BackgroundInsulin-like growth factor-II (IGF-II) promotes cell proliferation and survival and plays an important role in normal fetal development and placental function. IGF-II binds both the insulin-like growth factor receptor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. Interestingly both IGF-II and the IR-A are often upregulated in cancer and IGF-II acts via both receptors to promote cancer proliferation. There is relatively little known about the mechanism of ligand induced activation of the insulin (IR) and IGF-1R. The recently solved IR structure reveals a folded over dimer with two potential ligand binding pockets arising from residues on each receptor half. Site-directed mutagenesis has mapped receptor residues important for ligand binding to two separate sites within the ligand binding pocket and we have recently shown that the IGFs have two separate binding surfaces which interact with the receptor sites 1 and 2.Methodology/Principal FindingsIn this study we describe a series of partial IGF-1R and IR agonists generated by mutating Glu12 of IGF-II. By comparing receptor binding affinities, abilities to induce negative cooperativity and potencies in receptor activation, we provide evidence that residue Glu12 bridges the two receptor halves leading to receptor activation.Conclusions/SignificanceThis study provides novel insight into the mechanism of receptor binding and activation by IGF-II, which may be important for the future development of inhibitors of its action for the treatment of cancer.

Very little is known about the residues important for the interaction of insulin-like growth factor II (IGF-II) with the type 1 IGF receptor (IGF-1R) and the insulin receptor (IR). Insulin, to which IGF-II is homologous, is proposed to cross-link opposite halves of the IR dimer through two receptor binding surfaces, site 1 and site 2. In the present study we have analyzed the contribution of IGF-II residues equivalent to insulin's two binding surfaces toward the interaction of IGF-II with the IGF-1R and IR. Four "site 1" and six "site 2" analogues were produced and analyzed in terms of IGF-1R and IR binding and activation. Insulin-like growth factor II (IGF-II)2 is a single-chain polypeptide with homology to IGF-I and insulin. Its 67 amino acids are arranged, like those counterparts in IGF-I, in four domains in the order B, C, A, and D from the N terminus ( The mitogenic and metabolic activities of the IGFs and insulin result from their interaction with the type 1 IGF receptor (IGF-1R) and/or the exon 11-(IR-A) and exon 11ϩ (IR-B) isoforms of the insulin receptor (IR). These class II receptor tyrosine kinases exist at the membrane as preformed disulfidelinked homodimers composed of two ␣ and two ␤ subunits in a ␤-␣-␣-␤ arrangement (reviewed in Refs. 9 -11). The IR and IGF-1R share between 41 and 84% sequence similarity that is most pronounced in their tyrosine kinase domains. Despite the homology in sequence and structure between these receptors, each exhibits distinct ligand binding preferences. The IGF-1R and IR bind their cognate ligands with high affinity (IGF-I and insulin, respectively). Both the IGF-1R and IR-A bind IGF-II with high affinity and are capable of mediating IGF-II action (12). Interestingly, the IR-B has a low affinity for IGF-II. The discerning factors for this isoform discrimination are largely undefined but may involve steric hindrance between the 12 amino acids encoded by exon 11 of IR-B and the IGF-II C domain.There are currently no structures of any of these ligand⅐receptor complexes. The binding of insulin to the IR is certainly the best characterized of these interactions. Insulin has two IR binding surfaces: the "site 1" binding surface lies within the insulin dimerization surface, whereas "site 2" overlaps its hexamer-forming surface. Insulin is proposed to crosslink opposite halves of the insulin receptor dimer through these two receptor binding surfaces (13,14). The stoichiometry of binding at physiological insulin concentrations is 1:1. However, each IR has two potential ligand binding pockets both also consisting of two ligand binding surfaces (site 1 and site 2 of one monomer and site 1Ј and site 2Ј of the other, which combine to give two identical binding pockets, site 1/2Ј and site 1Ј/2). This putative arrangement is consistent with the three-dimensional crystal structure of the IR ectodomain (15). Evidence from ligand binding studies suggests that insulin may cross-link only one pair of binding surfaces at a time, with binding of a second molecule to an unoccupied site acce...

The structural transitions required for insulin to activate its receptor and initiate regulation of glucose homeostasis are only partly understood. Here, using ring-closing metathesis, we substitute the A6-A11 disulfide bond of insulin with a rigid, non-reducible dicarba linkage, yielding two distinct stereo-isomers (cis and trans). Remarkably, only the cis isomer displays full insulin potency, rapidly lowering blood glucose in mice (even under insulin-resistant conditions). It also posseses reduced mitogenic activity in vitro. Further biophysical, crystallographic and molecular-dynamics analyses reveal that the A6-A11 bond configuration directly affects the conformational flexibility of insulin A-chain N-terminal helix, dictating insulin’s ability to engage its receptor. We reveal that in native insulin, contraction of the Cα-Cα distance of the flexible A6-A11 cystine allows the A-chain N-terminal helix to unwind to a conformation that allows receptor engagement. This motion is also permitted in the cis isomer, with its shorter Cα-Cα distance, but prevented in the extended trans analogue. These findings thus illuminate for the first time the allosteric role of the A6-A11 bond in mediating the transition of the hormone to an active conformation, significantly advancing our understanding of insulin action and opening up new avenues for the design of improved therapeutic analogues.

The evolution of subterranean animals following multiple colonisation events from the surface has been well documented, but few studies have investigated the potential for species diversification within cavernicolous habitats. Isolated calcrete (carbonate) aquifers in central Western Australia have been shown to contain diverse assemblages of aquatic subterranean invertebrate species (stygofauna) and to offer a unique model system for exploring the mechanisms of speciation in subterranean ecosystems. In this paper, we investigated the hypothesis that microallopatric speciation processes (fragmentation and isolation by distance (IBD)) occur within calcretes using a comparative phylogeographic study of three stygobiontic diving beetle species, one amphipod species and a lineage of isopods. Specimens were sequenced for the mitochondrial cytochrome c oxidase 1 gene from three main sites: Quandong Well, Shady Well (SW) and Mt. Windarra (MW), spanning a 15 km region of the Laverton Downs Calcrete. Phylogenetic and haplotype network analyses revealed that each species possessed a single divergent clade of haplotypes that were present only at the southern MW site, despite the existence of other haplotypes at MW that were shared with SW. IBD between MW and SW was evident, but the common phylogeographic pattern most likely resulted from fragmentation, possibly by a salt lake adjacent to MW. These findings suggest that microallopatric speciation within calcretes may be a significant diversifying force, although the proportion of stygofauna species that may have resulted from in situ speciation in this system remains to be determined.

The development of fast-acting and highly stable insulin analogues is challenging. Insulin undergoes structural transitions essential for binding and activation of the insulin receptor (IR), but these conformational changes can also affect insulin stability. Previously, we substituted the insulin A6-A11 cystine with a rigid, non-reducible C=C linkage ("dicarba" linkage). A -alkene permitted the conformational flexibility of the A-chain N-terminal helix necessary for high-affinity IR binding, resulting in surprisingly rapid activity Here, we show that, unlike the rapidly acting LysPro insulin analogue (KP insulin), -dicarba insulin is not inherently monomeric. We also show that-dicarba KP insulin lowers blood glucose levels even more rapidly than KP insulin, suggesting that an inability to oligomerize is not responsible for the observed rapid activity onset of -dicarba analogues. Although rapid-acting, neither dicarba species is stable, as assessed by fibrillation and thermodynamics assays. MALDI analyses and molecular dynamics simulations of-dicarba insulin revealed a previously unidentified role of the A6-A11 linkage in insulin conformational dynamics. By controlling the conformational flexibility of the insulin B-chain helix, this linkage affects overall insulin structural stability. This effect is independent of its regulation of the A-chain N-terminal helix flexibility necessary for IR engagement. We conclude that high-affinity IR binding, rapid activity, and insulin stability can be regulated by the specific conformational arrangement of the A6-A11 linkage. This detailed understanding of insulin's structural dynamics may aid in the future design of rapid-acting insulin analogues with improved stability.

The mechanisms by which insulin activates the insulin receptor to promote metabolic processes and cellular growth are still not clear. Significant advances have been gained from recent structural studies in understanding how insulin binds to its receptor. However, the way in which specific interactions lead to either metabolic or mitogenic signalling remains unknown. Currently there are only a few examples of insulin receptor agonists that have biased signalling properties. Here we use novel insulin analogues that differ only in the chemical composition at the A6–A11 bond, as it has been changed to a rigid, non-reducible C=C linkage (dicarba bond), to reveal mechanisms underlying signaling bias. We show that introduction of an A6-A11 cis-dicarba bond into either native insulin or the basal/long acting insulin glargine results in biased signalling analogues with low mitogenic potency. This can be attributed to reduced insulin receptor activation that prevents effective receptor internalization and mitogenic signalling. Insight gained into the receptor interactions affected by insertion of an A6-A11 cis-dicarba bond will ultimately assist in the development of new insulin analogues for the treatment of diabetes that confer low mitogenic activity and therefore pose minimal risk of promoting cancer with long term use.

No abstract