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

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.

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

Biochemical Journal ◽

10.1042/bj3050599 ◽

1995 ◽

Vol 305 (2) ◽

pp. 599-604 ◽

Cited By ~ 4

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.

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The Role of Insulin Receptor Sulphydryl Groups in Insulin Binding and Cellular Response in Rat Adipocytes

Journal of Receptor Research ◽

10.3109/10799899009064657 ◽

1990 ◽

Vol 10 (1-2) ◽

pp. 45-59 ◽

Cited By ~ 4

Author(s):

Andre G. Douen ◽

Malcolm N. Jones

Keyword(s):

Insulin Receptor ◽

Cellular Response ◽

Insulin Binding ◽

Rat Adipocytes ◽

Sulphydryl Groups

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

Hormone and Metabolic Research ◽

10.1055/s-2007-1019013 ◽

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

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Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region.

The Journal of Cell Biology ◽

10.1083/jcb.115.6.1535 ◽

1991 ◽

Vol 115 (6) ◽

pp. 1535-1545 ◽

Cited By ~ 64

Author(s):

J M Backer ◽

S E Shoelson ◽

E Haring ◽

M F White

Keyword(s):

Insulin Receptor ◽

Cho Cells ◽

Insulin Receptors ◽

Receptor Occupancy ◽

Wild Type ◽

Coated Pits ◽

Intracellular Potassium ◽

Juxtamembrane Region ◽

High Insulin ◽

Mutant Receptors

The effect of receptor occupancy on insulin receptor endocytosis was examined in CHO cells expressing normal human insulin receptors (CHO/IR), autophosphorylation- and internalization-deficient receptors (CHO/IRA1018), and receptors which undergo autophosphorylation but lack a sequence required for internalization (CHO/IR delta 960). The rate of [125I]insulin internalization in CHO/IR cells at 37 degrees C was rapid at physiological concentrations, but decreased markedly in the presence of increasing unlabeled insulin (ED50 = 1-3 nM insulin, or 75,000 occupied receptors/cell). In contrast, [125I]insulin internalization by CHO/IRA1018 and CHO/IR delta 960 cells was slow and was not inhibited by unlabeled insulin. At saturating insulin concentrations, the rate of internalization by wild-type and mutant receptors was similar. Moreover, depletion of intracellular potassium, which has been shown to disrupt coated pit formation, inhibited the rapid internalization of [125I]insulin at physiological insulin concentrations by CHO/IR cells, but had little or no effect on [125I]insulin uptake by CHO/IR delta 960 and CHO/IRA1018 cells or wild-type cells at high insulin concentrations. These data suggest that the insulin-stimulated entry of the insulin receptor into a rapid, coated pit-mediated internalization pathway is saturable and requires receptor autophosphorylation and an intact juxtamembrane region. Furthermore, CHO cells also contain a constitutive nonsaturable pathway which does not require receptor autophosphorylation or an intact juxtamembrane region; this second pathway is unaffected by depletion of intracellular potassium, and therefore may be independent of coated pits. Our data suggest that the ligand-stimulated internalization of the insulin receptor may require specific saturable interactions between the receptor and components of the endocytic system.

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Role of arginine 86 of the insulin receptor in insulin binding and activation of glucose transport

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/s0167-4889(97)00145-6 ◽

1998 ◽

Vol 1402 (1) ◽

pp. 86-94 ◽

Cited By ~ 4

Author(s):

Nicola Longo ◽

Sharon D Langley ◽

Maria J Still

Keyword(s):

Insulin Receptor ◽

Glucose Transport ◽

Insulin Binding

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Hormone-triggered conformational changes within the insulin-receptor ectodomain: requirement for transmembrane anchors

Biochemical Journal ◽

10.1042/bj3600189 ◽

2001 ◽

Vol 360 (1) ◽

pp. 189-198 ◽

Cited By ~ 14

Author(s):

Ralf-Rudiger FLÖRKE ◽

Kerstin SCHNAITH ◽

Waltraud PASSLACK ◽

Marc WICHERT ◽

Lothar KUEHN ◽

...

Keyword(s):

Secondary Structure ◽

Insulin Receptor ◽

Conformational Changes ◽

Sedimentation Coefficient ◽

Insulin Binding ◽

Underlying Structure ◽

Structural Constraints ◽

Wild Type ◽

Recombinant Human Insulin ◽

High Affinity

Interaction between two αβ half-receptors within the (αβ)2 holoreceptor complex is required for insulin binding with high affinity and for insulin-triggered changes of size and shape. To understand the underlying structure–function relationship, two truncated receptor constructs have been characterized. Reduction in the Stokes radius and increase in the sedimentation coefficient, which are characteristic for wild-type receptors, were entirely lacking for the recombinant human insulin receptor (HIR) ectodomain (HIR-ED). Stokes radii of about 5.8nm and sedimentation coefficients of 10.2S were found for both insulin-bound and free HIR-EDs. However, attaching the membrane anchors to the ectodomain, as with the recombinant membrane-anchored ectodomain (HIR-MAED) construct, was sufficient to restore not only high-affinity hormone binding but also the marked insulin-inducible alterations in hydrodynamic properties. The Stokes radii of HIR-MAED complexes, as assessed by non-denaturing PAGE, decreased upon insulin binding from 9.5nm to 7.9nm. In parallel, the sedimentation coefficient was increased from 9.0S to 9.8S. CD and fluorescence spectroscopy of HIR-MAED revealed only minor insulin-induced changes in the secondary structure. Similarity with wild-type receptors has also been demonstrated by the differential insertion of insulin-bound and free HIR-MAED complexes into artificial bilayer membranes of Triton X-114. The results are consistent with a model of receptor function that ensures a global insulin-triggered reorientation of subdomains within the ectodomain moieties while the secondary structure is essentially retained. For the rearrangement of such subdomains, the transmembrane anchors confer essential structural constraints on the receptor ectodomain.

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

Biochemical Journal ◽

10.1042/bj2740173 ◽

1991 ◽

Vol 274 (1) ◽

pp. 173-179 ◽

Cited By ~ 11

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.

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Identification of serines-967/968 in the juxtamembrane region of the insulin receptor as insulin-stimulated phosphorylation sites

Biochemical Journal ◽

10.1042/bj2980471 ◽

1994 ◽

Vol 298 (2) ◽

pp. 471-477 ◽

Cited By ~ 17

Author(s):

F Liu ◽

R A Roth

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Insulin Receptor ◽

Kinase Activity ◽

Insulin Receptors ◽

Wild Type ◽

Phosphatidylinositol 3 Kinase ◽

Juxtamembrane Region ◽

Kinase C ◽

Mutant Receptors

A line of Chinese hamster ovary cells overexpressing protein kinase C alpha was transfected with cDNAs encoding either the wild-type human insulin receptor or one of two mutant insulin receptors with either Ser-967 and -968 or -974 and -976 in the juxtamembrane region changed to alanine. Both mutant receptors exhibited normal insulin-activated tyrosine kinase activity as assessed by either autophosphorylation or insulin-stimulated increases in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase. The wild-type and mutant insulin receptors were also examined for serine and threonine phosphorylation in response to insulin and activation of protein kinase C. To visualize Ser/Thr-phosphorylation sites of the receptor better in response to insulin, the receptor from in vivo-labelled insulin-treated cells was first treated with a tyrosine-specific phosphatase to remove all tyrosine phosphorylation. Phosphopeptides from the three receptors were analysed by high-percentage polyacrylamide/urea gel electrophoresis and two-dimensional t.l.c. The mutant receptor lacking Ser-967 and -968 but not the mutant lacking Ser-974 and -976 was found to be missing phosphorylated peptides in response to insulin and, to a lesser extent, after activation of protein kinase C. However, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase was inhibited to the same extent by activation of protein kinase C in cells expressing the two mutant receptors as in cells expressing the wild-type receptor. These results indicate that these four serine residues in the juxtamembrane region are not major regulatory sites of the intrinsic tyrosine kinase activity of the insulin receptor by protein kinase C, although Ser-967 and/or -968 appear to be phosphorylated in response to insulin.

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