Quaternary Structure of the Insulin-Insulin Receptor Complex

Luo, Robert Z.; Beniac, Daniel R.; Fernandes, Allan; Yip, Cecil C.; Ottensmeyer, F. P.

doi:10.1126/science.285.5430.1077

Cited by 124 publications

(97 citation statements)

References 24 publications

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“…In the crystal structure the C terminus of the L2 domain is facing away from the putative binding pocket, and therefore in IR468.CT, which binds ligand as a monomer (7) ,the position of the L2 domain must adopt a different orientation than found in the crystals of the non-binding L1-CYS-L2 fragment of the IGFIR. However, in the holoreceptor it may be that the nanomolar binding site comprises epitopes from L1 on one monomer and the CT peptide domain from the other receptor monomer, which would be consistent with an antiparallel association in the dimer, as suggested by some electron microscopy studies of the insulin receptor (22,23). In the present study we have only investigated IR fragments; therefore, further studies are needed to demonstrate whether reconstitution is feasible with the minimized IGFIR.…”

Section: Discussionmentioning

confidence: 90%

Functional Reconstitution of Insulin Receptor Binding Site from Non-binding Receptor Fragments

Kristensen¹,

Andersen²,

Østergaard³

et al. 2002

Journal of Biological Chemistry

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We have previously shown that a minimized insulin receptor (IR) consisting of the first 468 amino acids of the insulin receptor fused to 16 amino acids from the C terminus of the ␣-subunit (CT domain) bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Schä ffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780 -17786). In the present study, we show that a smaller construct that has the first 308 residues fused to the CT domain also binds insulin. Insulin receptor fragments consisting of the first 468 or 308 residues did not bind insulin. However, when these fragments were mixed with a synthetic peptide corresponding to the CT domain, insulin binding was detectable. At concentrations of 10 M CT peptide, insulin binding was fully reconstituted yielding apparent affinities of 9 -11 nM. To further investigate the minimum requirement for the length of the N terminus of IR, we tested smaller receptor fragments for insulin binding in the presence of the CT peptide and found that a fragment consisting of the first 255 amino acids of IR was able to fully reconstitute the insulin binding site, yielding an apparent affinity of 11 ؎ 4 nM for insulin.Insulin mediates its effects by binding to tyrosine kinase receptors in the plasma membrane of targets cells. The IR 1 protein is a dimer of two identical ␣␤ monomers covalently linked by disulfide bonds in a ␤-␣-␣-␤ conformation (1). The IR structure function has recently been reviewed (2). Predictions of the structure of the IR ectodomain have been based on sequence alignments with homologous domains in other receptors, reviewed by . The consensus from these alignments is that the first 468 residues of the ␣-subunit contain two large homologous domains L1 and L2 separated by a cysteine-rich (CYS) region (4, 5). A crystal structure of the L1-CYS-L2 region of the homologous IGFI receptor (IGFIR) has been solved, confirming this domain structure (6). However, this construct does not bind ligand, whereas studies on minimized receptors show that, when fusing the L1-CYS-L2 domain to either of the C-terminal ␣-subunit sequences, IGFIR residues 691-706 or IR residues 704 -719 (CT domains), 2 the construct does bind ligand (7). The affinity of the minimized insulin receptor (mIR) for insulin is ϳ5-10 nM or similar to what is found for the soluble IR ectodomain (sIR) (8). The solubilized holoreceptor (hIR) binds insulin with an affinity that is almost 1000-fold better than mIR and sIR (9). Recently, we reported that, when expressing a receptor construct in which 48 residues of the exon 9 region of the ␣-subunit were deleted, we obtained a dimeric ␣-␣ receptor fragment that bound insulin with full holoreceptor affinity. The data showed that the picomolar affinity was associated with the dimeric structure of the ␣-subunits, whereas monomers had nanomolar affinity for insulin (9). Moreover the dimeric construct (mIR.Fn0/Ex10) also displayed accelerated dissociation of labeled insulin in the presence of excess unlabeled insulin, which is another characteristic of ...

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Section: Discussionmentioning

confidence: 90%

Functional Reconstitution of Insulin Receptor Binding Site from Non-binding Receptor Fragments

Kristensen¹,

Andersen²,

Østergaard³

et al. 2002

Journal of Biological Chemistry

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“…The signaling proteins in this pathway affect cellular metabolism, growth and survival. The insulin receptor is a heterotetrameric protein that has two extracellular α subunits (120 kD) and two transmembrane β subunits (90 kD) (Luo et al, 1999). Insects only have a single insulin-like receptor that is responsible for modulating both growth and nutritional cues.…”

Section: Insulin Signaling Pathway In Insectsmentioning

confidence: 99%

Nutritional sensitivity of fifth instar prothoracic glands in the tobacco hornworm, Manduca sexta

Walsh

Smith

2011

Journal of Insect Physiology

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The tobacco hornworm, Manduca sexta, has been a useful model for studying insect endocrinology. In M. sexta the majority of growth occurs during the fifth (final) larval stage. This growth is concurrent with endocrine changes that lead to a metamorphic molt. Past research has focused on the interplay of three growth hormones: juvenile hormone (JH), prothoracicotropic hormone (PTTH) and ecdysone, in regulating metamorphosis. The prothoracic glands, source of ecdysteroid molting hormones in insects, play a crucial role in coordinating growth. Insulin/insulin-like growth factor (IGF) signaling has been shown to regulate growth and secretion of steroidogenic tissues in both vertebrates and invertebrates, but it had yet to be well-characterized in M. sexta.In this dissertation I examined the role of insulin-directed growth in prothoracic glands during both normal larval development and in response to nutrient deprivation. In particular, I predicted that in response to a reduction in nutrients, the prothoracic glands would stop growing and ecdysone secretion would be reduced. Changes were assessed in actin abundance and in two nutritionally sensitive transcripts, insulin receptor and 4EBP.Actin abundance in prothoracic glands increased in the first four days of this stage, demo nstrating significant growth. Actin decreased in glands from starved larvae, indicating a cessation of growth. Both insulin receptor and 4EBP transcript were present during normal larval development. As expected, both insulin receptor and 4EBP transcript increased in response to starvation. This research was the first demonstration of insulin signaling in prothoracic glands of fifth instar M. sexta larvae, and revealed nutritional sensitivity of the prothoracic glands. 5Finally, ecdysone assays were conducted to determine the impact of nutrition on glandular secretory capacity. Starved larvae were unable to secrete as much ecdysone as fed controls. Injections of insulin were unable to rescue the ability of the glands to secrete high levels of ecdysone. This demonstrated that although insulin signaling was correlated with normal growth of the glands, insulin is not sufficient to impact the ability of the glands to secrete ecdysone in the absence of nutrie nts. 6 This thesis is dedicated to my parents,Robert and Angela Walsh, whose love and support made this dissertation possible.

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“…Insulin action is initiated by insulin binding to the extracellular ␣-subunit of the insulin receptor (IR), which transmits a transmembrane signal activating the intracellular tyrosine kinase domain of the ␤-subunits (12,13). Following IR autophosphorylation, the tyrosine kinase domain of the ␤-subunit tyrosine phosphorylates a number of cel-lular proteins, including members of the insulin receptor substrate (IRS1/2/3/4) family (14).…”

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confidence: 99%

R-α-Lipoic Acid Action on Cell Redox Status, the Insulin Receptor, and Glucose Uptake in 3T3-L1 Adipocytes

Moini

Tirosh

Park

et al. 2002

Archives of Biochemistry and Biophysics

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The insulin signaling pathway has been reported to mediate R-␣-lipoic acid-(R-LA-)-stimulated glucose uptake into 3T3-L1 adipocytes and L6 myotubes. We investigated the role of the thiol antioxidant dihydrolipoic acid (DHLA) and intracellular glutathione (GSH) in R-LA-stimulated glucose transport and explored the hypothesis that R-LA could increase glucose uptake into 3T3-L1 adipocytes in an oxidant-mimetic manner. R-LA pretreatment of 3T3-L1 cells stimulated glucose transport at early time points (30 min-6 h), whereas it inhibited glucose uptake at later time points. Analysis of the oxidized and reduced content of LA in cells and medium showed that >90% of lipoic acid present was in its oxidized form. Furthermore, all oxidized forms of LA (S-, R-, and racemic LA) stimulated glucose uptake, whereas the reduced form, dihydrolipoic acid, was ineffective. Intracellular GSH levels were not changed at the early time points (before 12 h), while longer preincubation (24 -48 h) of cells with R-LA significantly increased intracellular GSH. Pretreatment of adipocytes with R-LA increased intracellular peroxide levels at early time points (30 min-6 h), after which it was decreased (12-48 h). R-LA also increased tyrosine phosphorylation of immunoprecipitated insulin receptors from 3T3-L1 adipocytes. These results indicate that (i) 3T3-L1 adipocytes have a low capacity to reduce R-LA and the oxidized form of lipoic acid is responsible for stimulating glucose uptake, (ii) R-LA modulates glucose uptake by changing the intracellular redox status, and (iii) the insulin receptor is a potential cellular target for R-LA action.

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Quaternary Structure of the Insulin-Insulin Receptor Complex

Cited by 124 publications

References 24 publications

Functional Reconstitution of Insulin Receptor Binding Site from Non-binding Receptor Fragments

Functional Reconstitution of Insulin Receptor Binding Site from Non-binding Receptor Fragments

Nutritional sensitivity of fifth instar prothoracic glands in the tobacco hornworm, Manduca sexta

R-α-Lipoic Acid Action on Cell Redox Status, the Insulin Receptor, and Glucose Uptake in 3T3-L1 Adipocytes

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