Insulin Receptor Kinase Domain Research Articles

Biochemical characterization of the Drosophila insulin receptor kinase and longevity-associated mutants.

Drosophila melanogaster (fruit fly) insulin receptor (D-IR) is highly homologous to the human counterpart. Like the human pathway, D-IR responds to numerous insulin-like peptides to activate cellular signals that regulate growth, development, and lipid metabolism in fruit flies. Allelic mutations in the D-IR kinase domain elevate life expectancy in fruit flies. We developed a robust heterologous expression system to express and purify wild-type and longevity-associated mutant D-IR kinase domains to investigate enzyme kinetics and substrate specificities. D-IR exhibits remarkable similarities to the human insulin receptor kinase domain but diverges in substrate preferences. We show that longevity-associated mutations reduce D-IR catalytic activity. Deletion of the unique kinase insert domain portion or mutations proximal to activating tyrosines do not influence kinase activity, suggesting their potential role in substrate recruitment and downstream signaling. Through biochemical investigations, this study enhances our comprehension of D-IR's role in Drosophila physiology, complementing genetic studies and expanding our knowledge on the catalytic functions of this conserved signaling pathway.

Dual Anti-/Prooxidant Behaviors of Flavonoids Pertaining to Cu(II)-Catalyzed Tyrosine Nitration of the Insulin Receptor Kinase Domain in an Antidiabetic Study.

Flavonoid, as a potent antioxidant, exerts many beneficial effects in type 2 diabetes, whereas the prooxidative property may be also important in vivo if copper is involved. Here, we chose an insulin receptor kinase domain fragment (KK-1, residues 1126-1165), containing the A-loop of the receptor as well as three key autophosphorylation sites (Tyr1158, Tyr1162, and Tyr1163) associated with receptor signal transduction to investigate the roles and the structure-activity relationship of three antidiabetic flavonoids (kaempferol, luteolin, and apigenin) and two others with a similar structure (diosmetin and genistein), on modulation of Cu(II)-mediated tyrosine nitration and the corresponding effect on its functional phosphorylation in the Cu2+/H2O2/NO2- system. We found that both properties of flavonoid played roles on inhibition of Cu(II)-mediated protein nitration in the H2O2/NO2- system: (1) on the one hand, flavonoid scavenged free radicals as antioxidants, inhibited tyrosine nitration, and thus inhibited the reduction of tyrosine phosphorylation caused by tyrosine nitration; and (2) on the other hand, flavonoid promoted •OH production as a prooxidant, which increased 3,3'-dityrosine formation. The formation of 3,3'-dityrosine decreased Cu2+-induced tyrosine nitration and thus interfered with its phosphorylation. This study confirms that the weight relationship between antioxidation and prooxidation of a flavonoid needs to be studied clearly before nutritional and medical applications.

Stepwise autophosphorylation regulates biased agonism of the insulin receptor

Insulin is a key regulator of energy metabolism in peripheral tissues but also functions as a growth factor. Insulin binding to the insulin receptor (IR) leads to autophosphorylation of intracellular tyrosine residues, which simultaneously initiates a multitude of signals and functions. In contrast, some artificial (non‐insulin) ligands for the IR result in biased agonism, selectively activating the PI3K/AKT pathway and metabolic effects without activating mitogen‐activated protein kinase (MAPK) pathway and mitogenic effects. However, the precise mechanism of biased agonism at the receptor level remains unclear. The biased agonist IR‐A48 aptamer selectively induces mono‐phosphorylation of Tyr1150 residue (m‐pY1150) in the kinase domain of IR. Hence, we hypothesized that IR autophosphorylation is a stepwise process in which formation of m‐pY1150 represents an intermediate step. To explore this idea, we used hybrid receptors in which insulin can bind only one of the two binding sites of the dimeric receptor. Asymmetric insulin binding selectively induced symmetric m‐pY1150 in both kinase domains. Moreover, the juxtamembrane domain, which interacts with the kinase domain, restricted the full activation of IR, and symmetric m‐pY1150 played a crucial role in the rearrangement of intracellular domains to release this restriction. Our findings demonstrate that the symmetry of insulin binding to a dimeric receptor determines the stepwise autophosphorylation of IR. Furthermore, considering that the degree of ligand symmetry on a receptor depends mainly on ligand concentration, our results suggest that IR may play metabolic‐biased roles in peripheral tissues dependent on local insulin concentrations.Support or Funding InformationThis research was supported by the Global Research Laboratory (GRL) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (No. NRF‐2016K1A1A2912722).

Type A Insulin Resistance Syndrome- Novel insulin receptor gene mutation and familiar phenotypic variability

Type A Insulin Resistance Syndrome is due to heterozygous mutations in the insulin receptor (INSR) gene or its signaling pathway. We present a premenarcheal 14 year-old girl with normal BMI, severe hirsutism, acanthosis nigricans, clitoral hypertrophy, deep voice, enlarged polycystic ovaries, severe hyperinsulinemia and biochemical hyperandrogenism. We identified a novel heterozygous missense variant in the tyrosine kinase domain of INSR (p.Leu1150Pro) and an heterozygous missense variant in SH2B adapter protein 1 involved in the insulin pathway (p.Ala663Val). Interestingly, the patients’ mother and brother had the same INSR mutation although of a milder phenotype, reason why their IR went undiagnosed.

Hydrogen Attachment/Abstraction Dissociation (HAD) of Gas-Phase Peptide Ions for Tandem Mass Spectrometry.

Dissociation of gas-phase peptide ions through interaction with low-energy hydrogen (H) radical (∼0.15 eV) was observed with a quadrupole ion trap mass spectrometry. The H radical generated by thermal dissociation of H2 molecules passing through a heated tungsten capillary (∼2000 °C) was injected into the ion trap containing target peptide ions. The fragmentation spectrum showed abundant c-/z- and a-/x-type ions, attributable to H attachment/abstraction to/from peptide ion. Because the low-energy neutral H radical initiated the fragmentation, the charge state of the precursor ion was maintained during the dissociation. As a result, precursor ions of any charge state, including singly charged positive and negative ions, could be analyzed for amino acid sequence. The sequence coverage exceeding 90% was obtained for both singly protonated and singly deprotonated substance P peptide. This mass spectrometry also preserved labile post-translational modification bonds. The modification sites of triply phosphorylated peptide (kinase domain of insulin receptor) were identified with the sequence coverage exceeding 80%.

Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation.

Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors.

Human biliverdin reductase‐based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor

Insulin binding changes conformation of the insulin receptor kinase (IRK) domain and initiates glucose uptake through the insulin, IGF-1, phosphatidyl inositol 3-kinase (PI3K), and MAPK pathways; human biliverdin reductase (hBVR) is an IRK substrate and pathway effector. This is the first report on hBVR peptide-mediated IRK activation and conformational change. (290)KYCCSRK, which increased IRK V(max) without changing K(m), stimulated glucose uptake and potentiated insulin and IGF-1 stimulation in 4 cell lines. KYCCSRK in native hBVR was necessary for the hBVR and IRK cross-activation. Peptide treatment also activated PI3K downstream effectors, Akt and ERK, phosphorylation, and Elk transcriptional activity. In cells transfected with CMV-regulated EGFP-VP-peptide plasmid, C(292)→A mutant did not stimulate glucose uptake; K(296)→A decreased uptake and kinase activity. KEDQYMKMTV, corresponding to hBVR's SH2-binding domain, was a potent inhibitor of glucose uptake and IRK. The mechanism of action of peptides was examined using cells expressing IRK (aa 988-1263) activated by coexpressed KYCCSRK. Three active cys-mutants of IRK, with fluorophore coupled to cysteines, C(1056), C(1138), or C(1234), were examined for changes in fluorescence emission spectra in the presence of peptides. KYCCSRK and KEDQYMKMTV bound to different sites in IRK. The findings identify novel agents for activating or inhibiting insulin signaling and offer a new approach for treatment of type 2 diabetes and hypoglycemia.

“DFG-Flip” in the Insulin Receptor Kinase Is Facilitated by a Helical Intermediate State of the Activation Loop

We have characterized a large-scale inactive-to-active conformational change in the activation-loop of the insulin receptor kinase domain at the atomistic level via untargeted temperature-accelerated molecular dynamics (TAMD) and free-energy calculations using the string method. TAMD simulations consistently show folding of the A-loop into a helical conformation followed by unfolding to an active conformation, causing the highly conserved DFG-motif (Asp1150, Phe1151, and Gly1152) to switch from the inactive “D-out/F-in” to the nucleotide-binding-competent “D-in/F-out” conformation. The minimum free-energy path computed from the string method preserves these helical intermediates along the inactive-to-active path, and the thermodynamic free-energy differences are consistent with previous work on various other kinases. The mechanisms revealed by TAMD also suggest that the regulatory spine can be dynamically assembled/disassembled either by DFG-flip or by movement of the αC-helix. Together, these findings both broaden our understanding of kinase activation and point to intermediates as specific therapeutic targets.

Helpful introduction to signal transduction

![Figure][1] Structure and Function in Cell Signalling By John Nelson Wiley (2008) 410 pages, ISBN 978-0-470-02551-2, £34.95 (paperback) When studying cell signaling, it is easy to get overwhelmed with the complexity of each pathway and with the crosstalk between them. Teaching the

Insulin resistance and increased pancreatic β-cell proliferation in mice expressing a mutant insulin receptor (P1195L)

Several mutations of the tyrosine kinase domain of insulin receptor (IR) have been clinically reported to lead insulin resistance and insulin hypersecretion in humans. However, it has not been completely clarified how insulin resistance and pancreatic beta-cell function affect each other under the expression of mutant IR. We investigated the response of pancreatic beta-cells in mice carrying a mutation (P1195L) in the tyrosine kinase domain of IR beta-subunit. Homozygous (Ir(P1195L/P1195L)) mice showed severe ketoacidosis and died within 2 days after birth, and heterozygous (Ir(P1195L/wt)) mice showed normal levels of plasma glucose, but high levels of plasma insulin in the fasted state and after glucose loading, and a reduced response of plasma glucose lowering effect to exogenously administered insulin compared with wild type (Ir(wt/wt)) mice. There were no differences in the insulin receptor substrate (IRS)-2 expression and its phosphorylation levels in the liver between Ir(P1195L/wt) and Ir(wt/wt) mice, both before and after insulin injection. This result may indicate that IRS-2 signaling is not changed in Ir(P1195L/wt) mice. The beta-cell mass increased due to the increased numbers of beta-cells in Ir(P1195L/wt) mice. More proliferative beta-cells were observed in Ir(P1195L/wt) mice, but the number of apoptotic beta-cells was almost the same as that in Ir(wt/wt) mice, even after streptozotocin treatment. These data suggest that, in Ir(P1195L/wt) mice, normal levels of plasma glucose were maintained due to high levels of plasma insulin resulting from increased numbers of beta-cells, which in turn was due to increased beta-cell proliferation rather than decreased beta-cell apoptosis.

Interdependent Regulation of Insulin Receptor Kinase Activity by ADP and Hydrogen Peroxide

Insulin signaling requires autophosphorylation of the insulin receptor kinase (IRK) domain. Using purified recombinant IRK fragments and the isolated intact insulin receptor, we show here that autophosphorylation is inhibited by ADP and that this effect is essentially reversed by hydrogen peroxide. Autophosphorylation was inhibited by hydrogen peroxide (60 microM) in the absence of ADP but enhanced in the presence of inhibitory concentrations of ADP (67 microM). Enhancement by hydrogen peroxide required direct interaction of hydrogen peroxide with the kinase domain and was not seen in insulin receptor mutants C1245A and C1308A. A similar enhancement was obtained in intact cells in the absence of insulin upon treatment with 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea, indicating that IRK activity can be alternatively enhanced by a shift in the thiol/disulfide redox status. Molecular modeling of the IRK domain indicated that the ATP-binding site becomes distorted after releasing the nucleotide unless the IRK domain is oxidatively derivatized at Cys1245. Recent clinical studies suggest that these effects may play a role in obesity due to the fact that cytoplasmic creatine kinase in combination with phosphocreatine normally ensures rapid removal of ADP in muscle cells but not in fat cells.

A Family of Evolution–Entropy Hybrid Methods for Ranking Protein Residues by Importance

In order to identify the amino acids that determine protein structure and function it is useful to rank them by their relative importance. Previous approaches belong to two groups; those that rely on statistical inference, and those that focus on phylogenetic analysis. Here, we introduce a class of hybrid methods that combine evolutionary and entropic information from multiple sequence alignments. A detailed analysis in insulin receptor kinase domain and tests on proteins that are well-characterized experimentally show the hybrids' greater robustness with respect to the input choice of sequences, as well as improved sensitivity and specificity of prediction. This is a further step toward proteome scale analysis of protein structure and function.

Multiple Activation Loop Conformations and Their Regulatory Properties in the Insulin Receptor's Kinase Domain

Low catalytic efficiency of protein kinases often results from intrasteric inhibition caused by the activation loop blocking the active site. In the insulin receptor's kinase domain, Asp-1161 and Tyr-1162 in the peptide substrate-like sequence of the unphosphorylated activation loop can interact with four invariant residues in the active site: Lys-1085, Asp-1132, Arg-1136, and Gln-1208. Contributions of these six residues to intrasteric inhibition were tested by mutagenesis, and the unphosphorylated kinase domains were characterized. The mutations Q1208S, K1085N, and Y1162F each relieved intrasteric inhibition, increasing catalytic efficiency but without changing the rate-limiting step of the reaction. The mutants R1136Q and D1132N were virtually inactive. Steric accessibility of the active site was ranked by relative changes in iodide quenching of intrinsic fluorescence, and A-loop conformation was ranked by limited tryptic cleavage. Together these ranked the openness of the active site cleft as R1136Q approximately D1132N > or = D1161A > Y1162F approximately K1085N > Q1208S > or = wild-type. These findings demonstrate the importance of specific invariant residues for intrasteric inhibition and show that diverse activation loop conformations can produce similar steady-state kinetic properties. This suggests a broader range of regulatory properties for the activation loop than expected from a simple off-versus-on switch for kinase activation.

Vanadium(V) complexes in enzyme systems: aqueous chemistry, inhibition and molecular modeling in inhibitor design.

Vanadate in aqueous solution is known to influence a number of enzyme-catalyzed reactions. Such effects are well known to carry over to living systems where numerous responses to the influence of vanadium have been well-documented; perhaps the most studied being the insulin-mimetic effect. Studies of the aqueous chemistry of vanadate provide an insight into the mechanisms by which vanadate affects enzyme systems and suggests methods for the elucidation of specific types of responses. Studies of the corresponding enzymes provide complementary information that suggests model vanadate systems be studied and provides clues as to functional groups that might be utilized in the development of selective enzyme inhibition. The insulin-mimetic effect is thought by many workers to originate in the effectiveness of vanadium as an inhibitor of protein tyrosine phosphatase (PTPase) activity. One, or more PTPases regulate the phosphotyrosine levels of the insulin receptor kinase domain. Appropriate ligands allow modification of the reactivity and function of vanadate. For instance, although the complex, ((CH(3))(2)NO)(2)V(O)OH, is not quite as good an inhibitor of PTPase activity as is vanadate, it is much more effective in cell cultures for increasing glucose transport and glycogen synthesis. Studies of the chemistry of this complex provide an explanation of the efficacy of this compound as a PTPase inhibitor that is supported by computer modeling studies. Computer calculations using X-ray data of known PTPases as a basis for homology modeling then suggests functionality that needs to be addressed in developing selective PTPase inhibitors.

Activation of the insulin receptor's kinase domain changes the rate-determining step of substrate phosphorylation.

The insulin receptor and many other protein kinases are activated by relief of intrasteric inhibition that is regulated by reversible phosphorylation. The changes accompanying activation of the insulin receptor's kinase domain were analyzed using steady-state kinetics, viscometric analysis, and equilibrium binding measurements. Peptide phosphorylation catalyzed by the unphosphorylated basal-state kinase is limited by a slow rate of the chemical step, and the activated enzyme is limited by product release rates. Underlying these changes were a 36-fold increase in the rate constant for the chemical step of the enzyme-catalyzed reaction, a 5-fold increase in the affinity for MgATP, and an 8-fold increase in the affinity for peptide substrate. This results in binding of substrates that is 2.2 kcal/mol more favorable and a free energy barrier for transition state formation that is lowered by 2.1 kcal/mol in the activated enzyme. Therefore, the change in conformational free energy inherent in the protein after autophosphorylation [Bishop, S. M., Ross, J. B. A., and Kohanski, R. A. (1999) Biochemistry 38, 3079-3089] is equally distributed between formation of the substrate ternary complex and formation of the transition state complex.

Stable plasma membrane expression of the soluble domain of the human insulin receptor in yeast

The soluble cytoplasmic kinase domain of the human insulin receptor was N-terminally equipped with either an N-acetylation or a dual-acylation motif (MGC box, to allow myristoylation/palmitoylation) and expressed in yeast cells under the control of the inducible CUP1 promoter. Although the cellular concentration was about the same in both instances (reflecting similar stability against proteolysis), only the myristoylated protein was capable of autophosphorylation to a significant extent and was active to phosphorylate endogenous yeast proteins at tyrosine residues in vivo. Cellular subfractionation showed that the insulin receptor was associated with plasma membranes, from where it was not extractable with high salt or alkali, but a significant fraction was also localized in the nuclear fraction. The myristoylated protein is absent from the cytoplasm. No effect of expression of either the acetylated or the myristoylated version on growth and respiration on various carbon sources was detected, suggesting a failure of the active insulin receptor kinase domain to couple to yeast (glucose) signalling cascades.

Naturally occurring amino acid substitutions at Arg1174 in the human insulin receptor result in differential effects on receptor biosynthesis and hybrid formation, leading to discordant clinical phenotypes.

Missense mutations in the tyrosine kinase domain of the human insulin receptor frequently result in a dominantly inherited form of insulin resistance. We noted a marked disparity in the clinical phenotypes of our study subjects with different missense mutations at the same residue (Arg1174) of the insulin receptor. Subjects with a tryptophan substitution (W) were only moderately hyperinsulinemic, whereas those with a glutamine substitution (Q) had severe clinical and biochemical insulin resistance. Studies were undertaken to explore the molecular mechanisms underlying these differences. Both W and Q mutant receptors bound insulin normally but were kinase inactive. The W mutation resulted in more rapid degradation of newly synthesized mutant receptor, which contrasted with the near-normal biosynthesis of the Q receptor. The propensity of the W receptor to form hybrids with the cotransfected wild-type (WT) receptor was also markedly impaired compared with the Q receptor, to an extent greater than could be explained by lower steady-state expression. Thus, the more clinically benign consequences of the heterozygous W mutant receptor are likely to relate to its impaired biosynthesis and/or reduced capacity to form hybrids with WT receptors. In addition to providing an explanation for the milder phenotype of 1174W versus 1174Q carriers, these studies provide further support for the notion that the dominant-negative effect of insulin receptor tyrosine kinase mutations involves the competition between inactive mutant homodimers and WT/mutant hybrids with active WT homodimers for both ligands and intracellular substrates.

Alternative splicing, gene localization, and binding of SH2-B to the insulin receptor kinase domain.

The SH2-B protein is an SH2-domain-containing molecule that interacts with a number of phosphorylated kinase and receptor molecules including the insulin receptor. Two isoforms of the SH2-B have been identified and have been proposed to arise through alternate splicing. Here we have identified a third isoform of the SH2-B protein, SH2-Bgamma, that interacts specifically with the insulin receptor. This interaction required phosphorylation of residue Y1146 in the triple tyrosine motif within the activation loop of the IR kinase and is one of only two signaling molecules shown to interact directly with this residue of the insulin receptor kinase domain. The intron/exon structure of the SH2-B gene was determined. Alternate splice sites utilized to generate the different isoforms of the SH2-B protein were identified in the 3' end of the SH2-B gene immediately downstream of the exon encoding the core of the SH2 domain. Additionally, the chromosomal location of the SH2-B gene was determined to be the distal arm of mouse Chromosome (Chr) 7 in a region linked to obesity in mice.

Modeling Studies of the Interactions between the Insulin Receptor Kinase Domain and Protein Tyrosine Phosphatase 1B

The sulfotyrosine peptide, IRK1154, is based on the corresponding phosphotyrosine segment of the insulin receptor kinase domain (IRK) and is a known inhibitor of the function of the protein tyrosine phosphatase, PTP1B. Two-dimensional NMR spectroscopy, in the transferred nuclear Overhauser effect (NOE) enhancement experiment, was used to obtain information concerning the bound structure of this peptide. Computer-simulated docking experiments, followed by molecular dynamics simulations in a fully hydrated model, provided information concerning the site-specific interactions influencing the bound peptide. Using the structural and orientational information from the NMR studies as a guide, together with the X-ray coordinates for PTP1B and IRK, a detailed model of the binding of these two proteins was developed. The interface between the two entities is described, and the sites of positive interactions are identified. Potential sources of destabilizing interactions, necessary for dissociation of the two enzyme...

Autophosphorylation dependent destabilization of the insulin receptor kinase domain: tryptophan-1175 reports changes in the catalytic cleft.

Protein kinases are regulated by conformational or chemical changes which facilitate access of substrates to the active site and promote correct orientations of catalytically essential residues and water molecules. The switch between basal and activated states of the insulin receptor's kinase domain (IRKD) results from autophosphorylation. We investigated the effects of IRKD autophosphorylation on the conformational stability by guanidine hydrochloride (GdnHCl) dependent denaturation and by iodide quenching of intrinsic fluorescence. Tryptophan residues of the recombinant soluble IRKD (residues R953-S1355) were excited at a lambdaex of 295 nm, and emission spectra were analyzed for centroid (a characteristic of average polarity of the indole rings' environments) and integrated fluorescence intensity over the lambdaem range of 310-420 nm. Denaturation profiles of both apo- and phospho-IRKD forms are complex with at least three distinct unfolding transitions. The first and last transitions were reversible and cooperative and had midpoints at 0.4 or 0.7 M GdnHCl and 2.4 or 2.7 M GdnHCl, respectively; transitions of phospho-IRKD occurred at lower GdnHCl concentrations. Calculations of free energy of unfolding suggested a loss of approximately 2.3 kcal/mol of stabilization for the first transition and approximately 1.5 kcal/mol for the third transition. Circular dichroism showed subtle changes in secondary structure over the first transition and global unfolding over the last transition. The first transition reports changes primarily in the local environment of W1175, which is near the catalytic loop and is conserved among protein tyrosine kinases. W1175 is also the dominant fluorophore of the native emission spectrum. Iodide quenching of W1175 was virtually undetectable in the apo-IRKD but significant in the phospho-IRKD, suggesting that W1175 exposure to small solutes is strongly dependent on the conformation of the activation loop. These studies indicate that autophosphorylation, while exposing the catalytic center, also produces a conformer less stable than the apoenzyme.