scholarly journals Changes in insulin-receptor tyrosine, serine and threonine phosphorylation as a result of substitution of tyrosine-1162 with phenylalanine

1991 ◽  
Vol 274 (1) ◽  
pp. 173-179 ◽  
Author(s):  
J M Tavaré ◽  
M Dickens
Keyword(s):  
Insulin Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Serine Phosphorylation ◽  
Beta Subunit ◽  
Intact Cells ◽  
Ovary Cells ◽  
Tyrosine Residues

Previous studies, by ourselves and others, have shown that tyrosine residues 1158, 1162 and 1163 are very rapidly autophosphorylated on the human insulin receptor after insulin binding and that this is followed by the autophosphorylation of tyrosine residues 1328 and 1334. The autophosphorylation of these tyrosine residues, and their role in transmembrane signalling, were examined by using Chinese-hamster ovary cells transfected with either normal intact insulin receptors or receptors in which tyrosine residues 1162 or 1162/1163 were substituted with phenylalanine. These studies show the following. (1) Tyrosine-1158 could still be autophosphorylated when tyrosine-1162 and -1163 were substituted with phenylalanine. (2) Insulin-stimulated insulin-receptor tyrosine phosphorylation in intact cells was complete within 30 s and was accompanied, after a lag of 2-5 min, by a rise in serine and threonine phosphorylation the beta-subunit. (3) Replacement of tyrosine-1162 with phenylalanine blocked insulin-stimulated threonine phosphorylation of the insulin receptor in intact cells. (4) Insulin-stimulated serine phosphorylation of the beta-subunit was found in both intact cells and partially purified receptor preparations incubated with [gamma-32P]ATP and was still apparent after the replacement of tyrosine-1162 with phenylalanine. (5) Our data strongly suggest that insulin-stimulated insulin-receptor serine and threonine phosphorylations are initiated through two distinct pathways, with only the latter showing a strict dependence on autophosphorylation of tyrosine-1162.

scholarly journals The extracellular portion of the insulin receptor beta-subunit regulates the cellular trafficking of the insulin-insulin receptor complex. Studies on Chinese hamster ovary cells carrying the Cys 860-->Ser insulin receptor mutation

2003 ◽  
pp. 365-371 ◽  
Author(s):  
L Benzi ◽  
P Cecchetti ◽  
AM Ciccarone ◽  
S Novelli ◽  
A Paoli ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Insulin Binding ◽  
Beta Subunit ◽  
Insulin Degradation ◽  
Ovary Cells ◽  
Intracellular Targeting

OBJECTIVE: Chinese hamster ovary (CHO) cells transfected with human engineered insulin receptor (IR) cDNA to mutate Cys 860 to Ser (CHO-IR(C860S)) showed a defective insulin internalization without affecting insulin binding and IR autophosphorylation. Moreover, this mutation reduces insulin receptor substrate (IRS)-1 tyrosine phosphorylation and insulin-induced metabolic and mitogenic effects. Altogether, these observations support a role of the extracellular domain of IR beta-subunit in insulin and receptor intracellular targeting as well as in insulin signaling. DESIGN AND METHODS: This study assesses in more details the effect of IR(C860S) mutation on the trafficking of the insulin-IR complex. In particular, IR internalization, phosphorylation, dissociation and recycling, as well as insulin degradation and retroendocytosis have been investigated in CHO cells overexpressing either wild type (CHO-IR(WT)) or mutated IRs. RESULTS: the C860S mutation significantly decreases IR internalization both insulin stimulated and constitutive. In spite of a similar dissociation of internalized insulin-IR complex, recycling of internalized IR was significantly faster (half life (t(1/2)): 21 min vs 40 min, P<0.001) and more extensive (P<0.01) for IR(C860S) than for IR(WT). On the other hand, insulin degradation and retroendocytosis were superimposable in both cell lines. As expected, insulin-induced phosphorylation was similar in both IRs, however dephosphorylation was much more rapid and was greater (P<0.01) in CHO-IR(WT) as compared with CHO-IR(C860S) cells. CONCLUSIONS: Transmembrane and intracellular domain of IR seem to be determinants for IR internalization. Now we report that Cys 860 in the IR beta-subunit ectodomain may be of relevance in ensuring a proper internalization and intracellular trafficking of the insulin-IR complex.

scholarly journals Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells

1988 ◽  
Vol 250 (1) ◽  
pp. 95-101 ◽  
Author(s):  
O Koshio ◽  
Y Akanuma ◽  
M Kasuga
Keyword(s):  
Insulin Receptor ◽  
Kinase Activity ◽  
Intact Cell ◽  
Insulin Receptors ◽  
Beta Subunit ◽  
Receptor Kinase ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Insulin Receptor Kinase ◽  
Insulin Receptor Kinase Activity

H-35 rat hepatoma cells were labelled with [32P]orthophosphate and their insulin receptors isolated on wheat germ agglutinin (WGA)-agarose and anti-(insulin receptor) serum. The incubation of these cells with 10 mM-H2O2 for 10 min increased the phosphorylation of both the serine and tyrosine residues of the beta subunit of the insulin receptor. Next, insulin receptors were purified on WGA-agarose from control and H2O2-treated H-35 cells and the purified fractions incubated with [gamma-32P]ATP and Mn2+. Phosphorylation of the beta subunit of insulin receptors obtained from H2O2-treated cells was 150% of that of control cells. The kinase activity of the WGA-purified receptor preparation obtained from H2O2-treated cells, as measured by phosphorylation of src-related synthetic peptide, was increased about 4-fold over control cells. These data suggest that in intact cell systems, H2O2 may increase the insulin receptor kinase activity by inducing phosphorylation of the beta subunit of insulin receptor.

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 Functional characteristics of insulin receptors with a Thr-->Ser1200 mutation overexpressed in Chinese hamster ovary cells

2000 ◽  
pp. 125-131 ◽  
Author(s):  
M Schutt ◽  
H Benecke ◽  
M Drenckhan ◽  
HH Klein
Keyword(s):  
Insulin Receptor ◽  
Kinase Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Insulin Receptors ◽  
Receptor Kinase ◽  
Ovary Cells ◽  
Insulin Receptor Kinase

OBJECTIVE: To investigate the functional properties of insulin receptors with a Thr-->Ser(1200)-mutation that is associated with severe insulin resistance in humans. DESIGN AND METHODS: The effect of in situ insulin-stimulation on insulin receptor kinase activity was studied in Chinese hamster ovary cells with overexpressed human Ser(1200)-mutated, non-mutated, and ATP-binding site-mutated (Lys-->Arg(1030)) receptors using a microwell-based assay that only detects human (and not hamster) insulin receptors. Moreover, the fraction of anti-phosphotyrosine antibody-binding receptors following in situ stimulation was separated, and autophosphorylation and kinase activity resulting from in situ and/or in vitro activation evaluated in this fraction. RESULTS: Although insulin-stimulated kinase activity of human-specific anti-insulin receptor antibody-binding receptors in cells with Ser(1200)-mutated insulin receptors represented only 3.3% of that reached in cells with non-mutated receptors, a clear insulin-induced increase in kinase activity was observed (3.4-fold; P<0.05). This increase was associated with a 2.3+/-0.6% (P<0.05) increase in anti-phosphotyrosine-binding receptors with a kinase activity representing 43+/-8% of that found in activated non-mutated receptors. In vitro autophosphorylation and kinase activation proceeded much more slowly in Ser(1200)-mutated receptors (t(1/2)): 100 min) compared with non-mutated receptors (t(1/2)): 1 min) and were inhibitable by lower alkaline phosphatase concentrations (EC(50): 3 U/ml and 70 U/ml respectively). No activation of insulin receptor kinase was observed with Arg(1030)-mutated receptors. CONCLUSIONS: Overexpressed Ser(1200)-mutated human insulin receptors possess insulin-stimulated kinase activity and can be activated in situ and in vitro. They are characterized by a markedly slower autophosphorylation reaction, which, in a phosphatase-containing environment, results in a small fraction of phosphorylated and activated receptors.

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.

Antiapoptotic Signaling by the Insulin Receptor in Chinese Hamster Ovary Cells

Biochemistry ◽  
1998 ◽  
Vol 37 (45) ◽  
pp. 15747-15757 ◽  
Author(s):  
Whaseon Lee-Kwon ◽  
Doekbae Park ◽  
Padmavathi V. Baskar ◽  
Sutapa Kole ◽  
Michel Bernier
Keyword(s):  
Insulin Receptor ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells
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