Understanding insulin and its receptor from their three-dimensional structures.

BACKGROUND: Insulin's discovery 100 years ago and its ongoing use since that time to treat diabetes belies the molecular complexity of its structure and that of its receptor. Advances in single-particle cryo-electron microscopy have over the past three years revolutionized our understanding of the atomic detail of insulin-receptor interactions. SCOPE OF REVIEW: This review describes the three-dimensional structure of insulin and its receptor and details on how they interact. This review also highlights the current gaps in our structural understanding of the system. MAJOR CONCLUSIONS: A near-complete picture has been obtained of the hormone receptor interactions, providing new insights into the kinetics of the interactions and necessitating a revision of the extant two-site cross-linking model of hormone receptor engagement. How insulin initially engages the receptor and the receptor's traversed trajectory as it undergoes conformational changes associated with activation remain areas for future investigation.

Large Stokes-shift bioorthogonal probes for STED, 2P-STED and multi-color STED nanoscopy.

Synthesis and multiple STED imaging applications of four, red-emitting (610-670 nm), tetrazine-functionalized fluorescent probes (CBRD = Chemical Biology Research group Dye 1-4) with large Stokes-shift is presented. Present studies revealed the super-resolution microscopy applicability of the probes as demonstrated through bioorthogonal labeling scheme of cytoskeletal proteins actin and keratin-19, and mitochondrial protein TOMM20. Furthermore, super-resolved images of insulin receptors in live-cell bioorthogonal labeling schemes through a genetically encoded cyclooctynylated non-canonical amino acid are also presented. The large Stokes-shifts and the wide spectral bands of the probes enabled the use of two common depletion lasers (660 nm and 775 nm). The probes were also found suitable for super-resolution microscopy in combination with two-photon excitation (2P-STED) resulting in improved spatial resolution. One of the dyes was also used together with two commercial dyes in the three-color STED imaging of intracellular structures.

The signalling conformation of the insulin receptor ectodomain.

Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy structure of a single insulin bound to a physiologically relevant, high-affinity version of the receptor ectodomain, the latter generated through attachment of C-terminal leucine zipper elements to overcome the conformational flexibility associated with ectodomain truncation. The resolution of the cryo-electron microscopy maps is 3.2 Å in the insulin-binding region and 4.2 Å in the membrane-proximal region. The structure reveals how the membrane proximal domains of the receptor come together to effect signalling and how insulin's negative cooperativity of binding likely arises. Our structure further provides insight into the high affinity of certain super-mitogenic insulins. Together, these findings provide a new platform for insulin analog investigation and design.

Reducing effects of particle adsorption to the air-water interface in cryo-EM.

Most protein particles prepared in vitreous ice for single-particle cryo-electron microscopy (cryo-EM) are adsorbed to air-water or substrate-water interfaces, which can cause the particles to adopt preferred orientations. By using a rapid plunge-freezing robot and nanowire grids, we were able to reduce some of the deleterious effects of the air-water interface by decreasing the dwell time of particles in thin liquid films. We demonstrated this by using single-particle cryo-EM and cryo-electron tomography (cryo-ET) to examine hemagglutinin, insulin receptor complex, and apoferritin.

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

The insulin receptor is a dimeric protein that has a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels. Insulin receptor dysfunction has been associated with many diseases, including diabetes, cancer and Alzheimer's disease. The primary sequence of the receptor has been known since the 1980s, and is composed of an extracellular portion (the ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Binding of insulin to the dimeric ECD triggers auto-phosphorylation of the tyrosine kinase domain and subsequent activation of downstream signalling molecules. Biochemical and mutagenesis data have identified two putative insulin-binding sites, S1 and S2. The structures of insulin bound to an ECD fragment containing S1 and of the apo ectodomain have previously been reported, but details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single-particle cryo-electron microscopy reconstructions of the 1:2 (4.3 Å) and 1:1 (7.4 Å) complexes of the insulin receptor ECD dimer with insulin. The symmetrical 4.3 Å structure shows two insulin molecules per dimer, each bound between the leucine-rich subdomain 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 binding interactions at S1 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 subdomain orientations and triggers downstream signal propagation.

Visualization of ligand-induced transmembrane signaling in the full-length human insulin receptor.

Insulin receptor (IR) signaling plays a critical role in the regulation of metabolism and growth in multicellular organisms. IRs are unique among receptor tyrosine kinases in that they exist exclusively as covalent (αß)2 homodimers at the cell surface. Transmembrane signaling by the IR can therefore not be based on ligand-induced dimerization as such but must involve structural changes within the existing receptor dimer. In this study, using glycosylated full-length human IR reconstituted into lipid nanodiscs, we show by single-particle electron microscopy that insulin binding to the dimeric receptor converts its ectodomain from an inverted U-shaped conformation to a T-shaped conformation. This structural rearrangement of the ectodomain propagates to the transmembrane domains, which are well separated in the inactive conformation but come close together upon insulin binding, facilitating autophosphorylation of the cytoplasmic kinase domains.

Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development.

BACKGROUND: The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host's liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood. RESULTS: Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite's glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin. CONCLUSIONS: Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs.

Ultrastructural immunolocalization of IGF-1 and insulin receptors in rat pituitary culture: evidence of a functional interaction between gonadotroph and lactotroph cells.

We have investigated the expression of receptors for insulin and insulin-like growth factor 1 (IGF-1) in rat pituitary cells in vitro and examined the morphological and proliferative changes induced in adenohypophyseal cells by insulin and IGF-1. The proliferation of lactotrophs was determined by double-immunostaining for bromodeoxyuridine and prolactin. Incubation with insulin (10, 100 or 1000 ng/ml) or IGF-1 (5, 30 or 100 ng/ml) for 48 or 72 h significantly increased the number of lactotrophs undergoing mitosis. Co-incubation of insulin or IGF-1 with genistein (25 microM), an inhibitor of the tyrosine kinase receptor, reduced the proliferation of lactotrophs elicited by the hormone and the growth factor. The receptors for insulin and IGF-1 were localized in intact pituitary cells by ultrastructural immunocytochemistry with the colloidal gold-protein A technique. Gonadotrophs expressed both receptors, specific labelling being restricted to this cell type. Electron-microscopical observations of pituitary cell cultures incubated with insulin or IGF-1 revealed gonadotroph cells exhibiting the fine-structural features of enhanced protein synthetic activity. These findings suggest that both insulin and IGF-1 are able to induce the proliferation of lactotrophs through an indirect mechanism mediated by a factor synthesized by gonadotroph cells, in addition to stimulating the biosynthetic activity of the gonadotroph in a direct manner.

Structure of the insulin receptor ectodomain reveals a folded-over conformation.

The insulin receptor is a phylogenetically ancient tyrosine kinase receptor found in organisms as primitive as cnidarians and insects. In higher organisms it is essential for glucose homeostasis, whereas the closely related insulin-like growth factor receptor (IGF-1R) is involved in normal growth and development. The insulin receptor is expressed in two isoforms, IR-A and IR-B; the former also functions as a high-affinity receptor for IGF-II and is implicated, along with IGF-1R, in malignant transformation. Here we present the crystal structure at 3.8 A resolution of the IR-A ectodomain dimer, complexed with four Fabs from the monoclonal antibodies 83-7 and 83-14 (ref. 4), grown in the presence of a fragment of an insulin mimetic peptide. The structure reveals the domain arrangement in the disulphide-linked ectodomain dimer, showing that the insulin receptor adopts a folded-over conformation that places the ligand-binding regions in juxtaposition. This arrangement is very different from previous models. It shows that the two L1 domains are on opposite sides of the dimer, too far apart to allow insulin to bind both L1 domains simultaneously as previously proposed. Instead, the structure implicates the carboxy-terminal surface of the first fibronectin type III domain as the second binding site involved in high-affinity binding.

Single molecule imaging of supported planar lipid bilayer--reconstituted human insulin receptors by in situ scanning probe microscopy.

A 480-kDa disulfide-linked heterodimer single-pass transmembrane protein, the insulin receptor, is autophosphorylated upon insulin binding to its extracellular domain. Remarkably, the structural basis for this activation process remained largely unknown until the recent cryoelectron microscopy studies of the insulin-insulin receptor complex by Luo et al. [Science 285 (1999) 1077]. We report here the results of an in situ study by high-resolution scanning probe microscopy of the full-length insulin receptor reconstituted within supported planar lipid bilayers. Our preliminary studies confirm that (1) the intact receptor can be reconstituted constitutively within a lipid vesicle and (2) fusion of the receptor-containing vesicles to mica resulted in the formation of molecular flat 5.5-nm-thick supported planar bilayers populated by two populations of protrusions, the shape and size of which are consistent with those of the insulin receptor's intra- and extracellular domains as modeled by the cryo-EM data of Ottensmeyer et al. [Biochemistry 39 (2000) 12103]. These results establish a framework for real-time studies of insulin-insulin receptor binding by in situ SPM and single molecule force spectroscopy.

Structural studies of the detergent-solubilized and vesicle-reconstituted insulin receptor.

Insulin binding to the insulin receptor initiates a cascade of cellular events that are responsible for regulating cell metabolism, proliferation, and growth. We have investigated the structure of the purified, functionally active, human insulin receptor using negative stain and cryo-electron microscopy. Visualization of the detergent-solubilized and vesicle-reconstituted receptor shows the alpha(2)beta(2) heterotetrameric insulin receptor to be a three-armed pinwheel-like complex that exhibits considerable variability among individual receptors. The alpha-subunit of the receptor was labeled with an insulin analogue.streptavidin gold conjugate, which facilitated the identification of the receptor arm responsible for insulin binding. The gold label was localized to the tip of a single receptor arm of the three-armed complex. The beta-subunit of the insulin receptor was labeled with a maleimide-gold conjugate, which allowed orientation of the receptor complex in the membrane bilayer. The model derived from electron microscopic studies displays a "Y"-like morphology representing the predominant species identified in the reconstituted receptor images. The insulin receptor dimensions are approximately 12.2 nm by 20.0 nm, extending 9.7 nm above the membrane surface. The beta-subunit-containing arm is approximately 13.9 nm, and each alpha-subunit-containing arm is 8.6 nm in length. The model presented is the first description of the insulin receptor visualized in a fully hydrated state using cryo-electron microscopy.

Single-molecule imaging of human insulin receptor ectodomain and its Fab complexes.

The insulin receptor (IR) is a four-chain, transmembrane dimer held together by disulfide bonds. To gain information about the molecular envelope and the organization of its domains, single-molecule images of the IR ectodomain and its complexes with three Fabs have been analyzed by electron microscopy. The data indicate that the IR ectodomain resembles a U-shaped prism of approximate dimensions 90 x 80 x 120 A. The width of the cleft (assumed membrane-distal) between the two side arms is sufficient to accommodate ligand. Fab 83-7, which recognizes the cys-rich region of IR, bound halfway up one end of each side arm in a diametrically opposite manner, indicating a twofold axis of symmetry normal to the membrane surface. Fabs 83-14 and 18-44, which have been mapped respectively to the first fibronectin type III domain (residues 469-592) and residues 765-770 in the insert domain, bound near the base of the prism at opposite corners. These images, together with the data from the recently determined 3D structure of the first three domains of the insulin-like growth factor type I receptor, suggest that the IR dimer is organized into two layers with the L1/cys-rich/L2 domains occupying the upper (membrane distal) region of the U-shaped prism and the fibronectin type III domains and the insert domains located predominantly in the membrane-proximal region.

How do protein kinases discriminate between serine/threonine and tyrosine? Structural insights from the insulin receptor protein-tyrosine kinase.

The eukaryotic protein kinases that directly phosphorylate proteins are divided into two major classes: those that phosphorylate tyrosine and those that phosphorylate serine and threonine. Until recently, the similarities between these two classes of enzymes, which now total more than 400, were based primarily on sequence alignments. A recent report of the structure of the kinase domain (IRK) of the insulin receptor protein-tyrosine kinase now allows the features of these two families to be compared at the structural level. We review here this first tyrosine-specific protein kinase structure, and compare and contrast it to the structure of the serine/threonine-specific cAMP-dependent protein kinase. Although the general fold of the polypeptide backbone is conserved as predicted, unique features at the IRK active site provide a basis for understanding the differences in specificity for the phosphate acceptor amino acid. The structure of this inactive, dephosphorylated protein-tyrosine kinase also defines for the first time how activation might be achieved.

Membrane topology of insulin receptors reconstituted into lipid vesicles.

Insulin receptors were incorporated into liposomes by two different procedures, one using dialysis and one using detergent removal by Bio-Beads. Receptor incorporation was analyzed by gradient centrifugation and electron microscopy. Reconstituted receptors projected up to 12 nm above the membrane and exhibited a T-shaped structure compatible with that previously described for the solubilized receptor. Insulin binding and autophosphorylation experiments indicated that approx. 50% of the receptors were incorporated right-side out. Such random orientation was confirmed by immunogold labeling of the alpha- and the beta-subunit of the receptor. Immunogold labeling of the C-terminus of the beta-subunit indicates that it resides about 6 nm off the membrane, while two alpha-subunit epitopes were labeled at about twice this distance, confirming that the alpha-subunit is harbored in the cross-bar of the T-structure.

Sorting and recycling efficiency of apical insulin binding sites during endocytosis in proximal tubule cells.

Intracellular traffic and recycling of apical insulin binding sites are examined in isolated, perfused proximal tubules. The endocytic binding sites were specific as revealed by 90% reduction in 125I-labeled insulin binding by 10(-5) M insulin. The traffic was followed by developing a chemical cross-linking method to covalently label the binding sites. Only 3% of cross-linked insulin-gold was transported to the lysosomes, reflecting high sorting and recycling efficiency. Correspondingly, only 4% of cross-linked 125I-insulin was degraded, and only 5% of the electron microscopy-autoradiographic grains was associated with lysosomes. No label was transferred to the Golgi apparatus; thus neither lysosomes nor Golgi apparatus is involved in the recycling. In contrast, approximately 40% of non-cross-linked ligand was transferred to the lysosomes. Tubules first pretreated with cross-linker and then perfused with insulin-gold or 125I-labeled insulin-like growth factor I revealed lysosomal accumulation and degradation at control levels. Thus the cross-linker does not interfere with membrane or protein processing. The study also provides evidence for a vesicular transtubular transport because insulin-gold was transcytosed to the basolateral part of the cells and to the intracellular spaces (0.5%). In contrast cross-linked label was never observed in intercellular spaces, suggesting sorting of apical binding sites, a mechanism contributing to maintenance of cell polarity. In conclusion, traffic, sorting, and recycling of binding sites take place with high efficiency.

A truncated human insulin receptor missing the COOH-terminal 365 amino acid residues does not undergo insulin-mediated receptor migration or aggregation.

A previous study of tyrosine kinase-defective insulin receptors demonstrated that receptor autophosphorylation or tyrosine kinase activity was required for concentrating insulin receptors in coated pits, but not for their migration or aggregation on the cell surface. Furthermore, receptor migration and aggregation on the cell surface were not sufficient to cause internalization of the occupied receptors in coated pits. In the present study, biochemical and ultrastructural techniques were used to compare insulin receptor mobility and internalization in Rat 1 fibroblasts expressing wild-type human insulin receptors (HIRc) with those in cells expressing receptors truncated at residues 978 (HIR delta 978) or 1301 of the carboxyl-terminus (HIR delta CT). There were no significant differences in the mobility or internalization of insulin receptors on HIR delta CT cells compared to those of insulin receptors on HIRc cells. Ultrastructural analysis revealed that truncated insulin receptors on HIR delta 978 cells failed to migrate from their initial location on the microvilli, move to the plasma membrane, and aggregate in coated pits. Receptor-mediated insulin internalization in HIR delta 978 cells was markedly decreased due entirely to a decrease in ATP-dependent, coated pit-mediated internalization. ATP-independent endocytosis in non-coated pinocytotic invaginations was not affected by receptor truncations. These results provide evidence of the roles that regions of the beta-subunit play in the processing of occupied insulin receptors. 1) The carboxyl-terminus of the insulin receptor is not involved in the events leading to receptor internalization, i.e. migration, aggregation, and concentration in coated pits. 2) Internalization of insulin receptors by the ATP-independent noncoated invagination pathway is not regulated by residues in the insulin receptor beta-subunit distal to 978. 3) Sequences in the beta-subunit between 978-1300, but not the autophosphorylation and kinase domains, are involved in insulin-induced receptor migration and aggregation.

[Regulation of insulin receptor expression and its gene]. / Régulation de l'expression du récepteur de l'insuline et de son gène.

The insulin receptor is a membrane macromolecule whose expression on the cell surface is essential for cell sensitivity to insulin. Current knowledge on the regulation of expression of the insulin receptor and its gene in human and animal cells is presented. Although ubiquitously distributed, the insulin receptor and its messenger RNA (mRNA) are mainly expressed in metabolically active cells such as hepatocytes and adipocytes. Two receptor isoforms, generated by alternative splicing of exon 11, have been identified. Isoform B (exon 11+) predominates in liver and adipocytes, and isoform A (exon 11-) in brain, spleen and leukocytes. In vivo and in several cell models, the expression of the insulin receptor and/or its mRNA is under positive regulation by glucocorticoid hormones and negative regulation by insulin. Glucocorticoid hormones stimulate receptor gene transcription and receptor protein synthesis. Insulin stimulates receptor protein degradation and, in certain cell types, decreases receptor mRNA level. Vanadate (an insulinomimetic agent) corrects, in vivo, the hyperexpression of the liver receptor observed in experimental insulinopenic diabetes, but its effects on receptor expression in vitro are complex and vary with the cell type. In vivo the insulin receptor and/or its mRNA are expressed early in fetal development with a high level, in liver, of isoform A. Maximal expression is reached at the end of gestation and then decreases after birth. In several cell models, receptor protein and/or mRNA expression is affected by cell growth and/or differentiation. Several cis- and trans-acting factors regulating the expression of the human insulin receptor gene and its response to glucocorticoid hormones have been identified.

Structural organization of the human insulin receptor ectodomain.

To provide an experimental system amenable to a detailed biochemical and structural investigation of the extracellular (ligand binding) domain of the insulin receptor, we developed a mammalian heterologous cell expression system from which tens of milligrams of the soluble secreted ectodomain (the IR921 protein) can be routinely purified using methods that do not require harsh elution conditions. The purified IR921 protein has a Stokes radius of 6.8 nm and a sedimentation coefficient of 9.8 S, from which we calculate a hydro-dynamic mass of 281 kDa. Electron microscopic images, using both rotary shadowing and negative staining techniques, demonstrate a characteristic substructure for the IR921 protein consisting of two elongated arms, with a globular domain at each end, connected to each other at a point somewhat off-center to form a Y structure. Analysis using circular dichroism and fluorescence spectroscopy illustrate that insulin binding results in conformational changes in the ectodomain. Furthermore, fluorescence anisotropy decay data reveal segmental mobility within the IR921 protein that is successively frozen as a result of insulin binding, in contrast to results obtained in a previous study of the epidermal growth factor receptor ectodomain. This result suggests a divergence in hormone-induced signaling mechanisms used by the insulin and epidermal growth factor receptors.

Transmembrane signaling by the human insulin receptor kinase. Relationship between intramolecular beta subunit trans- and cis-autophosphorylation and substrate kinase activation.

To examine the role of intramolecular beta subunit trans- and cis-autophosphorylation in signal transduction, the vaccinia virus/bacteriophage T7 expression system was used to generate insulin holoreceptors composed of a kinase-defective half-receptor precursor (alpha beta A/K or alpha beta A/K.delta CT) and a kinase-active half-receptor precursor (alpha beta delta CT or alpha beta WT). In the alpha beta A/K-alpha beta delta CT hybrid insulin receptor, insulin stimulated a 20-fold increase in intramolecular beta subunit trans-phosphorylation, whereas cis-phosphorylation increased only 3-fold over the basal state. Similarly, in the alpha beta WT-alpha beta A/K.delta CT hybrid insulin receptor, insulin stimulated trans-phosphorylation approximately 30-fold and cis-phosphorylation only 3-fold over the basal state. Although cis-phosphorylation of the kinase-functional alpha beta half-receptor was observed within these hybrid receptor species, this was not sufficient to stimulate exogenous substrate kinase activity. These data demonstrate that insulin primarily activates an intramolecular beta subunit trans-phosphorylation reaction within the insulin holoreceptor and suggest that this reaction is necessary for activation of the holoreceptor. Furthermore, our results suggest a molecular basis for the dominant-negative phenotype observed in insulin-resistant patients possessing one kinase-defective insulin receptor allele.

Internalization of the human insulin receptor. The insulin-independent pathway.

Internalization of the human insulin receptor requires the activation by insulin of the intrinsic kinase of the receptor. However, even in the absence of kinase activation, insulin receptors slowly enter the cells. In the present study, we addressed the question of this insulin-independent pathway of internalization. To that end, we traced insulin receptor internalization with a monoclonal antibody (mAb 83-14) directed against the alpha-subunit of the human insulin receptor. Internalization of this antibody was followed in Chinese hamster ovary (CHO) cells transfected with either normal (CHO.HIRC2) or kinase-deficient (CHO.A1018) human insulin receptors. The internalization rate of 125I-mAb 83-14 was comparable in CHO cells expressing kinase-active or kinase-inactive receptors and was similar to that observed for 125I-insulin in CHO.A1018 cells. Moreover, in CHO.HIRC2 cells, the internalization of 125I-mAb 83-14 was identical with that of its 125I-Fab fragments. Thus, mAb 83-14 represents an appropriate tool to study the constitutive internalization of the insulin receptor. Internalization of insulin receptors tagged with 125I-mAb 83-14 was unaffected by cytochalasin B, which excluded a macropinocytotic process. By contrast, internalization was sensitive to hypertonia, which abrogates clathrin-coated pits-mediated endocytosis. The implication of clathrin-coated pits in this internalization process was directly demonstrated by quantitative electron microscopic autoradiography, which showed that 125I-mAb 83-14 present on the nonvillous domain of the cell surface preferentially associate with clathrin-coated pits at all time points.