways are activated upon interactions between the intracellular domains of the receptor and its downstream effectors. Insulin receptor substrate proteins (IRS-1, -2, -3 and -4) are the best-studied substrates for the insulin receptor kinase (IRK). We have previously shown that IRS-1 and IRS-2 interact with the juxtamembrane (JM) but not with the carboxyl-terminal (CT) region of the insulin receptor (IR) in vitro. However, the precise role of these IR regions in mediating insulin's bioeffects is still unresolved. In the present work we made use of vaccinia virus as a vector for quantitative expression of the JM and CT domains within the cytoplasm of physiologically insulin-responsive primary rat adipocytes and rat hepatoma Fao cells. We could demonstrate that overexpression of either the JM or the CT domains did not inhibit either insulin binding or insulin-stimulated receptor autophosphorylation. In contrast, metabolic effects such as insulin-induced glucose utilization in adipocytes, and insulin-induced amino acid utilization in Fao hepatoma cells were inhibited (70-80%) in cells overexpressing the JM but not the CT domains of IR. The inhibitory effects of the overexpressed JM domain were accompanied by inhibition of insulin-stimulated IRS-1 phosphorylation, decreased IRS-1-associated PI3K activity, and decreased phosphorylation of the downstream effectors of PI3K, PKB and p70 S6K. Insulin-stimulated thymidine incorporation in Fao cells was also inhibited (40%) upon overexpression of the JM but not the CT region of IR. Our findings suggest that interactions between the JM region of IR and its downstream effectors are obligatory for insulin-stimulated metabolic functions in physiologically relevant insulin responsive cells. They also rule out the possibility that interaction of proteins, including PI3K, with the CT domain can provide an alternative pathway.

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