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.

Porcine IGF-1R synonymous mutations in the extracellular domain affect proliferation and differentiation of skeletal muscle cells.

OBJECTIVE: Miniature pigs are considered ideal organ donors for xenotransplantation in humans, but the mechanism underlying their dwarfism remains to be elucidated. IGF-1R is a crucial factor in body size formation in mammals, including skeletal muscle formation and development. The extracellular domain (ECD) binds to the ligand, a phenomenon that results in the activation of downstream pathways. METHODS: In this study, the coding sequences of two IGF-1R ECD haplotypes of the large Landrace (LP) pig and the small Bama Xiang (BM) pig were cloned into pcDNA3.1 vectors to generate pcDNA3.1-LP and pcDNA3.1-BM. The two recombinant vectors were then transfected into skeletal muscle cells. RESULTS: IGF-1R transcript was found to be expressed at higher levels in the pcDNA3.1-LP group than in the pcDNA3.1-BM group. The IGF-1R ECD from LP promoted cell proliferation and CyclinD1 expression, and promoted the phosphorylation of protein kinase B (to yield p-AKT). Moreover, the IGF-1R ECD from LP increased cell differentiation and the expression of myogenic determination factor (MyoD). CONCLUSION: Our data indicated that the IGF-1R ECD haplotypes between pig breeds with different body sizes affect IGF-1R expression, in turn affecting the proliferation and differentiation of skeletal muscle cells by activating downstream signalling pathways.

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.

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.

Cryo-EM studies of the apo states of human IGF1R.

IGF1R plays an important role in regulating cellular metabolism and growth. As a single transmembrane protein, its structure is flexible. Although previous studies revealed some structures of IGF1R, the cryo-EM apo structures of the receptor have never been reported. Herein, we reported four distinct cryo-EM structures that reveal the apo states of IGF1R. These conformations were classified as "Resting states" and "Active states", according to the orientation of α-CT helices and structural symmetry. In addition, a "Ligand-pocket" was formed in the active conformations, which presented a new view of conformational changes of apo-IGF1R. These results suggest a new dynamic change model to show the details of why and how ligands can bind to IGF1R.

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.

Cryo-EM Structure Reveals Polymorphic Ligand-bound States of IGF1R.

Type 1 insulin-like growth factor receptor (IGF1R) plays an important role in regulating cellular metabolism and cell growth and has been identified as an anticancer drug target. Although previous studies have revealed some structures of IGF1R with different ligands, the continuous dynamic conformation change remains unclear. Here, we report 10 distinct structures (7.9-3.6 Å) of IGF1R bound to IGF1 or insulin to reveal the polymorphic conformations of ligand-bound IGF1R. These results showed that the α-CT2, disulfide bond (C670-C670'), and FnIII-2 domains had the most flexible orientations for the conformational change that occurs when ligands bind to the receptor. In addition, we found one special conformation (tentatively named the diverter-switch state) in both complexes, which may be one of the apo-IGF1R forms under ligand-treatment conditions. Hence, these results illustrated the mechanism of how different ligands could bind to human IGF1R and provided a rational template for drug design.

A promising strategy for investigating the anti-aging effect of natural compounds: a case study of caffeoylquinic acids.

Caffeoylquinic acids, as plant-derived polyphenols, exhibit multiple biological activities such as antioxidant, anti-inflammatory, and neuroprotective activities. However, only limited information about their effect on longevity is available. In the current study, molecular docking was employed to explore the interactions between six representative caffeoylquinic acids and the insulin-like growth factor-1 receptor (IGFR), which is an important target protein for longevity. The results indicated that all six compounds were embedded well in the active pocket of IGFR, and that 3,5-diCQA exhibited the strongest affinity to IGFR. Moreover, ASP1153, GLU1080, ASP1086, and ARG1003 were the key amino acid residues during the interaction of these 6 compounds with IGFR. Furthermore, the lifespan extension effect of caffeoylquinic acids was evaluated in a Caenorhabditis elegans (C. elegans) model. The results revealed that all the caffeoylquinic acids significantly extended the lifespan of wild-type worms, of which 3,5-diCQA was the most potent compound. Meanwhile, 3,5-diCQA enhanced the healthspan by increasing the body bending and pharyngeal pumping rates and reducing the intestinal lipofuscin level. Further studies demonstrated that 3,5-diCQA induced longevity effects by downregulating the insulin/insulin-like growth factor signaling (IIS) pathway. This study suggested that the combination of molecular docking and genetic analysis of specific worm mutants could be a promising strategy to reveal the anti-aging mechanisms of small molecule natural compounds.

The Effects of Marine Algal Polyphenols, Phlorotannins, on Skeletal Muscle Growth in C2C12 Muscle Cells via Smad and IGF-1 Signaling Pathways.

Skeletal muscle is an important tissue in energy metabolism and athletic performance. The use of effective synthetic supplements and drugs to promote muscle growth is limited by various side effects. Moreover, their use is prohibited by anti-doping agencies; hence, natural alternatives are needed. Therefore, we evaluated the muscle growth effect of substances that can act like synthetic supplements from edible marine algae. First, we isolated six marine algal polyphenols belonging to the phlorotannin class, namely dieckol (DK), 2,7″-phloroglucinol-6,6'-bieckol (PHB), phlorofucofuroeckol A (PFFA), 6,6'-bieckol (6,6-BK), pyrogallol-phloroglucinol-6,6'-bieckol (PPB), and phloroglucinol (PG) from an edible brown alga, Ecklonia cava and evaluated their effects on C2C12 myoblasts proliferation and differentiation. Of the six phlorotannin isolates evaluated, DK and PHB induced the highest degree of C2C12 myoblast proliferation. In addition, DK and PHB regulates myogenesis by down-regulating the Smad signaling, a negative regulator, and up-regulating the insulin-like growth factor-1 (IGF-1) signaling, a positive regulator. Interestingly, DK and PHB bind strongly to myostatin, which is an inhibitor of myoblast proliferation, while also binding to IGF-1 receptors. Moreover, they bind to IGF-1 receptor. These results suggest that DK and PHB are potential natural muscle building supplements and could be a safer alternative to synthetic drugs.

An innovative fluorescent probe targeting IGF1R for breast cancer diagnosis.

Breast cancer is the most dangerous, among all malignant tumors that threaten women's lives and health. Surgical resection can effectively prolong the survival time of patients with early breast cancer. Insulin-like growth factor type 1 receptor (IGF1R) is a member of the large family of receptor tyrosine kinases, and it's significantly overexpressed in breast cancer cells, which make them ideal biomarkers for the diagnosis and surgery navigation of breast cancer. Herein, we developed a series of IGF1R-targeted probes (YQ-L) for fluorescent imaging in breast cancer based on the strategy of drug repositioning. YQ-L exhibited specific IGF1R binding both in vitro and in vivo, especially probe 5d exhibited higher tumor uptake with a high tumor/normal ratio in the MCF-7 tumor bearing mouse. The maximum T/N ratio of probe 5d was 4.9, which was about 3 times that of indocyanine green (ICG). Meanwhile, probe 5d displayed more favorable in vivo pharmacokinetic properties than that of ICG with less hepatic and intestinal uptake. Convenient preparation, excellent IGF1R specificity in breast cancer, rapid clearance from normal organs and good biosafety profiles of probe 5d warrant further investigations for clinical translation in detection and surgery navigation of breast cancer.

Defining the epitope of a blood-brain barrier crossing single domain antibody specific for the type 1 insulin-like growth factor receptor.

Ligand-activated signaling through the type 1 insulin-like growth factor receptor (IGF1R) is implicated in many physiological processes ranging from normal human growth to cancer proliferation and metastasis. IGF1R has also emerged as a target for receptor-mediated transcytosis, a transport phenomenon that can be exploited to shuttle biotherapeutics across the blood-brain barrier (BBB). We employed differential hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) to characterize the interactions of the IGF1R ectodomain with a recently discovered BBB-crossing single-domain antibody (sdAb), VHH-IR5, in comparison with IGF-1 binding. HDX-MS confirmed that IGF-1 induced global conformational shifts in the L1/FnIII-1/-2 domains and α-CT helix of IGF1R. In contrast, the VHH-IR5 sdAb-mediated changes in conformational dynamics were limited to the α-CT helix and its immediate vicinity (L1 domain). High-resolution NMR spectroscopy titration data and linear peptide scanning demonstrated that VHH-IR5 has high-affinity binding interactions with a peptide sequence around the C-terminal region of the α-CT helix. Taken together, these results define a core linear epitope for VHH-IR5 within the α-CT helix, overlapping the IGF-1 binding site, and suggest a potential role for the α-CT helix in sdAb-mediated transcytosis.

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.

Analyzing structural differences between insulin receptor (IR) and IGF1R for designing small molecule allosteric inhibitors of IGF1R as novel anti-cancer agents.

IR and insulin-like growth factor-1 receptor (IGF-1R) share high degree of sequence and structural similarity that hinders the development of anticancer drugs targeting IGF1R, which is dysregulated in many cancers. Although IR and IGF1R mediate their activities through similar signalling pathways, yet they show different physiological effects. The exact molecular mechanism(s) how IR and IGF1R exert their distinct functions remain largely unknown. Here, we performed in silico analysis and generated GFP-fusion proteins of wild type IR and its K1079R mutant to analyze their subcellular localization, cytoplasmic and nuclear activities in comparison to IGF1R and its K1055R mutant. We showed that, like K1055R mutation in IGF1R, K1079R mutation does not impede the subcellular localization and nuclear activities of IR. Although K1079R mutation significantly decreases the kinase activity of IR but not as much as K1055R mutation, which was seen to drastically reduce the kinase activity of IGF1R. Moreover, K1079 residue in IR is seen to be sitting in a pocket which is different than the allosteric inhibitor binding pocket present in its homologue (IGF1R). This is for the first time such a study has been conducted to identify structural differences between these receptors that could be exploited for designing small molecule allosteric inhibitor(s) of IGF1R as novel anti-cancer drugs.

A computational strategy to understand structure-activity relationship of 1,3-disubstituted imidazole [1,5-α] pyrazine derivatives described as ATP competitive inhibitors of the IGF-1 receptor related to Ewing sarcoma.

We followed a comprehensive computational strategy to understand and eventually predict the structure-activity relationship of thirty-three 1,3-disubstituted imidazole [1,5-α] pyrazine derivatives described as ATP competitive inhibitors of the IGF-1 receptor related to Ewing sarcoma. The quantitative structure-activity relationship model showed that the inhibitory potency is correlated with the molar volume, a steric descriptor and the net charge calculated value on atom C1 (q1) and N4 (q4) of the pharmacophore, all of them appearing to give a positive contribution to the inhibitory activity. According to experimental and calculated values, the most potent compound would be 3-[4-(azetidin-2-ylmethyl) cyclohexyl]-1-[3-(benzyloxy) phenyl] imidazo [1,5-α]pyrazin-8-amine (compound 23). Docking was used to guess important residues involved in the ATP-competitive inhibitory activity. It was validated by 200 ns of molecular dynamics (MD) simulation using improved linear interaction energy (LIE) method. MD of previously preferred structures by docking shows that the most potent ligand could establish hydrogen bonds with the ATP-binding site of the receptor, and the Ser979 and Ser1059 residues contribute favourably to the binding stability of compound 23. MD simulation also gave arguments about the chemical structure of the compound 23 being able to fit in the ATP-binding pocket, expecting to remain stable into it during the entire simulation and allowing us to hint the significant contribution expected to be given by electrostatic and hydrophobic interactions to the ligand-receptor complex stability. This computational combined strategy here described could represent a useful and effective prime approach to guide the identification of tyrosine kinase inhibitors as new lead compounds.

Integrated bioinformatics analysis to decipher molecular mechanism of compound Kushen injection for esophageal cancer by combining WGCNA with network pharmacology.

Compound Kushen injection (CKI), a medicine in widespread clinical use in China, has proven therapeutic effects on cancer. However, few molecular mechanism analyses have been carried out. To address this problem, bioinformatics approaches combining weighted gene co-expression network analysis with network pharmacology methods were undertaken to elucidate the underlying molecular mechanisms of CKI in the treatment of esophageal cancer (ESCA). First, the key gene modules related to the clinical traits of ESCA were analysed by WCGNA. Based on the results, the hub genes related to CKI treatment for ESCA were explored through network pharmacology. Molecular docking simulation was performed to recognize the binding activity of hub genes with CKI compounds. The results showed that the potential hub targets, including EGFR, ErbB2, CCND1 and IGF1R, are therapeutic targets of CKI for the treatment of ESCA. Moreover, these targets were significantly enriched in many pathways related to cancer and signalling pathways, such as the PI3K-Akt signalling pathway and ErbB signalling pathway. In conclusion, this research partially highlighted the molecular mechanism of CKI in the treatment of ESCA, offering great potential in the identification of the effective compounds in CKI and biomarkers for ESCA treatment.

Dimeric states of transmembrane domains of insulin and IGF-1R receptors: Structures and possible role in activation.

Despite the biological significance of insulin signaling, the molecular mechanisms of activation of the insulin receptor (IR) and other proteins from its family remain elusive. Current hypothesis on signal transduction suggests ligand-triggered structural changes in the extracellular domain followed by transmembrane (TM) domains closure and dimerization leading to trans-autophosphorylation and kinase activity in intracellular segments of the receptor. Using NMR spectroscopy, we detected dimerization of isolated TM segments of IR in different membrane-mimicking environments and observed multiple signals of NH groups of protein backbone possibly corresponding to several dimer conformations. Taking available experimental data as constraints, several atomistic models of dimeric TM domains of IR and insulin-like growth factor 1 (IGF-1R) receptors were elaborated. Molecular dynamics simulations of IR ectodomain revealed noticeable collective movements potentially responsible for closure of the C-termini of FnIII-3 domains and spatial approaching of TM helices upon insulin-induced receptor activation. In addition, we demonstrated that the intracellular part of the receptor does not impose restrictions on the positioning of TM helices in the membrane. Finally, we used two independent structure prediction methods to generate a series of dimer conformations followed by their cluster analysis and dimerization free energy estimation to select the best dimer models. Biological relevance of the later was further tested via comparison of the hydrophobic organization of TM helices for both wild-type receptors and their mutants. Based on these data, the ability of several segments from other proteins to functionally replace IR and/or IGF-1R TM domains was explained.

How IGF-II Binds to the Human Type 1 Insulin-like Growth Factor Receptor.

Human type 1 insulin-like growth factor receptor (IGF-1R) signals chiefly in response to the binding of insulin-like growth factor I. Relatively little is known about the role of insulin-like growth factor II signaling via IGF-1R, despite the affinity of insulin-like growth factor II for IGF-1R being within an order of magnitude of that of insulin-like growth factor I. Here, we describe the cryoelectron microscopy structure of insulin-like growth factor II bound to a leucine-zipper-stabilized IGF-1R ectodomain, determined in two conformations to a maximum average resolution of 3.2 Å. The two conformations differ in the relative separation of their respective points of membrane entry, and comparison with the structure of insulin-like growth factor I bound to IGF-1R reveals long-suspected differences in the way in which the critical C domain of the respective growth factors interact with IGF-1R.

IGF-1 receptor activity in the Golgi of migratory cancer cells depends on adhesion-dependent phosphorylation of Tyr1250 and Tyr1251.

Although insulin-like growth factor 1 (IGF-1) signaling promotes tumor growth and cancer progression, therapies that target the IGF-1 receptor (IGF-1R) have shown poor clinical efficacy. To address IGF-1R activity in cancer cells and how it differs from that of the closely related insulin receptor (IR), we focused on two tyrosines in the IGF-1R C-terminal tail that are not present in the IR and are essential for IGF-1-mediated cancer cell survival, migration, and tumorigenic growth. We found that Tyr1250 and Tyr1251 (Tyr1250/1251) were autophosphorylated in a cell adhesion-dependent manner. To investigate the consequences of this phosphorylation, we generated phosphomimetic Y1250E/Y1251E (EE) and nonphosphorylatable Y1250F/Y1251F (FF) mutant forms of IGF-1R. Although fully competent in kinase activity and signaling, the EE mutant was more rapidly internalized and degraded than either the wild-type or FF receptor. IGF-1 promoted the accumulation of wild-type and EE IGF-1R within the Golgi apparatus, whereas the FF mutant remained at the plasma membrane. Golgi-associated IGF-1R signaling was a feature of migratory cancer cells, and Golgi disruption impaired IGF-1-induced signaling and cell migration. Upon the formation of new cell adhesions, IGF-1R transiently relocalized to the plasma membrane from the Golgi. Thus, phosphorylation at Tyr1250/1251 promoted IGF-1R translocation to and signaling from the Golgi to support an aggressive cancer phenotype. This process distinguishes IGF-1R from IR signaling and could contribute to the poor clinical efficacy of antibodies that target IGF-1R on the cell surface.

CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.

Cancer genomics studies have identified thousands of putative cancer driver genes1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.

Visualization of Ligand-Bound Ectodomain Assembly in the Full-Length Human IGF-1 Receptor by Cryo-EM Single-Particle Analysis.

Tyrosine kinase receptor of insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) bind to hormones, such as insulin, IGF-1, and IGF-2, and transduces the signals across the cell membrane. However, the complete structure of the receptor and the signal transduction mechanism remains unclear. Here, we report the cryo-EM structure of the ligand-bound ectodomain in the full-length human IGF-1R. We reconstructed the IGF-1R/insulin complex at 4.7 Å and the IGF-1R/IGF-1 complex at 7.7 Å. Our structures reveal that only one insulin or one IGF-1 molecule binds to and activates the full-length human IGF-1R receptor.