A stepwise activation model for the insulin receptor.

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.

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

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.

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.

An aptamer agonist of the insulin receptor acts as a positive or negative allosteric modulator, depending on its concentration.

Aptamers are widely used as binders that interact with targets with high affinity or as inhibitors of the function of target molecules. However, they have also been used to modulate target protein function, which they achieve by activating the target or stabilizing its conformation. Here, we report a unique aptamer modulator of the insulin receptor (IR), IR-A62. Alone, IR-A62 acts as a biased agonist that preferentially induces Y1150 monophosphorylation of IR. However, when administered alongside insulin, IR-A62 shows variable binding cooperativity depending on the ligand concentration. At low concentrations, IR-A62 acts as a positive allosteric modulator (PAM) agonist that enhances insulin binding, but at high concentrations, it acts as a negative allosteric modulator (NAM) agonist that competes with insulin for IR. Moreover, the concentration of insulin affects the binding of IR-A62 to IR. Finally, the subcutaneous administration of IR-A62 to diabetic mice reduces blood glucose levels with a longer-lasting effect than insulin administration. These findings imply that aptamers can elicit various responses from receptors beyond those of a simple agonist or inhibitor. We expect further studies of IR-A62 to help reveal the mechanism of IR activation and greatly expand the range of therapeutic applications of aptamers.

Formation of cellular close-ended tunneling nanotubes through mechanical deformation.

Membrane nanotubes or tunneling nanotubes (TNTs) that connect cells have been recognized as a previously unidentified pathway for intercellular transport between distant cells. However, it is unknown how this delicate structure, which extends over tens of micrometers and remains robust for hours, is formed. Here, we found that a TNT develops from a double filopodial bridge (DFB) created by the physical contact of two filopodia through helical deformation of the DFB. The transition of a DFB to a close-ended TNT is most likely triggered by disruption of the adhesion of two filopodia by mechanical energy accumulated in a twisted DFB when one of the DFB ends is firmly attached through intercellular cadherin-cadherin interactions. These studies pinpoint the mechanistic questions about TNTs and elucidate a formation mechanism.

A hotspot for enhancing insulin receptor activation revealed by a conformation-specific allosteric aptamer.

Aptamers are single-stranded oligonucleotides that bind to a specific target with high affinity, and are widely applied in biomedical diagnostics and drug development. However, the use of aptamers has largely been limited to simple binders or inhibitors that interfere with the function of a target protein. Here, we show that an aptamer can also act as a positive allosteric modulator that enhances the activation of a receptor by stabilizing the binding of a ligand to that receptor. We developed an aptamer, named IR-A43, which binds to the insulin receptor, and confirmed that IR-A43 and insulin bind to the insulin receptor with mutual positive cooperativity. IR-A43 alone is inactive, but, in the presence of insulin, it potentiates autophosphorylation and downstream signaling of the insulin receptor. By using the species-specific activity of IR-A43 at the human insulin receptor, we demonstrate that residue Q272 in the cysteine-rich domain is directly involved in the insulin-enhancing activity of IR-A43. Therefore, we propose that the region containing residue Q272 is a hotspot that can be used to enhance insulin receptor activation. Moreover, our study implies that aptamers are promising reagents for the development of allosteric modulators that discriminate a specific conformation of a target receptor.

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

Metformin is the first-line therapy for type 2 diabetes, but there are large inter-individual variations in responses to this drug. Its mechanism of action is not fully understood, but activation of AMP-activated protein kinase (AMPK) and changes in the gut microbiota appear to be important. The inhibitory role of microbial metabolites on metformin action has not previously been investigated. Here, we show that concentrations of the microbial metabolite imidazole propionate are higher in subjects with type 2 diabetes taking metformin who have high blood glucose. We also show that metformin-induced glucose lowering is not observed in mice pretreated with imidazole propionate. Furthermore, we demonstrate that imidazole propionate inhibits AMPK activity by inducing inhibitory AMPK phosphorylation, which is dependent on imidazole propionate-induced basal Akt activation. Finally, we identify imidazole propionate-activated p38γ as a novel kinase for Akt and demonstrate that p38γ kinase activity mediates the inhibitory action of imidazole propionate on metformin.

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.

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 ß 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 ß 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.

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.

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-ß-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.