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.

Characterization data of gilthead sea bream (Sparus aurata) IGF-I receptors (IGF-IRa/Rb).

In this data article we describe the coding sequence of two IGF-IR paralogues (IGF-IRa and IGF-IRb) obtained from gilthead sea bream embryos. The putative protein architecture (domains and other important motifs) was determined and, amino acid sequences alignment and phylogenetic analysis of both receptors together with IGF-IR orthologues from different vertebrates was performed. Additionally, a semi-quantitative conventional PCR was done to analyze the mRNA expression of both receptors in different tissues of gilthead sea bream. These data will assist in further physiological studies in this species. In this sense, the expression of both receptors during ontogeny in muscle as well as the differential effects of IGF-I and IGF-II on their regulation during in vitro myogenesis has been recently studied (doi: 10.1016/j.ygcen.2015.11.011; [1]).

IGF-I and IGF-II effects on local IGF system and signaling pathways in gilthead sea bream (Sparus aurata) cultured myocytes.

The insulin-like growth factors (IGFs) have a fundamental role in a vast range of functions acting through a tyrosine-kinase receptor (IGF-IR). IGFs in muscle can affect the expression of components of the local IGF system, myogenic regulatory factors (MRFs), proliferating (proliferating cell nuclear antigen, PCNA) or differentiating molecules (myosin heavy chain, MHC) and, lead to the activation of different signaling pathways. The response of all these genes to IGFs incubation at two different times in day 4 cultured myocytes of gilthead sea bream was analyzed. Both IGFs increased the expression of IGF-I and IGFBP-5, but showed different effects on the receptors, with IGF-I suppressing the expression of both isoforms (IGF-IRa and IGF-IRb) and IGF-II up-regulating only IGF-IRb. Moreover, the protein levels of PCNA and target of rapamycin (TOR) increased after IGF-II incubation, although a decline in Myf5 and a rise in MHC gene expression was caused by IGF-I. Taken together, these results provide evidence for the importance of IGFs on controlling muscle development and growth in gilthead sea bream and suggest that each IGF may be preferentially acting through a specific IGF-IR. Moreover, the data support the hypothesis that IGF-II has a more important role during proliferation, whereas IGF-I seems to be relevant for the differentiation phase of myogenesis.

Effects of sustained exercise on GH-IGFs axis in gilthead sea bream (Sparus aurata).

The endocrine system regulates growth mainly through the growth hormone (GH)/insulin-like growth factors (IGFs) axis and, although exercise promotes growth, little is known about its modulation of these factors. The aim of this work was to characterize the effects of 5 wk of moderate sustained swimming on the GH-IGFs axis in gilthead sea bream fingerlings. Plasma IGF-I/GH ratio and tissue gene expression of total IGF-I and three splice variants, IGF-II, three IGF binding proteins, two GH receptors, two IGF-I receptors, and the downstream molecules were analyzed. Fish under exercise (EX) grew more than control fish (CT), had a higher plasma IGF-I/GH ratio, and showed increased hepatic IGF-I expression (mainly IGF-Ia). Total IGF-I expression levels were similar in the anterior and caudal muscles; however, IGF-Ic expression increased with exercise, suggesting that this splice variant may be the most sensitive to mechanical action. Moreover, IGFBP-5b and IGF-II increased in the anterior and caudal muscles, respectively, supporting enhanced muscle growth. Furthermore, in EX fish, hepatic IGF-IRb was reduced together with both GHRs; GHR-II was also reduced in anterior muscle, while GHR-I showed higher expression in the two muscle regions, indicating tissue-dependent differences and responses to exercise. Exercise also increased gene and protein expression of target of rapamycin (TOR), suggesting enhanced muscle protein synthesis. Altogether, these data demonstrate that moderate sustained activity may be used to increase the plasma IGF-I/GH ratio and to potentiate growth in farmed gilthead sea bream, modulating the gene expression of different members of the GH-IGFs axis (i.e., IGF-Ic, IGF-II, IGFBP-5b, GHR-I, and TOR).

Protective hinge in insulin opens to enable its receptor engagement.

Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in ß cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 ß-turn, coupling reorientation of Phe(B24) to a 60° rotation of the B25-B28 ß-strand away from the hormone core to lie antiparallel to the receptor's L1-ß2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile(A2), Val(A3), Val(B12), Phe(B24), and Phe(B25)) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.

How insulin engages its primary binding site on the insulin receptor.

Insulin receptor signalling has a central role in mammalian biology, regulating cellular metabolism, growth, division, differentiation and survival. Insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus and the onset of Alzheimer's disease; aberrant signalling occurs in diverse cancers, exacerbated by cross-talk with the homologous type 1 insulin-like growth factor receptor (IGF1R). Despite more than three decades of investigation, the three-dimensional structure of the insulin-insulin receptor complex has proved elusive, confounded by the complexity of producing the receptor protein. Here we present the first view, to our knowledge, of the interaction of insulin with its primary binding site on the insulin receptor, on the basis of four crystal structures of insulin bound to truncated insulin receptor constructs. The direct interaction of insulin with the first leucine-rich-repeat domain (L1) of insulin receptor is seen to be sparse, the hormone instead engaging the insulin receptor carboxy-terminal α-chain (αCT) segment, which is itself remodelled on the face of L1 upon insulin binding. Contact between insulin and L1 is restricted to insulin B-chain residues. The αCT segment displaces the B-chain C-terminal ß-strand away from the hormone core, revealing the mechanism of a long-proposed conformational switch in insulin upon receptor engagement. This mode of hormone-receptor recognition is novel within the broader family of receptor tyrosine kinases. We support these findings by photo-crosslinking data that place the suggested interactions into the context of the holoreceptor and by isothermal titration calorimetry data that dissect the hormone-insulin receptor interface. Together, our findings provide an explanation for a wealth of biochemical data from the insulin receptor and IGF1R systems relevant to the design of therapeutic insulin analogues.

Structural resolution of a tandem hormone-binding element in the insulin receptor and its implications for design of peptide agonists.

The C-terminal segment of the human insulin receptor alpha-chain (designated alphaCT) is critical to insulin binding as has been previously demonstrated by alanine scanning mutagenesis and photo-cross-linking. To date no information regarding the structure of this segment within the receptor has been available. We employ here the technique of thermal-factor sharpening to enhance the interpretability of the electron-density maps associated with the earlier crystal structure of the human insulin receptor ectodomain. The alphaCT segment is now resolved as being engaged with the central beta-sheet of the first leucine-rich repeat (L1) domain of the receptor. The segment is alpha-helical in conformation and extends 11 residues N-terminal of the classical alphaCT segment boundary originally defined by peptide mapping. This tandem structural element (alphaCT-L1) thus defines the intact primary insulin-binding surface of the apo-receptor. The structure, together with isothermal titration calorimetry data of mutant alphaCT peptides binding to an insulin minireceptor, leads to the conclusion that putative "insulin-mimetic" peptides in the literature act at least in part as mimics of the alphaCT segment as well as of insulin. Photo-cross-linking by novel bifunctional insulin derivatives demonstrates that the interaction of insulin with the alphaCT segment and the L1 domain occurs in trans, i.e., these components of the primary binding site are contributed by alternate alpha-chains within the insulin receptor homodimer. The tandem structural element defines a new target for the design of insulin agonists for the treatment of diabetes mellitus.

The A-chain of insulin contacts the insert domain of the insulin receptor. Photo-cross-linking and mutagenesis of a diabetes-related crevice.

The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of Val(A3), which projects within a crevice between the A- and B-chains. Engineered receptor alpha-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a Pap(B25)-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and alpha-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (Gly(A3) and Ser(A3)) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of Aba(A3) and allo-Thr(A3) analogs are reduced at least 10-fold; the activity of Thr(A3)-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (Aba(A3)) or by altered polarity (allo-Thr(A3) and Thr(A3)). Our results provide evidence that Val(A3), a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the alpha-subunit in a hormone-receptor complex.

Complementation analysis demonstrates that insulin cross-links both alpha subunits in a truncated insulin receptor dimer.

The insulin receptor is a homodimer composed of two alphabeta half receptors. Scanning mutagenesis studies have identified key residues important for insulin binding in the L1 domain (amino acids 1-150) and C-terminal region (amino acids 704-719) of the alpha subunit. However, it has not been shown whether insulin interacts with these two sites within the same alpha chain or whether it cross-links a site from each alpha subunit in the dimer to achieve high affinity binding. Here we have tested the contralateral binding mechanism by analyzing truncated insulin receptor dimers (midi-hIRs) that contain complementary mutations in each alpha subunit. Midi-hIRs containing Ala(14), Ala(64), or Gly(714) mutations were fused with Myc or FLAG epitopes at the C terminus and were expressed separately by transient transfection. Immunoblots showed that R14A+FLAG, F64A+FLAG, and F714G+Myc mutant midi-hIRs were expressed in the medium but insulin binding activity was not detected. However, after co-transfection with R14A+FLAG/F714G+Myc or F64A+FLAG/F714G+Myc, hybrid dimers were obtained with a marked increase in insulin binding activity. Competitive displacement assays revealed that the hybrid mutant receptors bound insulin with the same affinity as wild type and also displayed curvilinear Scatchard plots. In addition, when hybrid mutant midi-hIR was covalently cross-linked with (125)I(A14)-insulin and reduced, radiolabeled monomer was immunoprecipitated only with anti-FLAG, demonstrating that insulin was bound asymmetrically. These results demonstrate that a single insulin molecule can contact both alpha subunits in the insulin receptor dimer during high affinity binding and this property may be an important feature for receptor signaling.

Gene duplication and functional divergence of the zebrafish insulin-like growth factor 1 receptors.

Insulin-like growth factor (IGF) 1 receptor (IGF1R)-mediated signaling plays key roles in growth, development, and physiology. Recent studies have shown that there are two distinct ig f1r genes in zebrafish, termed ig f1ra and ig f1rb. In this study, we tested the hypothesis that zebrafish ig f1ra and ig f1rb resulted from a gene duplication event at the ig f1r locus and that this has led to their functional divergence. The genomic structures of zebrafish ig f1ra and ig f1rb were determined and their loci mapped. While zebrafish ig f1ra has 21 exons and is located on linkage group (LG) 18, zebrafish ig f1rb has 22 exons and mapped to LG 7. There is a strong syntenic relationship between the two zebrafish genes and the human IG F1R gene. Using a MO-based loss-of-function approach, we show that both Igf1ra and Igf1rb are required for zebrafish embryo viability and proper growth and development. Although Igf1ra and Igf1rb demonstrated a large degree of functional overlap with regard to cell differentiation in the developing eye, inner ear, heart, and muscle, they also exhibited functional distinction involving a greater requirement for Igf1rb in spontaneous muscle contractility. These findings suggest that the duplicated zebrafish ig f1r genes play largely overlapping but not identical functional roles in early development and provide novel insight into the functional evolution of the IGF1R/insulin receptor gene family.

Autoradiographic and immunohistochemical localization of insulin-like growth factor-I receptor binding sites in brain of the brown trout, Salmo trutta.

Insulin-like growth factor-I (IGF-I), a peptide closely related to insulin, is known to play crucial roles in brain development. While the central sites of action of IGF-I in higher vertebrates are now well established, surprisingly little is known in the teleost model where the brain undergoes continual, indeterminate, growth. In this study, we have mapped the distribution of putative IGF-I receptor (IGF-IR) binding sites in the brain of the brown trout using both ligand binding in vitro autoradiography and immunohistochemistry. The presence of IGF binding proteins (IGFBPs) was further studied by competitive inhibition using unlabelled IGF-I and des-(1-3)-IGF-I. In both juvenile and adult trout brain, [125I]IGF-I binding was highest in cerebellum and optic tectum, both regions of the teleost brain known to grow the most actively throughout life. At the cellular level, IGF-IR immunoreactivity was confirmed on cell bodies and dendrites, particularly of larger presumptive neurons including purkinje cells and dendritic fibres throughout the molecular layer of the cerebellum. Abundant IGF-IR expression in hypothalamic regions may further be related to neuron growth while a possible hypophysiotropic role will require further investigation. Competitive inhibition studies employing des-(1-3)-IGF-I also suggest IGFBPs are present in all regions exhibiting high [125I]IGF-I ligand binding and confirms the presence of this important regulatory component of the IGF-I system in the teleost brain. The importance of the IGF-I system in brain development, particularly in regions such as the cerebellum, together with the continual lifetime growth of the fish central nervous system, suggest the teleost brain is an extremely useful site for studying the actions of IGF-I in relation to neuron proliferation, growth, and survival in an adult brain.

Islet amyloid polypeptide is expressed in the pancreatic islet parenchyma of the teleostean fish, Myoxocephalus (cottus) scorpius.

The comparative endocrinology of the 37-amino-acid-residue islet amyloid polypeptide (IAPP) is poorly known, possibly due to the fact that available antisera, raised against mammalian IAPP, fail to give immunoreactivity with islet parenchymal cells of non-mammalian vertebrates. Using reverse transcriptase-linked polymerase chain reaction with degenerate primers, IAPP was identified, and its deduced amino-acid sequence partially characterized, in three species of teleostean fish, i.e. Danio rerio (zebrafish), Salmo salar (Atlantic salmon), and Myoxocephalus (cottus) scorpius (daddy sculpin). The daddy sculpin is a species where the histophysiology of the pancreatic islet parenchyma has previously been comprehensively studied. From the deduced amino-acid sequence, a synthetic peptide, corresponding to positions 20-29 of Salmo IAPP, was synthesized. A mouse antiserum to this peptide gave a distinct immunoreactivity with the insulin-producing beta cells of the sculpin Brockmann bodies and salmon endocrine pancreas. Thus, IAPP belongs to the group of peptide hormones expressed by the islet parenchymal cells in both mammals and non-mammalian vertebrates. Salmo salar IAPP(20-29) was found to give rise to amyloid-like fibrils in vitro.

Structural, biochemical, and expression analysis of two distinct insulin-like growth factor I receptors and their ligands in zebrafish.

We have cloned and characterized cDNAs encoding the zebrafish IGF ligands and receptors. Sequence comparison showed that the primary structures of zebrafish IGF-I, IGF-II, and IGF-I receptors (IGF-IRs) have been highly conserved in vertebrates. In contrast to the presence of a single IGF-IR gene in mammals, two distinct IGF-IR genes, termed igf-1ra and igf-1rb, were found in zebrafish. Structural and phylogenetic analyses indicated that both genes are orthologous to the human igf-1r gene. Immunoprecipitation studies with specific antibodies showed that both IGF-IR genes are expressed and both receptors bind to IGFs and des(1-3)IGF-I, but not to insulin. The spatio-temporal expression patterns of the two IGF-IRs and their ligands were determined using a combination of RT-PCR, whole mount in situ hybridization, and immunocytochemistry. Transcripts for both IGF-I and -II mRNAs were found throughout embryogenesis in a ubiquitous manner. In adult tissues, IGF-I mRNA was more abundant in liver and testis, and its level was increased after GH treatment, whereas IGF-II mRNA was not regulated by GH. IGF-IRa and IGF-IRb mRNAs and proteins were expressed in overlapping spatial domains, but exhibited distinct temporal expression patterns. In particular, the relative level of IGF-IRa mRNA was low during early embryogenesis and increased in the hatched larva, whereas the situation was reversed for IGF-IRb mRNA. In adult zebrafish, the overall tissue distribution patterns of the two IGF-IRs were similar, but there were differences in their cellular localization and relative abundance in defined cells/regions. The differential expression pattern of IGF-IRa and IGF-IRb suggest that they may play distinct roles in regulating the growth and development of zebrafish.