scholarly journals An extracellular domain of the β subunit is essential for processing, transport and kinase activity of insulin receptor

1995 ◽  
Vol 305 (2) ◽  
pp. 599-604 ◽  
Author(s):  
T Haruta ◽  
T Sawa ◽  
Y Takata ◽  
T Imamura ◽  
Y Takada ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Critical Role ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Extracellular Domain ◽  
Immunoblot Analysis ◽  
Beta Subunit ◽  
Lentil Lectin ◽  
Mutant Receptors

The extracellular portion of the insulin receptor (IR) beta-subunit has four cysteine and four asparagine residues which are potentially involved in disulphide bond formation between the alpha- and beta-subunits and N-linked glycosylation respectively. However, the function of this portion is not fully understood. In order to investigate the role of the extracellular domain of beta-subunit, we created a deletion mutant of IR cDNA which lacked 47 amino acid residues encoded by 141 bp corresponding to exon 13 of the IR gene. Insulin binding and surface labelling of COS 7 cells transiently expressing the mutant insulin receptors (IR delta Ex13) showed that the mutated receptors were not expressed on the cell surface. However, immunoblot analysis showed that uncleaved form (190 kDa) of the mutant receptors were intracellularly expressed. Deglycosylation with endoglycosidase H showed that the mutant receptors had mainly high-mannose oligosaccharide chains. The mutant IRs bound with high affinity to lentil lectin but with low affinity to wheat germ agglutinin. Therefore, it is suggested that misfolding of the mutant receptors inhibits transport to the Golgi apparatus where processing of oligosaccharide chains, as well as proteolytic cleavage into subunits, takes place. The binding affinity of the mutant receptors for insulin was 50% of normal. Furthermore, insulin-stimulated autophosphorylation of IR delta Ex13 was markedly impaired. These data provide the evidence for a critical role of the extracellular domain of IR beta-subunit for processing and transport as well as the intramolecular signal transduction to activate IR tyrosine kinase.

scholarly journals The insulin receptor juxtamembrane region contains two independent tyrosine/beta-turn internalization signals.

1992 ◽  
Vol 118 (4) ◽  
pp. 831-839 ◽  
Author(s):  
J M Backer ◽  
S E Shoelson ◽  
M A Weiss ◽  
Q X Hua ◽  
R B Cheatham ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Insulin Receptor ◽  
Insulin Binding ◽  
Beta Turns ◽  
Cho Cell ◽  
Beta Turn ◽  
Tyrosine Residues ◽  
Juxtamembrane Region ◽  
Mutant Receptors

We have investigated the role of tyrosine residues in the insulin receptor cytoplasmic juxtamembrane region (Tyr953 and Tyr960) during endocytosis. Analysis of the secondary structure of the juxtamembrane region by the Chou-Fasman algorithms predicts that both the sequences GPLY953 and NPEY960 form tyrosine-containing beta-turns. Similarly, analysis of model peptides by 1-D and 2-D NMR show that these sequences form beta-turns in solution, whereas replacement of the tyrosine residues with alanine destabilizes the beta-turn. CHO cell lines were prepared expressing mutant receptors in which each tyrosine was mutated to phenylalanine or alanine, and an additional mutant contained alanine at both positions. These mutations had no effect on insulin binding or receptor autophosphorylation. Replacements with phenylalanine had no effect on the rate of [125I]insulin endocytosis, whereas single substitutions with alanine reduced [125I]insulin endocytosis by 40-50%. Replacement of both tyrosines with alanine reduced internalization by 70%. These data suggest that the insulin receptor contains two tyrosine/beta-turns which contribute independently and additively to insulin-stimulated endocytosis.

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.

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 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.

NOTCH3 expression in metastatic TNBC cells.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e12123-e12123
Author(s):  
Antonino Bonaventura D'Assoro
Keyword(s):  
Critical Role ◽  
Progression Free Survival ◽  
Immunoblot Analysis ◽  
Causative Role ◽  
Clinical Database ◽  
Self Renewal ◽  
Free Survival ◽  
Notch Receptors ◽  
Genetic Targeting

e12123 Background: Onset of metastases involves a complex multistep biological process termed the invasion-metastasis cascade, which include dissemination of cancer cells from the primary tumor to secondary organs. NOTCH signaling plays a critical role in promoting Triple Negative Breast Cancer (TNBC) metastasis. Although all four NOTCH receptors show oncogenic properties, the unique role of each of these receptors in the sequential stepwise events that typify the invasion-metastasis cascade remains elusive. Methods: We established unique TNBC cells (TNBC-M14, -M25 and –M40), isolated from patient-derived brain metastasis xenografts (PD-BMXs). NOTCH3 genetic targeting was achieved using Lenti-vector shRNAs. Immunoblot analysis was used to assess NOTCH3 expression. ALDH1 activity was measured using the ALDEFLUOR Kit. To assess self-renewal capacity and resistance to docetaxel-based chemotherapy, TNBC cells were cultured under non-adherent conditions to form mammospheres (MPS). A publicly clinical database ( http://kmplot.com ) was employed to analyze NOTCH3 expression in a selected cohort of 107 lymph- node+ TNBC patients. Results: TNBC-M14, -M25 and -M40 MPS showed NOTCH3 overexpression and higher ALDH1 activity compared to MDA-MB 231 MPS used as control. To investigate the causative role of NOTCH3 in inducing ALDH1 activity, self-renewal capacity and chemoresistance, we infected TNBC MPS with Lenti-vector shRNAs targeting NOTCH3. NOTCH3 geneting targeting induced a significant reduction of ALDH1 activity compared to TNBC MPS infected with scrambled Lenti-shRNAs. NOTCH3 geneting targeting significantly impaired MPS formation and restored sensitivity to docetaxel. The correlation between aberrant NOTCH3 expression and poor outcome in patients with advanced TNBC was validated in a publicly clinical database. Conclusions: These findings provide a compelling preclinical rationale for the design of novel clinical trials that will selectively target NOTCH3 to restore chemosensitivity and to improve the progression-free survival of TNBC patients that lack FDA-approved targeted therapies.

scholarly journals Insulin receptor desensitization correlates with attenuation of tyrosine kinase activity, but not of receptor endocytosis

1987 ◽  
Vol 245 (2) ◽  
pp. 357-364 ◽  
Author(s):  
A D Blake ◽  
N S Hayes ◽  
E E Slater ◽  
C D Strader
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Glycogen Synthesis ◽  
Insulin Receptors ◽  
Beta Subunit ◽  
Tyrosine Kinase Activity ◽  
Down Regulation ◽  
Receptor Endocytosis

A model of insulin-receptor down-regulation and desensitization has been developed and described. In this model, both insulin-receptor down-regulation and functional desensitization are induced in the human HepG2 cell line by a 16 h exposure of the cells to 0.1 microM-insulin. Insulin-receptor affinity is unchanged, but receptor number is decreased by 50%, as determined both by 125I-insulin binding and by protein immunoblotting with an antibody to the beta-subunit of the receptor. This down-regulation is accompanied by a disproportionate loss of insulin-stimulated glycogen synthesis, yielding a population of cell-surface insulin receptors which bind insulin normally but which are unable to mediate insulin-stimulated glycogen synthesis within the cell. Upon binding of insulin, the desensitized receptors are internalized rapidly, with characteristics indistinguishable from those of control cells. In contrast, this desensitization is accompanied by a loss of the insulin-sensitive tyrosine kinase activity of insulin receptors isolated from these cells. Receptors isolated from control cells show a 5-25-fold enhancement of autophosphorylation of the beta-subunit by insulin; this insulin-responsive autophosphorylation is severely attenuated after desensitization to a maximum of 0-2-fold stimulation by insulin. Likewise, the receptor-mediated phosphorylation of exogenous angiotensin II, which is stimulated 2-10-fold by insulin in receptors from control cells, is completely unresponsive to insulin in desensitized cells. These data provide evidence that the insulin-receptor tyrosine kinase activity correlates with insulin stimulation of an intracellular metabolic event. The data suggest that receptor endocytosis is not sufficient to mediate insulin's effects, and thereby argue for a role of the receptor tyrosine kinase activity in the mediation of insulin action.

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.

Effect of L-Dopa Administration on Insulin Binding: Possible Role of Growth Hormone in Regulation of Insulin Receptor Affinity

1982 ◽  
Vol 14 (07) ◽  
pp. 342-345
Author(s):  
M. Iwasaki ◽  
M. Kobayashi ◽  
S. Ohgaku ◽  
H. Maegawa ◽  
Y. Shigeta
Keyword(s):  
Growth Hormone ◽  
Insulin Receptor ◽  
Insulin Binding ◽  
Receptor Affinity

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.

Sign in / Sign up

Export Citation Format

Share Document