The Disulfide Bonds in the C-terminal Domains of the Human Insulin Receptor Ectodomain

Sparrow, Lindsay G.; McKern, Neil M.; Gorman, Jeff J.; Strike, P. M.; Robinson, Christine P.; Bentley, John D.; Ward, Colin W.

doi:10.1074/jbc.272.47.29460

Cited by 116 publications

(103 citation statements)

References 37 publications

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“…Competitive displacement of the ␣CT segment from the L1 surface will dramatically reduce the affinity of the receptor for insulin as L1 itself has no measurable affinity for insulin (4). ␣CT displacement from the L1-␤ 2 surface may destabilize the receptor as a whole given that the ␣CT segments are coupled to each other via inter-␣-chain disulfide bond(s) within the Cys 682 -Cys 683 -Cys 685 motif and form a bridge across the pair of L1 domains (10,15).…”

Section: Discussionmentioning

confidence: 99%

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Lawrence

Margetts

Menting

et al. 2016

Journal of Biological Chemistry

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Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucinerich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor ␣-chain (termed ␣CT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective ␣CT residues Phe 701 and Phe 705 . The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor.In 2002, sets of synthetic peptides were identified that could compete for insulin binding to the human insulin receptor extracellular region and that had affinities in the high nanomolar to low micromolar range (1). Based on competition studies, the putative epitopes of these peptides were grouped into three non-overlapping sites, termed Sites 1, 2, and 3.3 Some Site 1 peptides were able to activate the receptor tyrosine kinase and act as agonists in an insulin-dependent fat cell assay, whereas Site 2 and Site 3 peptides were found to act as antagonists both in phosphorylation and fat cell assays. The highest affinity Site 1 peptides contained the motif FYXWF, whereas Site 2 peptides were characterized by either a short or long disulfide loop (1).Optimized versions of the Site 1 and Site 2 peptides were subsequently described (2), in particular those that were linked in tandem in various ways. Several of these linked peptides were found to function as either potent antagonists (e.g. peptide S661, a Site 1-Site 2 combination) or as potent agonists (e.g. peptide S597, a Site 2-Site 1 combination), depending on the order in which the individual Site 1 and Site 2 peptides were linked (2, 3). In earlier studies (4, 5), we provided isothermal titration calorimetry (ITC) 4 data that suggested how Site 1 peptides might bind to the insulin receptor. The insulin receptor (IR) itself is a disulfide-linked (␣␤) 2 homodimer; each ␣␤ monomer comprises, from its N terminus, two homologous leucine-rich repeat domains (L1 and L2) separated by a cysteine-rich region (CR) comprising eight disulfide-linked modules (6). These domains are followed by three fibronectin type III d...

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Section: Discussionmentioning

confidence: 99%

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Lawrence

Margetts

Menting

et al. 2016

Journal of Biological Chemistry

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“…The inter-monomer disulfide bonds 28,29 are shown as black lines; the intra-monomer “signaling bridge” 26 is shown as an orange line. b , CryoEM density map for Class 1 with the IR sub-domains fitted to the density; one monomer is yellow, the other is color coded as in a .…”

Section: Figurementioning

confidence: 99%

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

Scapin

Dandey

Zhang

et al. 2018

Nature

204

243

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Summary The insulin receptor (IR) is a dimeric protein that plays a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels1,2. IR dysfunctions have been associated with many diseases, including diabetes, cancer, and Alzheimer’s1,3,4. The primary sequence has been known since the 1980s5, and is composed of an extracellular portion (ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Insulin binding to the dimeric ECD triggers kinase domain auto-phosphorylation and subsequent activation of downstream signaling molecules. Biochemical and mutagenesis data have identified two putative insulin binding sites (S1 and S2)6. While insulin bound to an ECD fragment containing S1 and the apo ectodomain have been characterized structurally7,8, details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single particle cryoEM reconstructions for the 1:2 (4.3 Å) and 1:1 (7.4 Å) IR ECD dimer:Insulin complexes. The symmetric 4.3 Å structure shows two insulin molecules per dimer, each bound between the Leucine-rich sub domain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the S1 binding interactions and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative sub domain orientations and triggers downstream signal propagation.

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“…Oligosaccharide chains are added at specific sites of glycosylation. Disulfide bridges stabilize the interactions between the two alpha subunits, and between the alpha and beta subunits (Sparrow et al, 1997). Consequently, the single disulfide bond, which occurs between residues cysteine-647 in the alpha subunit and cysteine-872 in the beta subunit, provides a covalent link between the subunits (Cheatham and Kahn, 1992).…”

Section: Insulin Receptor and Irsmentioning

confidence: 99%

The role of intracellular signaling in insulin-mediated regulation of drug metabolizing enzyme gene and protein expression

Kim

Novak

2007

Pharmacology & Therapeutics

143

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Endogenous factors, including hormones, growth factors and cytokines, play an important role in the regulation of hepatic drug metabolizing enzyme expression in both physiological and pathophysiological conditions. Alterations of hepatic drug metabolizing enzymes gene and protein expression, observed in diabetes, fasting, obesity, protein-calorie malnutrition and long-term alcohol consumption alters the metabolism of xenobiotics, including procarcinogens, carcinogens, toxicants, and therapeutic agents and may also impact the efficacy and safety of therapeutic agents, as well as result in drug-drug interactions. Although the mechanisms by which xenobiotics regulate drug metabolizing enzymes have been studied intensively, less is known regarding the cellular signaling pathways and components which regulate drug metabolizing enzyme gene and protein expression in response to hormones and cytokines. Recent findings, however, have revealed that several cellular signaling pathways are involved in hormone-and growth factor-mediated regulation of drug metabolizing enzymes. Our laboratory, and others, have demonstrated that insulin and growth factors regulate drug metabolizing enzyme gene and protein expression, including cytochromes P450, glutathione S-transferases and microsomal epoxide hydrolase, through receptors which are members of the large receptor tyrosine kinase family, and by downstream effectors such as phosphatidylinositol 3-kinase, the mitogen activated protein kinase, Akt/protein kinase B, mTOR, and the p70S6 kinase. Here, we review current knowledge of the signaling pathways implicated in regulation of drug metabolizing enzyme gene and protein expression in response to insulin and growth factors, with the goal of increasing our understanding of how chronic disease affects these signaling pathways, components, and ultimately gene expression and translational control.

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The Disulfide Bonds in the C-terminal Domains of the Human Insulin Receptor Ectodomain

Cited by 116 publications

References 37 publications

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

The role of intracellular signaling in insulin-mediated regulation of drug metabolizing enzyme gene and protein expression

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