Differential sensitivity of the insulin-receptor kinase to thiol and oxidizing agents in the absence and presence of insulin

Wilden, Peter A.; Pessin, Jeffrey E.

doi:10.1042/bj2450325

Cited by 42 publications

(30 citation statements)

References 37 publications

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“…In addition, our studies provide a structural explanation for the widely observed stimulating effects of oxidants (28–32) and the effects of SH‐reactive reagents on the catalytic activity of the IRK (43–45). It may be important in this context that HP production can also be induced among others by insulin (46).…”

Section: Discussionmentioning

confidence: 67%

Phosphorylation of the insulin receptor kinase by phosphocreatine in combination with hydrogen peroxide: the structural basis of redox priming

et al. 1999

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Signaling by insulin requires autophosphorylation of the insulin receptor kinase (IRK) at Tyr1158, Tyr1162, and Tyr1163. Earlier experiments with (32)P-gamma-ATP indicated that the nonphosphorylated IRK (IRK-0P) is relatively inactive, and crystallographic data indicated that the ATP binding site of IRK-0P is blocked by its activation loop. We now show that phosphocreatine (PCr) in combination with hydrogen peroxide serves as an alternative phosphate donor and that ATP and PCr use distinct binding sites. Whereas phosphorylation of the IRK by ATP is inhibited by the nonhydrolyzable competitor adenylyl-imidodiphosphate, phosphorylation by PCr is enhanced. The IRK mutant Tyr1158Phe showed no phosphorylation with PCr but almost normal phosphorylation with ATP, whereas Tyr1162Phe was phosphorylated well with PCr but less then normal with ATP. 3-Dimensional models of IRK-0P revealed that the conversion of any of the four cysteine residues 1056, 1138, 1234, and 1245 into sulfenic acid produces structural changes that bring Tyr1158 into close contact with Asp1083 and render the well-known catalytic site at Asp1132 and Tyr1162 accessible from a direction that differs from the known ATP binding site. The mutant Cys1138Ala, in contrast, showed relatively inaccessible catalytic sites and weak catalytic activity in functional experiments. Taken together, these findings indicate that 'redox priming' of the IRK facilitates its autophosphorylation by PCr in the activation loop.

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Section: Discussionmentioning

confidence: 67%

Phosphorylation of the insulin receptor kinase by phosphocreatine in combination with hydrogen peroxide: the structural basis of redox priming

et al. 1999

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“…NEM inhibits autophosphorylation of the native insulin receptor (15)(16)(17)(18)(19), indicating that a free sulfhydryl group may be at or near the active site of the tyrosine kinase. It has been shown also that exposure of the insulin receptor to radioactive NEM results in incorporation of radioactivity into the /3 subunit (19).…”

Section: Resultsmentioning

confidence: 99%

Labile disulfide bonds in human placental insulin receptor.

Finn

Ridge

1990

Proc. Natl. Acad. Sci. U.S.A.

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The disulfide crosslinking pattern of human placental insulin receptor was investigated using selective reduction with tributylphosphine followed by alkylation with N-[3lHlethylmaleimide. Insulin receptor contains a single sulfhydryl group in each a3 subunit whose alkylation with N-[3H]ethylmaleimide inhibits receptor autophosphorylation. Alkylation is partially inhibited by ATP or the nonhydrolyzable substrate analog adenosine 5'-[li,-imidojtriphosphate when the nucleotides are added as Mn2+ complexes. Neither insulin nor 6 M guanidinium chloride renders additional sulfhydryl groups accessible to alkylation. When the receptor is reduced under drastic conditions with tributylphosphine in guanidinium chloride, 32 of the 37 sulfhydryl groups in the receptor's a subunit can be alkylated with N-[3Hjethylmaleimide. Surprisingly only three of the 10 cysteines in the 13 subunit become titratable under identical conditions. By using highly selective reducing conditions, we were able to determine quantitatively the maximum number of disulfide bridges that link the two ac8 halves to form the tetrameric structure and those that couple the a to the 13 subunits. Liberation of two sulfhydryl groups in the a and one in the (3 subunit resulted in formation of a.1 dimers. Free (3 subunit was formed when an additional disulfide bond was reduced. It is remarkable that the tetrameric structure of this highly complex receptor molecule, which contains a large number of cysteine residues, is maintained by such a small number of disulfide bonds. Three models of the arrangement of the labile disulfide bonds, consistent with these findings, are proposed.Human placental insulin receptor is a tetrameric glycoprotein composed of two heteromeric dimers, each dimer containing an a subunit (125 kDa) and a 8 subunit (90 kDa). The a subunits, located on the extracellular surface of the plasma membrane, contain the insulin binding site. The , subunits can be divided into three sections, an extracellular sequence, a membrane-spanning sequence, and an intracellular portion. When insulin combines with its receptor, the cytosolic portion of the 8 subunit undergoes autophosphorylation, activating it as a tyrosine kinase.The tetrameric structure of the receptor is maintained, in part, by disulfide bonds that possess differential sensitivity to reducing agents (1). At low concentrations of reducing agent, a,3 dimers are formed whereas higher concentrations of reductant are necessary to form individual subunits. Several models of the receptor have been proposed (2-4). The most familiar of these depicts the a subunits linked to one another by disulfide bonds to form the dimer with the , subunits linked by disulfide bonds only to the a subunits (,8-S-S-a-S-S-a-S-S-,B) (2, 3).The present study focuses on the quantitative determination of the disulfide linkages responsible for preserving the architecture of the insulin receptor. MATERIALS AND METHODSCrystalline bovine insulin was a gift from Eli Lilly. Triton X-100 and tributylphosphine were from Ald...

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“…The same laboratory (21) has shown that autophosphorylation is not inhibited when iodoacetamide is used to alkylate the receptor, suggesting that inhibition by NEM is due to steric interference.…”

Section: Methodsmentioning

confidence: 98%

ATP sensitizes the insulin receptor to insulin.

Ridge

Finn²

1988

Proc. Natl. Acad. Sci. U.S.A.

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Insulin receptor with high insulin binding and tyrosine kinase activities has been prepared from human placenta. Based on a molecular mass of 306 kDa for the receptor (the value obtained from the sum of the amino acid residues), this preparation is capable of binding 1.48 mol of insulin per mol of receptor. The receptor is free from phosphatase and ATPase activity and is not stimulated by sodium vanadate. Autophosphorylation is linear with respect to receptor concentration, and the 32p incorporated is stable even in the presence of a 100-fold excess of unlabeled ATP. The Km for ATP is 208 ,.M. N-Ethylmaleimide inhibits autophosphorylation. Alkylation with 3H-labeled N-ethylmaleimide results in the incorporation of 1.13 ± 0.37 mol of N-ethylmaleimide per mol of insulin binding activity exclusively into the f8 subunit of the receptor. The nonhydrolyzable ATP analog adenosine 5'-[fi,y-imido]triphosphate stimulates autophosphorylation of the receptor, an effect that is evident at ATP concentrations below 1 mM. The stimulatory effect of adenosine 5'-[j3,y-imido]triphosphate is the result of increasing the binding of insulin to the a subunit, and this reflects itself in a shift to the left of the insulin dose-response curve for autophosphorylation. The same is true for ATP. As a consequence, it is now possible to reconcile the concentration of insulin necessary for stimulating the autophosphorylation reaction with physiological levels and with the levels of insulin required for its classical biological effects.The tyrosine kinase activity of the human placental insulin receptor has been the subject of numerous investigations. The majority of these studies were performed with wheat germ agglutinin-purified receptor (wheat germ receptor) that contains only -0.3% of the insulin receptor (1). It appears that this preparation of the receptor was used rather than affinity-purified material because the latter, in spite of high insulin binding activity, exhibits very poor tyrosine kinase activity. However, the wheat germ receptor, which has relatively high tyrosine kinase activity, can be expected to contain phosphate-metabolizing enzymes such as ATPases and phosphatases, which are likely to interfere with accurate kinetic measurements. The disadvantages inherent in both of these preparations prompted the present investigation, which had as its primary goal the development of methodology for the preparation of highly purified receptor exhibiting tyrosine kinase activity comparable to that of the wheat germ receptor. This goal was achieved by the use of avidin-biotin technology and the development of an improved method for eluting the receptor from the affinity resin. The ensuing preparations, which exhibited a high level of insulin binding activity and a high degree of tyrosine kinase activity, appeared to be well suited for accurate kinetic measurements. Such studies are the subject of this paper. MATERIALS AND METHODSNa Bl_(6-biotinylamido)hexanoyl-Na.A1,B 9-bis(t-butyl-oxycarbonyl)insulin, succinoylavidin-Sepharose...

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Differential sensitivity of the insulin-receptor kinase to thiol and oxidizing agents in the absence and presence of insulin

Cited by 42 publications

References 37 publications

Phosphorylation of the insulin receptor kinase by phosphocreatine in combination with hydrogen peroxide: the structural basis of redox priming

Phosphorylation of the insulin receptor kinase by phosphocreatine in combination with hydrogen peroxide: the structural basis of redox priming

Labile disulfide bonds in human placental insulin receptor.

ATP sensitizes the insulin receptor to insulin.

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