Characterization of insulin/IGF hybrid receptors: contributions of the insulin receptor L2 and Fn1 domains and the alternatively spliced exon 11 sequence to ligand binding and receptor activation

The IR (insulin receptor) and IGFR (type I insulin-like growth factor receptor) are found as homodimers, but the respective pro-receptors can also heterodimerize to form insulin-IGF hybrid receptors. There are conflicting data on the ligand affinity of hybrids, and especially on the influence of different IR isoforms. To investigate further the contribution of individual ligand binding epitopes to affinity and specificity in the IR/IGFR family, we generated hybrids incorporating both IR isoforms (A and B) and IR/IGFR domain-swap chimaeras, by ectopic co-expression of receptor constructs in Chinese hamster ovary cells, and studied ligand binding using both radioligand competition and bioluminescence resonance energy transfer assays. We found that IR-A-IGFR and IR-B-IGFR hybrids bound insulin with similar relatively low affinity, which was intermediate between that of homodimeric IR and homodimeric IGFR. However, both IR-A-IGFR and IR-B-IGFR hybrids bound IGF-I and IGF-II with high affinity, at a level comparable with homodimeric IGFR. Incorporation of a significant fraction of either IR-A or IR-B into hybrids resulted in abrogation of insulin- but not IGF-I-stimulated autophosphorylation. We conclude that the sequence of 12 amino acids encoded by exon 11 of the IR gene has little or no effect on ligand binding and activation of IR-IGFR hybrids, and that hybrid receptors bind IGFs but not insulin at physiological concentrations regardless of the IR isoform they contained. To reconstitute high affinity insulin binding within a hybrid receptor, chimaeras in which the IGFR L1 or L2 domains had been replaced by equivalent IR domains were co-expressed with full-length IR-A or IR-B. In the context of an IR-A-IGFR hybrid, replacement of IR residues 325-524 (containing the L2 domain and part of the first fibronectin domain) with the corresponding IGFR sequence increased the affinity for insulin by 20-fold. We conclude that the L2 and/or first fibronectin domains of IR contribute in trans with the L1 domain to create a high affinity insulin-binding site within a dimeric receptor.     

Comparison of anti-apoptotic signalling by the insulin receptor and IGF-I receptor in preadipocytes and adipocytes

We compared the effectiveness of insulin receptor (IR) and type I insulin-like growth factor (IGF) receptor (IGFR) cytoplasmic domains in mediating anti-apoptotic effects in 3T3-L1 preadipocytes and adipocytes. We used TrkC/IR and TrkC/IGFR chimeras, stably expressed in these cells and stimulated with neurotrophin-3 (NT-3), so as to avoid interference from endogenous receptors. After 24-h serum deprivation, 10% of preadipocytes and 2% of adipocytes appeared apoptotic as determined by fluorescence-activated cell sorter (FACS) analysis of cells stained with propidium iodide (PI) and Annexin V. When NT-3 was added, the two chimeras inhibited apoptosis to the same extent by 80% in preadipocytes and 50% in adipocytes. Mutation of juxtamembrane tyrosines (IR Y960F, IGFR Y950F) abrogated these anti-apoptotic effects. Qualitatively similar results were obtained by determination of viable rather than apoptotic cells. We conclude that IR and IGFR have equal potential to inhibit apoptosis in cell backgrounds, which are normally responsive to either IGF-I or insulin.     

The insulin and EGF receptor structures: new insights into ligand-induced receptor activation

The insulin receptor (IR) and epidermal growth factor receptor (EGFR; also known as ErbB) families exhibit similarities in the composition of their ectodomains. The past five years have seen structures determined for all members of the EGFR family including some complexes with ligand or monoclonal antibody fragments. These structures have led to a clearer understanding of their mechanism of activation and inhibition. By contrast, obtaining equivalent understanding of the IR family has lagged behind. However, within the past year, structures of partial and complete ectodomains of the IR have been published that show that the extracellular region of the receptor adopts an unexpected 'inverted V' conformation relative to the cell membrane. This is very different from the folded-over (tethered) conformation of the unactivated EGFR and provides insight into the potential mechanism of activation of the IR.     

Distinctive effects of the carboxyl-terminal sequence of the insulin-like growth factor I receptor on its signaling functions.

We have shown previously that the extracellular sequences of the human insulin receptor (IR) and the insulin-like growth factor I receptor (IGFR) have an inhibitory effect on protein tyrosine kinase (PTK) activity and on the biological functions of their respective Gag-receptor fusion proteins. To study the role of IGFR carboxyl sequence in modulation of the Gag-IGFR PTK and biological activities, five mutants, CM1, CM2, CM3, CM4, and CM5, containing carboxyl deletions of 17, 27, 47, 67, and 88 amino acids (aa), respectively, were constructed from the parental virus UIGFR encoding the Gag-IGFR. Deletion of up to 27 aa had little effect on the cell-transforming and PTK activities of UIGFR. Deletions of 47 aa in CM3 abolished PTK and transforming activities. Surprisingly, a further deletion of 20 aa in CM4 beyond that in CM3 reactivated the kinase and transforming activities. CM5, containing a deletion of 20 aa beyond that in CM4, had only marginal transforming and PTK activities. We conclude that deletion of the carboxyl region of the Gag-IGFR inactivates, instead of activating as in the case with Gag-IR, its transforming activity and the amino acid sequence 1250 to 1310 is essential for PTK and transforming activities. Analysis of the ability of the full-length IGFR and its mutant receptors described above to associate with phosphatidylinositol 3 kinase indicated that the association required PTK activity and tyrosine phosphorylation of the receptors and correlated well with their transforming activities. The carboxyl 88 aa are not essential for the association.     

Increased IGFR activity and glucose transport in cultured skeletal muscle from insulin receptor null mice

We have studied the role of the insulin receptor (IR) in metabolic and growth-promoting effects of insulin on primary cultures of skeletal muscle derived from the limb muscle of IR null mice. Cultures of IR null skeletal muscle displayed normal morphology and spontaneous contractile activity. Expression of muscle-differentiating proteins was slightly reduced in myoblasts and myotubes of the IR null skeletal muscle cells, whereas that of the Na+/K+ pump appeared to be unchanged. Insulin-like growth factor receptor (IGFR) expression was higher in myoblasts from IR knockout (IRKO) than from IR wild-type (IRWT) mice but was essentially unchanged in myotubes. Expression of the GLUT-1 and GLUT-4 transporters appeared to be higher in IRKO than in IRWT myoblasts and was significantly greater in myotubes from IRKO than from IRWT cultures. Consistent with GLUT expression, both basal and insulin or insulin-like growth factor I (IGF-I)-stimulated glucose uptakes were higher in IR null skeletal myotubes than in wild-type skeletal myotubes. Interestingly, autophosphorylation of IGFR induced by insulin and IGF-I was markedly increased in IR null skeletal myotubes. These results indicate that, in the absence of IR, there is a compensatory increase in basal as well as in insulin- and IGF-I-induced glucose transport, the former being mediated via increased activation of the IGF-I receptor.     

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.     

A fully human recombinant IgG-like bispecific antibody to both the epidermal growth factor receptor and the insulin-like growth factor receptor for enhanced antitumor activity

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of cancers. Here we propose that simultaneous targeting of both receptors with a bispecific antibody would lead to enhanced antitumor activity. To this end, we produced a recombinant human IgG-like bispecific antibody, a Di-diabody, using the variable regions from two antagonistic antibodies: IMC-11F8 to EGFR and IMC-A12 to IGFR. The Di-diabody binds to both EGFR and IGFR and effectively blocked both EGF- and IGF-stimulated receptor activation and tumor cell proliferation. The Di-diabody also inherited the biological properties from both of its parent antibodies; it triggers rapid and significant IGFR internalization and degradation and mediates effective antibody-dependent cellular cytotoxicity in a variety of tumor cells. Finally, the Di-diabody strongly inhibited the growth of two different human tumor xenografts in vivo. Our results underscore the benefits of simultaneous targeting of two tumor targets with bispecific antibodies.     

Molecular variation and evolution of the tyrosine kinase domains of insulin receptor <Emphasis Type="Italic">IRa</Emphasis> and <Emphasis Type="Italic">IRb</Emphasis> genes in Cyprinidae

The insulin receptor (IR) gene plays an important role in regulating cell growth, differentiation and development. In the present study, DNA sequences of insulin receptor genes, IRa and IRb, were amplified and sequenced from 37 representative species of the Cyprinidae and from five outgroup species from non-cyprinid Cypriniformes. Based on coding sequences (CDS) of tyrosine kinase regions of IRa and IRb, molecular evolution and phylogenetic relationships were analyzed to better understand the characteristics of IR gene divergence in the family Cyprinidae. IRa and IRb were clustered into one lineage in the gene tree of the IR gene family, reconstructed using the unweighted pair group method with arithmetic mean (UPGMA). IRa and IRb have evolved into distinct genes after IR gene duplication in Cyprinidae. For each gene, molecular evolution analyses showed that there was no significant difference among different groups in the reconstructed maximum parsimony (MP) tree of Cyprinidae; IRa and IRb have been subjected to similar evolutionary pressure among different lineages. Although the amino acid sequences of IRa and IRb tyrosine kinase regions were highly conserved, our analyses showed that there were clear sequence variations between the tyrosine kinase regions of IRa and IRb proteins. This indicates that IRa and IRb proteins might play different roles in the insulin signaling pathway.     

Apposite insulin-like growth factor (IGF) receptor glycosylation is critical to the maintenance of vascular smooth muscle phenotype in the presence of factors promoting osteogenic differentiation and mineralization

Vascular calcification is strongly linked with increased morbidity and mortality from cardiovascular disease. Vascular calcification is an active cell-mediated process that involves the differentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. Several inhibitors of this process have been identified, including insulin-like growth factor-I (IGF-I). In this study, we examined the role of the IGF receptor (IGFR) and the importance of IGFR glycosylation in the maintenance of the VSMC phenotype in the face of factors known to promote osteogenic conversion. IGF-I (25 ng/ml) significantly protected VSMCs from β-glycerophosphate-induced osteogenic differentiation (p < 0.005) and mineral deposition (p < 0.01). Mevalonic acid depletion (induced by 100 nm cerivastatin) significantly inhibited these IGF protective effects (p < 0.01). Mevalonic acid depletion impaired IGFR processing, decreased the expression of mature IGFRs at the cell surface, and inhibited the downstream activation of Akt and MAPK. Inhibitors of N-linked glycosylation (tunicamycin, deoxymannojirimycin, and deoxynojirimycin) also markedly attenuated the inhibitory effect of IGF-I on β-glycerophosphate-induced mineralization (p < 0.05) and activation of Akt and MAPK. These results demonstrate that alterations in the glycosylation of the IGFR disrupt the ability of IGF-I to protect against the osteogenic differentiation and mineralization of VSMCs by several interrelated mechanisms: decreased IGFR processing, reduced IGFR cell-surface expression, and reduced downstream signaling via the Akt and MAPK pathways. IGF-I thus occupies a critical position in the maintenance of normal VSMC phenotype and protection from factors known to stimulate vascular calcification.     

Insulin and epidermal growth factor receptor family members share parallel activation mechanisms

Insulin receptor (IR) and the epidermal growth factor receptor (EGFR) were the first receptor tyrosine kinases (RTKs) to be studied in detail. Both are important clinical targets—in diabetes and cancer, respectively. They have unique extracellular domain compositions among RTKs, but share a common module with two ligand‐binding leucine‐rich‐repeat (LRR)‐like domains connected by a flexible cysteine‐rich (CR) domain (L1‐CR‐L2 in IR/domain, I‐II‐III in EGFR). This module is linked to the transmembrane region by three fibronectin type III domains in IR, and by a second CR in EGFR. Despite sharing this conserved ligand‐binding module, IR and EGFR family members are considered mechanistically distinct—in part because IR is a disulfide‐linked (αβ)₂ dimer regardless of ligand binding, whereas EGFR is a monomer that undergoes ligand‐induced dimerization. Recent cryo‐electron microscopy (cryo‐EM) structures suggest a way of unifying IR and EGFR activation mechanisms and origins of negative cooperativity. In EGFR, ligand engages both LRRs in the ligand‐binding module, “closing” this module to break intramolecular autoinhibitory interactions and expose new dimerization sites for receptor activation. How insulin binds the activated IR was less clear until now. Insulin was known to associate with one LRR (L1), but recent cryo‐EM structures suggest that it also engages the second LRR (albeit indirectly) to “close” the L1‐CR‐L2 module, paralleling EGFR. This transition simultaneously breaks autoinhibitory interactions and creates new receptor‐receptor contacts—remodeling the IR dimer (rather than inducing dimerization per se) to activate it. Here, we develop this view in detail, drawing mechanistic links between IR and EGFR.     

The effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody

In recent years a variety of recombinant methods have been developed for efficient production of bispecific antibodies (BsAb) in various formats. Bispecific diabody (bDAb), a 55-60 kDa molecule comprising two non-covalently associated cross-over single chain Fv (scFv) polypeptides, represents one of the most promising as well the most straightforward approaches to BsAb production. Here we constructed a bDAb, using two human scFv, 11F8 and A12, directed against the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR), respectively, as the building blocks. A total of 8 scFv and diabody constructs were prepared comprising the same two variable heavy (V(H)) and variable light (V(L)) chain domains but arranged in different orientations. V(H)/V(L) orientation, i.e., V(H)-linker-V(L) or V(L)-linker-V(H), showed significant effects on the expression and antigen-binding activity of scFv and monospecific diabody of both 11F8 and A12. Further, only 2 out of the 4 possible V(H)/V(L) orientations/arrangements in bDAb construction yielded active products that retain binding activity to both EGFR and IGFR. Both active bDAb preparations retained their original antigen-binding activity after incubation at 37 degrees C in mouse serum for up to 7 days, indicating excellent stability of the constructs. Taken together, our results underscore the importance of identifying/selecting optimal V(H)/V(L) orientation/arrangement for efficient production of active bDAb.     

Interaction of sea anemone <Emphasis Type="Italic">Heteractis crispa</Emphasis> Kunitz type polypeptides with pain vanilloid receptor TRPV1: In silico investigation

Using methods of molecular biology we defined the structures of the 31 sea anemone Heteractis crispa genes encoding polypeptides which are structurally homologous to the Kunitz protease inhibitor family. The identified sequences have single-point amino acid substitutions, a high degree of homology with sequences of known Kunitz family members from H. crispa, and represent a combinatorial library of polypeptides. We generated their three-dimensional structures by methods of homology modeling. Analysis of their molecular electrostatic potential allowed the division of the polypeptides into three clusters. One of them includes polypeptides APHC1, APHC2, and APHC3 which have been shown to possess, in addition to their trypsin inhibitory activity, a unique property of inhibiting the pain vanilloid receptor TRPV1 in vitro and providing the analgesic effects in vivo. The spatial structure of the polypeptide complexes with TRPV1, the nature of the interactions, as well as functionally important structural elements involved in the complex formation, were established by molecular docking technique. The designed models allowed us to propose a hypothesis contributing to the understanding of how APHC1-APHC3 affect the pain signals transduction by TRPV1: apparently, relaxation time of the receptor increases due to binding of its two chains with a polypeptide molecule which disrupts functioning of TRPV1 and leads to partial inhibition of the signal transduction in electrophysiological experiments.     

Cryptic plasmids from Lactobacillus helveticus and their evolutionary relationship

Abstract Three different cryptic plasmids from Lactobacillus helveticus have been identified and their DNA sequences determined. Analyses and comparisons of their primary structures revealed stretches of DNA with considerable homology. Thus, large portions of the plasmid non-coding sequences were conserved at 80–90% identity between the different plasmids identified so far in L. helveticus . Nevertheless, different plasmids found in a same host strain utilise different genes of replication, probably acquired during evolution from different replicons from Gram-positive bacterial origins. A remnant structure of such a possible genetic integration of a foreign replication gene into one of the plasmids of L. helveticus was identified.     

Prediction of a novel internal rearrangement of the insulin receptor

The insulin receptor (IR) plays critical roles in metabolism and growth, directed by the binding of insulin. Decades of research to understand the mechanism of insulin binding and activation of the IR have identified a region of the receptor, the C-terminal (CT) peptide, to be crucial for insulin binding. In particular, a truncated IR consisting of the first three domains fused to the CT peptide was found to bind insulin with nanomolar affinity, with undetectable binding in the absence of fused or soluble CT peptide. Problematically, all current crystal structures of the IR indicate the fusion point of the CT peptide to the three domains is located far from the position of the CT peptide as resolved in such structures. We have attempted to address this problem using molecular modelling and dynamics simulations. The results led to the identification of a potential inter-domain interaction between the L2 domain and the CT peptide that is not observed in any of the crystal structures of the IR. Investigations into this new interaction found a conformational change that could potentially be in response to insulin binding. Additionally, further simulation work with the new conformation demonstrated its compatibility with the position and orientation of insulin from the latest insulin-bound IR crystal structure.     

Bioinformatics Analysis Reveals Abundant Short Alpha-Helices as a Common Structural Feature of Oomycete RxLR Effector Proteins

RxLR effectors represent one of the largest and most diverse effector families in oomycete plant pathogens. These effectors have attracted enormous attention since they can be delivered inside the plant cell and manipulates host immunity. With the exceptions of a signal peptide and the following RxLR-dEER and C-terminal W/Y/L motifs identified from the sequences themselves, nearly no functional domains have been found. Recently, protein structures of several RxLRs were revealed to comprise alpha-helical bundle repeats. However, approximately half of all RxLRs lack obvious W/Y/L motifs, which are associated with helical structures. In this study, secondary structure prediction of the putative RxLR proteins was performed. We found that the C-terminus of the majority of these RxLR proteins, irrespective of the presence of W/Y/L motifs, contains abundant short alpha-helices. Since a large-scale experimental determination of protein structures has been difficult to date, results of the current study extend our understanding on the oomycete RxLR effectors in protein secondary structures from individual members to the entire family. Moreover, we identified less alpha-helix-rich proteins from secretomes of several oomycete and fungal organisms in which RxLRs have not been identified, providing additional evidence that these organisms are unlikely to harbor RxLR-like proteins. Therefore, these results provide additional information that will aid further studies on the evolution and functional mechanisms of RxLR effectors.     

Identification and characterization of an L1 family sequence with a very long open reading frame in the third intron of the human thymidylate synthase gene

A long L1 repetitive sequence (3.6 kilobase pairs) was found in the third intron of the human thymidylate synthase gene. This L1 family sequence is unique in that it possesses the longest open reading frame (1.7 kilobase pairs) of all L1 family members identified in sequences associated with specific genes that have been cloned thus far. Furthermore, the amino acid sequence deduced from the open reading frame of the L1 sequence was found to be highly homologous (90%) to that encoded by a known human teratocarcinoma L1 RNA species, and to contain several blocks of sequences homologous to ones in RNA-dependent DNA polymerases of various origins.