Insulin receptor Arg717 and IGF-1 receptor Arg704 play a key role in ligand binding and in receptor activation.

The insulin receptor (IR, with its isoforms IR-A and IR-B) and the insulin-like growth factor 1 receptor (IGF-1R) are related tyrosine kinase receptors. Recently, the portfolio of solved hormone-receptor structures has grown extensively thanks to advancements in cryo-electron microscopy. However, the dynamics of how these receptors transition between their inactive and active state are yet to be fully understood. The C-terminal part of the alpha subunit (αCT) of the receptors is indispensable for the formation of the hormone-binding site. We mutated the αCT residues Arg717 and His710 of IR-A and Arg704 and His697 of IGF-1R. We then measured the saturation binding curves of ligands on the mutated receptors and their ability to become activated. Mutations of Arg704 and His697 to Ala in IGF-1R decreased the binding of IGF-1. Moreover, the number of binding sites for IGF-1 on the His697 IGF-1R mutant was reduced to one-half, demonstrating the presence of two binding sites. Both mutations of Arg717 and His710 to Ala in IR-A inactivated the receptor. We have proved that Arg717 is important for the binding of insulin to its receptor, which suggests that Arg717 is a key residue for the transition to the active conformation.

Structural basis of the alkaline pH-dependent activation of insulin receptor-related receptor.

The insulin receptor (IR) family is a subfamily of receptor tyrosine kinases that controls metabolic homeostasis and cell growth. Distinct from IR and insulin-like growth factor 1 receptor, whose activation requires ligand binding, insulin receptor-related receptor (IRR)-the third member of the IR family-is activated by alkaline pH. However, the molecular mechanism underlying alkaline pH-induced IRR activation remains unclear. Here, we present cryo-EM structures of human IRR in both neutral pH inactive and alkaline pH active states. Combined with mutagenesis and cellular assays, we show that, upon pH increase, electrostatic repulsion of the pH-sensitive motifs of IRR disrupts its autoinhibited state and promotes a scissor-like rotation between two protomers, leading to a T-shaped active conformation. Together, our study reveals an unprecedented alkaline pH-dependent activation mechanism of IRR, opening up opportunities to understand the structure-function relationship of this important receptor.

The Activation Mechanism of the Insulin Receptor: A Structural Perspective.

The insulin receptor (IR) is a type II receptor tyrosine kinase that plays essential roles in metabolism, growth, and proliferation. Dysregulation of IR signaling is linked to many human diseases, such as diabetes and cancers. The resolution revolution in cryo-electron microscopy has led to the determination of several structures of IR with different numbers of bound insulin molecules in recent years, which have tremendously improved our understanding of how IR is activated by insulin. Here, we review the insulin-induced activation mechanism of IR, including (a) the detailed binding modes and functions of insulin at site 1 and site 2 and (b) the insulin-induced structural transitions that are required for IR activation. We highlight several other key aspects of the activation and regulation of IR signaling and discuss the remaining gaps in our understanding of the IR activation mechanism and potential avenues of future research.

Diversity of Structural, Dynamic, and Environmental Effects Explain a Distinctive Functional Role of Transmembrane Domains in the Insulin Receptor Subfamily.

Human InsR, IGF1R, and IRR receptor tyrosine kinases (RTK) of the insulin receptor subfamily play an important role in signaling pathways for a wide range of physiological processes and are directly associated with many pathologies, including neurodegenerative diseases. The disulfide-linked dimeric structure of these receptors is unique among RTKs. Sharing high sequence and structure homology, the receptors differ dramatically in their localization, expression, and functions. In this work, using high-resolution NMR spectroscopy supported by atomistic computer modeling, conformational variability of the transmembrane domains and their interactions with surrounding lipids were found to differ significantly between representatives of the subfamily. Therefore, we suggest that the heterogeneous and highly dynamic membrane environment should be taken into account in the observed diversity of the structural/dynamic organization and mechanisms of activation of InsR, IGF1R, and IRR receptors. This membrane-mediated control of receptor signaling offers an attractive prospect for the development of new targeted therapies for diseases associated with dysfunction of insulin subfamily receptors.

Structural models of viral insulin-like peptides and their analogs.

The insulin receptor (IR), the insulin-like growth factor-1 receptor (IGF1R), and the insulin/IGF1 hybrid receptors (hybR) are homologous transmembrane receptors. The peptide ligands, insulin and IGF1, exhibit significant structural homology and can bind to each receptor via site-1 and site-2 residues with distinct affinities. The variants of the Iridoviridae virus family show capability in expressing single-chain insulin/IGF1 like proteins, termed viral insulin-like peptides (VILPs), which can stimulate receptors from the insulin family. The sequences of VILPs lacking the central C-domain (dcVILPs) are known, but their structures in unbound and receptor-bound states have not been resolved to date. We report all-atom structural models of three dcVILPs (dcGIV, dcSGIV, and dcLCDV1) and their complexes with the receptors (µIR, µIGF1R, and µhybR), and probed the peptide/receptor interactions in each system using all-atom molecular dynamics (MD) simulations. Based on the nonbonded interaction energies computed between each residue of peptides (insulin and dcVILPs) and the receptors, we provide details on residues establishing significant interactions. The observed site-1 insulin/µIR interactions are consistent with previous experimental studies, and a residue-level comparison of interactions of peptides (insulin and dcVILPs) with the receptors revealed that, due to sequence differences, dcVILPs also establish some interactions distinct from those between insulin and IR. We also designed insulin analogs and report enhanced interactions between some analogs and the receptors.

Cryo-EM structure shows how two IGF1 hormones bind to the human IGF1R receptor.

IGF1R plays an important role in regulating cellular metabolism and cell growth, and has been identified as an anti-cancer and diabetes drug target. Although research have been reported many crystal and cryo-EM structures of IGF1R, the mechanism of ligand binding remains controversial, mainly because the structure differences among its cryo-EM, crystal and homologous protein insulin receptor structures. Here, we further determined one new high-resolution symmetric cryo-EM structure of ligand-bound IGF1R and be the first to prove that the receptor could bind to two IGFI molecules by single particle cryo-electron microscopy. And the structure is very different from its homologous protein insulin receptor: the two ligands just exist at the binding site 2 with saturating ligand conditions. Then, our findings resolved the major dispute about the comformational changes of IGF1R, and proposed a new theory how IGF1R binds to its ligands. Meanwhile, these findings imply more attention may be needed to study the relationship between the special conformation and their corresponding physiological functions in future.

Molecular basis for the role of disulfide-linked αCTs in the activation of insulin-like growth factor 1 receptor and insulin receptor.

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) control metabolic homeostasis and cell growth and proliferation. The IR and IGF1R form similar disulfide bonds linked homodimers in the apo-state; however, their ligand binding properties and the structures in the active state differ substantially. It has been proposed that the disulfide-linked C-terminal segment of α-chain (αCTs) of the IR and IGF1R control the cooperativity of ligand binding and regulate the receptor activation. Nevertheless, the molecular basis for the roles of disulfide-linked αCTs in IR and IGF1R activation are still unclear. Here, we report the cryo-EM structures of full-length mouse IGF1R/IGF1 and IR/insulin complexes with modified αCTs that have increased flexibility. Unlike the Γ-shaped asymmetric IGF1R dimer with a single IGF1 bound, the IGF1R with the enhanced flexibility of αCTs can form a T-shaped symmetric dimer with two IGF1s bound. Meanwhile, the IR with non-covalently linked αCTs predominantly adopts an asymmetric conformation with four insulins bound, which is distinct from the T-shaped symmetric IR. Using cell-based experiments, we further showed that both IGF1R and IR with the modified αCTs cannot activate the downstream signaling potently. Collectively, our studies demonstrate that the certain structural rigidity of disulfide-linked αCTs is critical for optimal IR and IGF1R signaling activation.

How insulin-like growth factor I binds to a hybrid insulin receptor type 1 insulin-like growth factor receptor.

Monomers of the insulin receptor and type 1 insulin-like growth factor receptor (IGF-1R) can combine stochastically to form heterodimeric hybrid receptors. These hybrid receptors display ligand binding and signaling properties that differ from those of the homodimeric receptors. Here, we describe the cryoelectron microscopy structure of such a hybrid receptor in complex with insulin-like growth factor I (IGF-I). The structure (ca. 3.7 Å resolution) displays a single IGF-I ligand, bound in a similar fashion to that seen for IGFs in complex with IGF-1R. The IGF-I ligand engages the first leucine-rich-repeat domain and cysteine-rich region of the IGF-1R monomer (rather than those of the insulin receptor monomer), consistent with the determinants for IGF binding residing in the IGF-1R cysteine-rich region. The structure broadens our understanding of this receptor family and assists in delineating the key structural motifs involved in binding their respective ligands.

Synergistic activation of the insulin receptor via two distinct sites.

Insulin receptor (IR) signaling controls multiple facets of animal physiology. Maximally four insulins bind to IR at two distinct sites, termed site-1 and site-2. However, the precise functional roles of each binding event during IR activation remain unresolved. Here, we showed that IR incompletely saturated with insulin predominantly forms an asymmetric conformation and exhibits partial activation. IR with one insulin bound adopts a Γ-shaped conformation. IR with two insulins bound assumes a Ƭ-shaped conformation. One insulin binds at site-1 and another simultaneously contacts both site-1 and site-2 in the Ƭ-shaped IR dimer. We further show that concurrent binding of four insulins to sites-1 and -2 prevents the formation of asymmetric IR and promotes the T-shaped symmetric, fully active state. Collectively, our results demonstrate how the synergistic binding of multiple insulins promotes optimal IR activation.

Structural Investigations of Full-Length Insulin Receptor Dynamics and Signalling.

Insulin regulates glucose homeostasis via binding and activation of the insulin receptor dimer at two distinct pairs of binding sites 1 and 2. Here, we present cryo-EM studies of full-length human insulin receptor (hIR) in an active state obtained at non-saturating, physiologically relevant insulin conditions. Insulin binds asymmetrically to the receptor under these conditions, occupying up to three of the four possible binding sites. Deletion analysis of the receptor together with site specific peptides and insulin analogs used in binding studies show that both sites 1 and 2 are required for high insulin affinity. We identify a homotypic interaction of the fibronectin type III domain (FnIII-3) of IR resulting in tight interaction of membrane proximal domains of the active, asymmetric receptor dimer. Our results show how insulin binding at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer. We propose an insulin binding and activation mechanism, which is sequential, exhibits negative cooperativity, and is based on asymmetry at physiological insulin concentrations with one to three insulin molecules activating IR.

Activation of neurotrophin signalling with light­inducible receptor tyrosine kinases.

Optogenetics combined with protein engineering based on natural light­sensitive dimerizing proteins has evolved as a powerful strategy to study cellular functions. The present study focused on tropomyosin kinase receptors (Trks) that have been engineered to be light­sensitive. Trk belongs to the superfamily of receptor tyrosine kinases (RTKs), which are single­pass transmembrane receptors that are activated by natural ligands and serve crucial roles in cellular growth, differentiation, metabolism and motility. However, functional variations exist among receptors fused with light­sensitive proteins. The present study proposed a signal transduction model for light­induced receptor activation. This model is based on analysis of previous light­induced Trk receptors reported to date and comparisons to the activation mechanism of natural receptors. In this model, quantitative differences on the dimerization induced from either top­to­bottom or bottom­to­up may lead to the varying amplitude of intracellular signals. We hypothesize that the top­to­bottom propagation is more favourable for activation and yields better results compared with the bottom­to­top direction. The careful delineation of the dimerization mechanisms fine­tuning activation will guide future design for an optimum cellular output with the precision of light.

Structures and interactions of insulin-like peptides from cone snail venom.

The venomous insulin-like peptides released by certain cone snails stimulate hypoglycemic shock to immobilize fish and catch the prey. Compared to human insulin (hIns), the cone snail insulins (Con-Ins) are typically monomeric and shorter in sequence, yet they exhibit moderate hIns-like biological activity. We have modeled six variants of Con-Ins (G3, K1, K2, T1A, T1B, and T2) and carried out explicit-solvent molecular dynamics (MD) simulations of eight types of insulins, two with known structures (hIns and Con-Ins-G1) and six Con-Ins with modeled structures, to characterize key residues of each insulin that interact with the truncated human insulin receptor (µIR). We show that each insulin/µIR complex is stable during explicit-solvent MD simulations and hIns interactions indicate the highest affinity for the "site 1" of IR. The residue contact maps reveal that each insulin preferably interacts with the αCT peptide than the L1 domain of IR. Through analysis of the average nonbonded interaction energy contribution of every residue of each insulin for the µIR, we probe the residues establishing favorable interactions with the receptor. We compared the interaction energy of each residue of every Con-Ins to the µIR and observed that γ-carboxylated glutamate (Gla), His, Thr, Tyr, Tyr/His, and Asn in Con-Ins are favorable substitutions for GluA4, AsnA21, ValB12, LeuB15, GlyB20, and ArgB22 in hIns, respectively. The identified insulin analogs, although lacking the last eight residues of the B-chain of hIns, bind strongly to µIR. Our findings are potentially useful in designing potent fast-acting therapeutic insulin.

Effects of tauroursodeoxycholic acid on glucose homeostasis: Potential binding of this bile acid with the insulin receptor.

AIMS: The bile acid (BA), tauroursodeoxycholic acid (TUDCA) regulates glucose homeostasis; however, it is not clear whether its effects on insulin signaling are due to its direct interaction with the insulin receptor (IR) or through activation of the G-coupled BA receptor, TGR5. We, herein, investigated whether the actions of TUDCA on glucose homeostasis occur via IR or TGR5 activation. MAIN METHODS: Glucose homeostasis was evaluated in high-fat diet (HFD)-obese or control (CTL) mice, after 30 days or one intraperitoneal (ip) injection of 300 mg/kg TUDCA, respectively. Molecular docking was performed to investigate the potential binding of TUDCA on the IR and TGR5. KEY FINDINGS: After 30 days of TUDCA treatment, HFD mice exhibited improvements in glucose tolerance and insulin sensitivity, which were abolished when these rodents received the IR antagonist, S961. Molecular docking experiments showed that TUDCA demonstrates high binding affinity for TGR5 and IR and strongly interacts with the insulin binding sites 1 and 2 of the IR. Consistent with this potential agonist activity of TUDCA on IR, CTL mice displayed increased hepatic phosphorylation of AKT after an ip injection of TUDCA. This effect was not associated with altered glycemia in CTL mice and was dependent on IR activation, as S961 prevented hepatic AKT activation by TUDCA. Furthermore, TUDCA activated the hepatic protein kinase A (PKA) and cAMP response element-binding protein (CREB) pathway in CTL mice, even after the administration of S961. SIGNIFICANCE: We provide novel evidence that TUDCA may be an agonist of the IR, in turn activating AKT and contributing, at least in part, to its beneficial effects upon glucose homeostasis.

Molecular Docking and Simulation Studies of Antidiabetic Agents Devised from Hypoglycemic Polypeptide-P of Momordica charantia.

Diabetes mellitus termed as metabolic disorder is a collection of interlinked diseases and mainly body's inability to manage glucose level which leads to cardiovascular diseases, renal failure, neurological disorders, and many others. The drugs contemporarily used for diabetes have many inevitable side effects, and many of them have become less responsive to this multifactorial disorder. Momordica charantia commonly known as bitter gourd has many bioactive compounds with antidiabetic properties. The current study was designed to use computational methods to discover the best antidiabetic peptides devised from hypoglycemic polypeptide-P of M. charantia. The binding affinity and interaction patterns of peptides were evaluated against four receptor proteins (i.e., as agonists of insulin receptor and inhibitors of sodium-glucose cotransporter 1, dipeptidyl peptidase-IV, and glucose transporter 2) using molecular docking approach. A total of thirty-seven peptides were docked against these receptors. Out of which, top five peptides against each receptor were shortlisted based on their S-scores and binding affinities. Finally, the eight best ligands (i.e., LIVA, TSEP, EKAI, LKHA, EALF, VAEK, DFGAS, and EPGGGG) were selected as these ligands strictly followed Lipinski's rule of five and exhibited good ADMET profiling. One peptide EPGGGG showed activity towards insulin and SGLT1 receptor proteins. The top complex for both these targets was subjected to 50 ns of molecular dynamics simulations and MM-GBSA binding energy test that concluded both complexes as highly stable, and the intermolecular interactions were dominated by van der Waals and electrostatic energies. Overall, the selected ligands strongly fulfilled the drug-like evaluation criterion and proved to have good antidiabetic properties.

Insulin Analogues with Altered Insulin Receptor Isoform Binding Specificities and Enhanced Aggregation Stabilities.

Insulin is a lifesaver for millions of diabetic patients. There is a need for new insulin analogues with more physiological profiles and analogues that will be thermally more stable than human insulin. Here, we describe the chemical engineering of 48 insulin analogues that were designed to have changed binding specificities toward isoforms A and B of the insulin receptor (IR-A and IR-B). We systematically modified insulin at the C-terminus of the B-chain, at the N-terminus of the A-chain, and at A14 and A18 positions. We discovered an insulin analogue that has Cα-carboxyamidated Glu at B31 and Ala at B29 and that has a more than 3-fold-enhanced binding specificity in favor of the "metabolic" IR-B isoform. The analogue is more resistant to the formation of insulin fibrils at 37 °C and is also more efficient in mice than human insulin. Therefore, [AlaB29,GluB31,amideB31]-insulin may be interesting for further clinical evaluation.

An overview of the insulin signaling pathway in model organisms Drosophila melanogaster and Caenorhabditis elegans.

The insulin/insulin-like growth factor signaling pathway is an evolutionary conserved pathway across metazoans and is required for development, metabolism and behavior. This pathway is associated with various human metabolic disorders and cancers. Thus, model organisms including Drosophila melanogaster and Caenorhabditis elegans provide excellent opportunities to examine the structure and function of this pathway and its influence on cellular metabolism and proliferation. In this review, we will provide an overview of human insulin and the human insulin signaling pathway and explore the recent discoveries in model organisms Drosophila melanogaster and Caenorhabditis elegans. Our review will provide information regarding the various insulin-like peptides in model organisms as well as the conserved functions of insulin signaling pathways. Further investigation of the insulin signaling pathway in model organisms could provide a promising opportunity to develop novel therapies for various metabolic disorders and insulin-mediated cancers.

Long-Chain Acylcarnitines Decrease the Phosphorylation of the Insulin Receptor at Tyr1151 Through a PTP1B-Dependent Mechanism.

The accumulation of lipid intermediates may interfere with energy metabolism pathways and regulate cellular energy supplies. As increased levels of long-chain acylcarnitines have been linked to insulin resistance, we investigated the effects of long-chain acylcarnitines on key components of the insulin signalling pathway. We discovered that palmitoylcarnitine induces dephosphorylation of the insulin receptor (InsR) through increased activity of protein tyrosine phosphatase 1B (PTP1B). Palmitoylcarnitine suppresses protein kinase B (Akt) phosphorylation at Ser473, and this effect is not alleviated by the inhibition of PTP1B by the insulin sensitizer bis-(maltolato)-oxovanadium (IV). This result indicates that palmitoylcarnitine affects Akt activity independently of the InsR phosphorylation level. Inhibition of protein kinase C and protein phosphatase 2A does not affect the palmitoylcarnitine-mediated inhibition of Akt Ser473 phosphorylation. Additionally, palmitoylcarnitine markedly stimulates insulin release by suppressing Akt Ser473 phosphorylation in insulin-secreting RIN5F cells. In conclusion, long-chain acylcarnitines activate PTP1B and decrease InsR Tyr1151 phosphorylation and Akt Ser473 phosphorylation, thus limiting the cellular response to insulin stimulation.

Role of surface-exposed charged basic amino acids (Lys, Arg) and guanidination in insulin on the interaction and stability of insulin-insulin receptor complex.

Naturally occurring proteins are emerging as novel therapeutics in the protein-based biopharmaceutical industry for the treatment of diabetes and obesity. However, proteins are not suitable for oral delivery due to short half-life, reduced physical and chemical stability and low permeability across the membrane. Chemical modification has been identified as a formulation strategy to enhance the stability and bioavailability of protein drugs. The present study aims to study the effect of charge-specific modification of basic amino acids (Lys, Arg) and guanidination on the interaction of insulin with its receptor using molecular modelling. Our investigation revealed that the guanidination of insulin (Lys-NHC = NHNH2) enhanced and exerted stronger binding of the protein to its receptor through electrostatic interaction than native insulin (Lys-NH3+). Point mutations of Lys and Arg (R22, K29; R22K, K29; R22, K29R; R22K, K29R) were attempted and the effects on the interaction and stability between insulin/modified insulins and insulin receptor were also analyzed in this study. The findings from the study are expected to provide a better understanding of the possible mechanism of action of the modified protein at a molecular level before advancing to real experiments.

Insulin-induced conformational changes in the full-length insulin receptor: structural insights gained from molecular modeling analyses.

Insulin receptor plays an important role in the regulation of energy metabolism. Dysfunction of insulin receptor (IR) can lead to many disease states, such as diabetes mellitus. Deciphering the complex dynamic structures of human IR and its mechanism of activation would greatly aid in understanding IR-mediated signaling pathways and also in designing new drugs (including nonpeptidal insulin analogs) to treat diabetes mellitus. Experimental evidence about IR structures has been gradually obtained by biologists over the past three decades. Based on available experimental structures of IR in different states, here we employ molecular modeling approach to construct the full-length IR structures in different states and model its structural and conformational changes during insulin-induced IR activation. Several key possible intermediate states are constructed based on structural alignment, rotation, and computational modeling. Based on the structures of the full-length IR in different states, it appears that there are two possible conformational transition pathways: one is symmetric and the other one is asymmetric. Structural changes and motions of different domains of the full-length IR along the pathways are analyzed. The role of insulin binding to IR in facilitating the conformational transition of the receptor is analyzed. Information and insights derived from our present structural modeling analyses may aid in understanding the complex dynamic, structural, and conformational changes during the process of IR activation.

Activity-dependent conformational transitions of the insulin receptor-related receptor.

The insulin receptor (IR), insulin-like growth factor 1 receptor (IGF-1R), and insulin receptor-related receptor (IRR) form a mini family of predimerized receptor-like tyrosine kinases. IR and IGF-1R bind to their peptide agonists triggering metabolic and cell growth responses. In contrast, IRR, despite sharing with them a strong sequence homology, has no peptide-like agonist but can be activated by mildly alkaline media. The spatial structure and activation mechanisms of IRR have not been established yet. The present work represents the first account of a structural analysis of a predimerized receptor-like tyrosine kinase by high-resolution atomic force microscopy in their basal and activated forms. Our data suggest that in neutral media, inactive IRR has two conformations, where one is symmetrical and highly similar to the inactive Λ/U-shape of IR and IGF-1R ectodomains, whereas the second is drop-like and asymmetrical resembling the IRR ectodomain in solution. We did not observe complexes of IRR intracellular catalytic domains of the inactive receptor forms. At pH 9.0, we detected two presumably active IRR conformations, Γ-shaped and T-shaped. Both of conformations demonstrated formation of the complex of their intracellular catalytic domains responsible for autophosphorylation. The existence of two active IRR forms correlates well with the previously described positive cooperativity of the IRR activation. In conclusion, our data provide structural insights into the molecular mechanisms of alkali-induced IRR activation under mild native conditions that could be valuable for interpretation of results of IR and IGF-IR structural studies.