Aptamer-based single-molecule imaging of insulin receptors in living cells

We present a single-molecule imaging platform that quantitatively explores the spatiotemporal dynamics of individual insulin receptors in living cells. Modified DNA aptamers that specifically recognize insulin receptors (IRs) with a high affinity were selected through the SELEX process. Using quantum dot-labeled aptamers, we successfully imaged and analyzed the diffusive motions of individual IRs in the plasma membranes of a variety of cell lines (HIR, HEK293, HepG2). We further explored the cholesterol-dependent movement of IRs to address whether cholesterol depletion interferes with IRs and found that cholesterol depletion of the plasma membrane by methyl-??-cyclodextrin reduces the mobility of IRs. The aptamer-based single-molecule imaging of IRs will provide better understanding of insulin signal transduction through the dynamics study of IRs in the plasma membrane.open1

Regulation of BRCA1 stability through the tandem UBX domains of isoleucyl-tRNA synthetase 1

Aminoacyl-tRNA synthetases possess unique domains. In this study the structure of the vertebrate IARS1 and EARS1 complex reveals that vertebrate IARS1 protects the DNA repair factor BRCA1 from proteolytic degradation via its UBX-fold domain. Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function

Stepwise autophosphorylation modulates biased agonism of the insulin receptor

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Blue-conversion of Organic Dyes Produces the Artifacts of Multi-color Fluorescent Imaging

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Sensitizing AptamerINSR-A43 Enhances Insulin Sensitivity and Insulin Receptor Signaling

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A stepwise activation model for the insulin receptor

Abstract The binding of insulin to the insulin receptor (IR) triggers a cascade of receptor conformational changes and autophosphorylation, leading to the activation of metabolic and mitogenic pathways. Recent advances in the structural and functional analyses of IR have revealed the conformations of the extracellular domains of the IR in inactive and fully activated states. However, the early activation mechanisms of this receptor remain poorly understood. The structures of partially activated IR in complex with aptamers provide clues for understanding the initial activation mechanism. In this review, we discuss the structural and functional features of IR complexed with various ligands and propose a model to explain the sequential activation mechanism. Moreover, we discuss the structures of IR complexed with biased agonists that selectively activate metabolic pathways and provide insights into the design of selective agonists and their clinical implications

Mechanistic understanding of insulin receptor modulation: Implications for the development of anti-diabetic drugs

The insulin receptor is an important regulator of metabolic processes in the body, and in particular of glucose homeostasis, including glucose uptake into peripheral tissues. Thus, insulin administration is an effective treatment for diabetes, which is characterized by chronic elevation of blood glucose. However, insulin is not only a metabolic regulator, but also functions as a growth hormone. Accordingly, studies of long-term insulin administration and of the hyperinsulinemia associated with type 2 diabetes have raised concerns about possible increases in the risks of cancer and atherosclerosis, due to excessive stimulation of cell proliferation. Interestingly, some insulin receptor ligands that have been developed based on a peptide, an antibody, and an aptamer selectively have metabolic effects exerted through the insulin receptor but do not cause significant cellular proliferation. Although these ligands therefore have potential as anti-diabetic drugs for advanced diabetes care, the mechanism whereby they specifically activate the insulin receptor is still unclear. Recently, studies of the structure of the insulin receptor have progressed considerably, and have provided further mechanistic understanding of insulin receptor activation. Based on this progress, we propose a mechanistic model of this specificity and discuss the potential for the development of novel anti-diabetic drugs that would not have the adverse effects caused by excessive mitogenic action.

Functional selectivity of insulin receptor revealed by aptamer-trapped receptor structures

Activation of insulin receptor (IR) initiates a cascade of conformational changes and autophosphorylation events. Herein, we determined three structures of IR trapped by aptamers using cryo-electron microscopy. The A62 agonist aptamer selectively activates metabolic signaling. In the absence of insulin, the two A62 aptamer agonists of IR adopt an insulin-accessible arrowhead conformation by mimicking site-1/site-2' insulin coordination. Insulin binding at one site triggers conformational changes in one protomer, but this movement is blocked in the other protomer by A62 at the opposite site. A62 binding captures two unique conformations of IR with a similar stalk arrangement, which underlie Tyr1150 mono-phosphorylation (m-pY1150) and selective activation for metabolic signaling. The A43 aptamer, a positive allosteric modulator, binds at the opposite side of the insulin-binding module, and stabilizes the single insulin-bound IR structure that brings two FnIII-3 regions into closer proximity for full activation. Our results suggest that spatial proximity of the two FnIII-3 ends is important for m-pY1150, but multi-phosphorylation of IR requires additional conformational rearrangement of intracellular domains mediated by coordination between extracellular and transmembrane domains. DNA aptamers can activate insulin receptor as selective agonists or positive allosteric modulators. Here, authors determine structures of the insulin receptor bound to aptamers and provide a basis for the selective activation or allosteric regulation of insulin receptor.11Ysciescopu

Microbial Imidazole Propionate Affects Responses to Metformin through p38γ-Dependent Inhibitory AMPK Phosphorylation

Koh et al. show that imidazole propionate, a microbial metabolite, impairs the glucose-lowering effect of the anti-diabetic drug metformin and inhibits metformin-induced AMPK activation by activating p38γ/Akt/inhibitory AMPK serine phosphorylation. They further show that metformin action is restored by blocking imidazole propionate-activated p38γ

PI3K-C2α Knockdown Results in Rerouting of Insulin Signaling and Pancreatic Beta Cell Proliferation

Insulin resistance is a syndrome that affects multiple insulin target tissues, each having different biological functions regulated by insulin. A remaining question is to mechanistically explain how an insulin target cell/tissue can be insulin resistant in one biological function and insulin sensitive in another at the same time. Here, we provide evidence that in pancreatic β cells, knockdown of PI3K-C2α expression results in rerouting of the insulin signal from insulin receptor (IR)-B/PI3K-C2α/PKB-mediated metabolic signaling to IR-B/Shc/ERK-mediated mitogenic signaling, which allows the β cell to switch from a highly glucose-responsive, differentiated state to a proliferative state. Our data suggest the existence of IR-cascade-selective insulin resistance, which allows rerouting of the insulin signal within the same target cell. Hence, factors involved in the rerouting of the insulin signal represent tentative therapeutic targets in the treatment of insulin resistance