The insulin-like growth factor 1 receptor (IGF1R) contributes to reduced size in dogs

Domestic dog breeds have undergone intense selection for a variety of morphologic features, including size. Among small-dog breeds, defined as those averaging less than?~15 in. at the withers, there remains still considerable variation in body size. Yet essentially all such dogs are fixed for the same allele at the insulin-like growth factor 1 gene, which we and others previously found to be a size locus of large effect. In this study we sought to identify additional genes that contribute to tiny size in dogs using an association scan with the single nucleotide polymorphism (SNP) dataset CanMap, in which 915 purebred dogs were genotyped at 60,968 SNP markers. Our strongest association for tiny size (defined as breed-average height not more than 10 in. at the withers) was on canine chromosome 3 (p?=?1.9?×?10^?70). Fine mapping revealed a nonsynonymous SNP at chr3:44,706,389 that changes a highly conserved arginine at amino acid 204 to histidine in the insulin-like growth factor 1 receptor (IGF1R). This mutation is predicted to prevent formation of several hydrogen bonds within the cysteine-rich domain of the receptor??s ligand-binding extracellular subunit. Nine of 13 tiny dog breeds carry the mutation and many dogs are homozygous for it. This work underscores the central importance of the IGF1 pathway in controlling the tremendous size diversity of dogs.     

The C region of human insulin-like growth factor (IGF) I is required for high affinity binding to the type 1 IGF receptor

We have produced and characterized the binding properties of three structural analogs of human insulin-like growth factor I (hIGF-I). These analogs are [1-62]hIGF-I, an analog lacking the carboxyl-terminal 8-amino acid D region of hIGF-I; [1-27, Gly4, 38-70]hIGF-I, an analog in which residues 28-37 of the C region of hIGF-I are replaced by a 4-reside glycine bridge; and [1-27,Gly4,38-62]hIGF-I, an analog with the C region glycine replacement and a D region deletion. The removal of the D region of hIGF-I has little effect on binding to the type 1 and type 2 insulin-like growth factor (IGF) receptors. [1-62]hIGF-I has 2-fold higher affinity for the insulin receptor and 4-fold higher affinity for IGF serum-binding proteins. The replacement of the C region of hIGF-I with a four-glycine span results in a 30-fold loss of affinity for the type 1 IGF receptor. However this analog has near normal affinity for the type 2 IGF receptor, the insulin receptor, and IGF serum-binding proteins. Incorporating the C region glycine replacement and the D region deletion into one analog does not affect binding to either the type 2 receptor or to IGF serum-binding proteins. As predicted from the single deletion analogs [1-27,Gly4,38-62]hIGF-I has reduced affinity for the type 1 IGF receptor (approximately 40-fold) and increased affinity for the insulin receptor (5-fold). These data indicate that determinants in the C region of hIGF-I are involved in maintaining high affinity binding to the type 1 IGF receptor and that neither the C region nor the D region are required for high affinity binding to the type 2 IGF receptor or to IGF serum-binding proteins.     

Free-ligand accelerated dissociation of insulin-like growth factor 1 (IGF-1) from the type I IGF receptor is reduced by insulin-like growth factor binding protein 3

Insulin-like growth factor binding proteins (IGFBP) can inhibit or accentuate the mitogenic activities of insulin-like growth factor 1 (IGF-1) depending upon the experimental model employed. Inhibitory effects may be attributed to sequestration of IGF-1 onto IGFBP rather than the type I IGF receptor. We have demonstrated that the presence of IGFBP in a simple equilibrium binding assay significantly reduces the total amount of IGF-1 bound to the type I IGF receptor and increases the IC₅₀ for IGF-1 binding. On the basis of such an experiment, performed at equilibrium, IGFBP should reduce the mitogenic activity of IGF-1. Recent work has demonstrated an inverse correlation between the dissociation rate of insulin-like molecules from their receptors and their mitogenic activity. It has also been suggested that the increased rate of dissociation of insulin and IGF-1 from their receptors at increased ligand concentrations serves as a ‘dampening’ mechanism to decrease mitogenic signalling. We have demonstrated increased rates of dissociation of IGF-1 from the type I IGF receptor with increasing concentrations of IGF-1. Furthermore, IGFBP-3 inhibits the acceleration of dissociation rates due to increased IGF-1 levels. Thus, under receptor saturating conditions IGFBP-3 may act to increase mitogenesis by increasing the residence time of individual molecules of IGF-1 upon the type I IGF receptor.     

Characteristics of binding of insulin-like growth factor (IGF)-I and IGF-II analogues to the type 1 IGF receptor determined by BIAcore analysis.

Insulin-like growth factor (IGF) binding to the type 1 IGF receptor (IGF1R) elicits mitogenic effects, promotion of differentiation and protection from apoptosis. This study has systematically measured IGF1R binding affinities of IGF-I, IGF-II and 14 IGF analogues to a recombinant high-affinity form of the IGF1R using BIAcore technology. The analogues assessed could be divided into two groups: (a) those designed to investigate binding of IGF-binding protein, which exhibited IGF1R-binding affinities similar to those of IGF-I or IGF-II; (b) those generated to probe IGF1R interactions with greatly reduced IGF1R-binding affinities. The relative binding affinities of IGF-I analogues and IGF-I for the IGF1R determined by BIAcore analysis agreed closely with existing data from receptor-binding assays using cells or tissue membranes, demonstrating that BIAcore technology is a powerful tool for measuring affinities of IGFs for IGF1R. In parallel studies, IGF1R-binding affinities were related to ability to protect against serum withdrawal-induced apoptosis in three different assays including Hoechst 33258 staining, cell survival, and DNA fragmentation assays using the rat pheochromocytoma cell line, PC12. In this model system, IGF-I and IGF-II at low nanomolar concentrations are able to prevent apoptosis completely. We conclude that ability to protect against apoptosis is directly related to ability to bind the IGF1R.     

Signalling through the type 1 insulin-like growth factor receptor (IGF1R) interacts with canonical Wnt signalling to promote neural proliferation in developing brain

Signalling through the IGF1R [type 1 IGF (insulin-like growth factor) receptor] and canonical Wnt signalling are two signalling pathways that play critical roles in regulating neural cell generation and growth. To determine whether the signalling through the IGF1R can interact with the canonical Wnt signalling pathway in neural cells in vivo, we studied mutant mice with altered IGF signalling. We found that in mice with blunted IGF1R expression specifically in nestin-expressing neural cells (IGF1R^Nestin−KO mice) the abundance of neural β-catenin was significantly reduced. Blunting IGF1R expression also markedly decreased: (i) the activity of a LacZ (β-galactosidase) reporter transgene that responds to Wnt nuclear signalling (LacZ^TCF reporter transgene) and (ii) the number of proliferating neural precursors. In contrast, overexpressing IGF-I (insulin-like growth factor I) in brain markedly increased the activity of the LacZ^TCF reporter transgene. Consistently, IGF-I treatment also markedly increased the activity of the LacZ^TCF reporter transgene in embryonic neuron cultures that are derived from LacZ^TCF Tg (transgenic) mice. Importantly, increasing the abundance of β-catenin in IGF1R^Nestin−KO embryonic brains by suppressing the activity of GSK3β (glycogen synthase kinase-3β) significantly alleviated the phenotypic changes induced by IGF1R deficiency. These phenotypic changes includes: (i) retarded brain growth, (ii) reduced precursor proliferation and (iii) decreased neuronal number. Our current data, consistent with our previous study of cultured oligodendrocytes, strongly support the concept that IGF signalling interacts with canonical Wnt signalling in the developing brain to promote neural proliferation. The interaction of IGF and canonical Wnt signalling plays an important role in normal brain development by promoting neural precursor proliferation.     

Study of the differential transcription in liver of growth hormone receptor (GHR), insulin-like growth factors (IGF1, IGF2) and insulin-like growth factor receptor (IGF1R) genes at different postnatal developmental ages in pig breeds

The objective of this study was to determine hepatic expression levels of GHR, IGF1R, IGF1 and IGF2 genes in young growing gilts at different developmental ages (60–210 days) in five pig breeds: Polish Large White (PLW), Polish Landrace (PL), Pulawska (Pul), Duroc (Dur) and Pietrain (Pie). We studied the differences among pig breeds as well as within each breed for pigs in different developmental ages. Obtained results revealed major differences among breeds in hepatic gene expression of porcine GHR, IGF1R, IGF1 and IGF2 genes in different developmental ages. The differences among breeds of GHR expression were significantly higher in PLW, PL at the age of 60, 90, 120 days as compared to Pul, Dur and Pie. In turn, the highest level of IGF1R expression was observed in PL at age of 150, 180 and 210 days, whereas in case of IGF1 the highest level was recorded in Pie gilts at the age of 60 and 90 days. Moreover trait associated study revealed highly significant correlations between hepatic expressions of IGF1R and IGF2 genes and carcass composition traits (P < 0.01) The results of study suggest that porcine GHR, IGF1R, IGF1 and IGF2 genes may be potential candidate genes for postnatal growth and carcass composition traits. Therefore, the implementation of the hepatic expression of GH/IGF genes into the pig breeding and gene assisted selection program in different pig breeds should be considered. However, further population wide study is needed to clarify the hepatic expression association with economic traits, such as body growth, meat quality and carcass composition traits.     

IGF-1对细胞凋亡的抑制调控

胰岛素样生长因子-1（insulin—like growth factor,IGF—1）是胰岛素样生长因子家族中的一种,通过与IGF-1受体相结合产生生物学效应,是通过内分泌、自分泌和旁分泌的三种途径分泌的低分子多肽。近些年来,研究发现IGF-1不仅具有胰岛素类似的功能以及介导生长激素的作用,还是多种类型细胞凋亡的一个重要抑制因子。本文就IGF-1抑制细胞凋亡的信号转导途径和IGF-1对Bcl-2家族、caspases家族以及关键转录因子的调控机制作一综述。     

Analysis of polymorphisms in the insulin-like growth factor 1 receptor (IGF1R) gene from Japanese quail selected for body weight

Insulin-like growth factor 1 receptor ( IGF1R ) is essential for the signalling of growth. In this study, we performed single nucleotide polymorphism (SNP) detection in the Japanese quail IGF1R coding region and an association study between SNPs and body weight in two lines (SS and LL) selected for large and small body weight. Of 21 SNPs obtained, a SNP at position AB292766:c.2293G>A led to the replacement of a valine with an isoleucine (V765I). The two lines were fixed for alternate alleles, with allele encoding valine fixed in the LL line. A significant effect of the SNP genotype was found on 10-week body weight ( P  < 0.01) and on 4- to 10-week and 6- to 10-week average daily gain ( P  < 0.05) in the F₂ family obtained from lines LL and SS. In six populations maintained in Japan or France, the frequency of allele encoding valine was higher than the allele encoding isoleucine.     

Understanding the mechanism of insulin and insulin-like growth factor (IGF) receptor activation by IGF-II

Background

Insulin-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 Findings

In 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/Significance

This 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.     

The binding sites of insulin-like growth factor I (IGF I) to type I IGF receptor and to a monoclonal antibody. Mapping by chemical modification of tyrosine residues

The surface topography of IGF I(insulin-like growth factor I) was investigated by chemical modification of amino acid residues in free IGF I and bound to type I IGF receptor or to monoclonal antibody MAB43. Tyrosine residues were modified either by chloramine-T or lactoperoxidase catalyzed iodination. In the free IGF I molecule, all 3 tyrosine residues, A19 (Tyr-60), B25 (Tyr-24), and C2 (Tyr-31), were iodinated. Monoclonal antibody MAB43 protected IGF I against modification at tyrosine residue A19, and in the type I IGF receptor-IGF I complex, all 3 tyrosine residues were shielded against iodine incorporation. These results allow the prediction of the binding domains in the IGF I molecule. The minimal receptor binding site in IGF I would include amino acid residues B25 to C2 and, possibly, the C-terminal part of the A-domain with tyrosine residue A19.     

Association of two SNPs in the coding region of the insulin-like growth factor 1 receptor (IGF1R) gene with growth-related traits in Angus cattle

The insulin-like growth factor 1 (IGF-1) is considered to be a factor that mainly regulates growth, differentiation, and the maintenance of various function in numerous tissues through binding to a family of transmembrane tyrosine kinase receptors, signaling primarily through the insulin-like growth factor 1 receptor (IGF-1R) encoded by the IGF1R gene. The objectives of the present study were to estimate the allele and genotype frequencies of the IGF1R/MspI (silent mutation within exon 12) and the IGF1R/TaqI (within the 3′ untranslated region, 3′UTR) gene polymorphisms in beef cattle and to determine associations between these polymorphisms and growth traits. In a preliminary study on 310 Angus calves, association analyses with three production traits (birth weight, BWT; weaning weight adjusted to 210 days, WWT₂₁₀; and average daily gain, ADG) were conducted. The GG genotype of the IGF1R/e12/MspI polymorphism was significantly associated (P?≤?0.05) with a higher WWT₂₁₀ (+5.06 kg) compared to the AG genotype. Polymorphism within the 3′UTR had no significant effect on growth traits. The effect of combined genotypes was also examined. At WWT₂₁₀, calves with the GG/AA and GG/AG combinations were heavier than calves with the AG/AA and AG/AG combined genotypes (P?≤?0.05). To our knowledge, this is the first report of a polymorphism within the coding region of the Bos taurus IGF1R gene.     

Targeted morphoproteomic profiling of Ewing's sarcoma treated with insulin-like growth factor 1 receptor (IGF1R) inhibitors: response/resistance signatures

Background

Insulin-like growth factor 1 receptor (IGF1R) targeted therapies have resulted in responses in a small number of patients with advanced metastatic Ewing''s sarcoma. We performed morphoproteomic profiling to better understand response/resistance mechanisms of Ewing''s sarcoma to IGF1R inhibitor-based therapy.

Methodology/Principal Findings

This pilot study assessed two patients with advanced Ewing''s sarcoma treated with IGF1R antibody alone followed by combined IGF1R inhibitor plus mammalian target of rapamycin (mTOR) inhibitor treatment once resistance to single-agent IGF1R inhibitor developed. Immunohistochemical probes were applied to detect p-mTOR (Ser2448), p-Akt (Ser473), p-ERK1/2 (Thr202/Tyr204), nestin, and p-STAT3 (Tyr 705) in the original and recurrent tumor. The initial remarkable radiographic responses to IGF1R-antibody therapy was followed by resistance and then response to combined IGF1R plus mTOR inhibitor therapy in both patients, and then resistance to the combination regimen in one patient. In patient 1, upregulation of p-Akt and p-mTOR in the tumor that relapsed after initial response to IGF1R antibody might explain the resistance that developed, and the subsequent response to combined IGF1R plus mTOR inhibitor therapy. In patient 2, upregulation of mTOR was seen in the primary tumor, perhaps explaining the initial response to the IGF1R and mTOR inhibitor combination, while the resistant tumor that emerged showed activation of the ERK pathway as well.

Conclusion/Significance

Morphoproteomic analysis revealed that the mTOR pathway was activated in these two patients with advanced Ewing''s sarcoma who showed response to combined IGF1R and mTOR inhibition, and the ERK pathway in the patient in whom resistance to this combination emerged. Our pilot results suggests that morphoproteomic assessment of signaling pathway activation in Ewing''s sarcoma merits further investigation as a guide to understanding response and resistance signatures.     

A chimeric receptor of the insulin-like growth factor receptor type 1 (IGFR1) and a single chain antibody specific to myelin oligodendrocyte glycoprotein activates the IGF1R signalling cascade in CG4 oligodendrocyte progenitors

In order to generate neural stem cells with increased ability to survive after transplantation in brain parenchyma we developed a chimeric receptor (ChR) that binds to myelin oligodendrocyte glycoprotein (MOG) via its ectodomain and activates the insulin-like growth factor receptor type 1 ‎‎(IGF1R) signalling cascade. Activation of this pro-survival pathway in response to ligand broadly available in the brain might increase neuroregenerative potential of transplanted precursors. The ChR was produced by fusing a MOG-specific single ‎chain antibody with the extracellular boundary of the IGF1R transmembrane segment. The ChR is expressed on the cellular surface, predominantly as a monomer, and is not N-glycosylated. To show MOG-dependent functionality of the ChR, neuroblastoma cells B104 expressing this ChR were stimulated with monolayers of cells expressing recombinant MOG. The ChR undergoes MOG-dependent tyrosine phosphorylation and homodimerisation. It promotes insulin and IGF-independent growth of the oligodendrocyte progenitor cell line CG4. The proposed mode of the ChR activation is by MOG-induced dimerisation which promotes kinase domain transphosphorylation, by-passing the requirement of conformation changes known to be important for IGF1R activation. Another ChR, which contains a segment of the β-chain ectodomain, was produced in an attempt to recapitulate some of these conformational changes, but proved non-functional.     

The insulin-like growth factor 1 (IGF-1) receptor is a substrate for gamma-secretase-mediated intramembrane proteolysis

Several type-1 membrane proteins undergo regulated intramembrane proteolysis resulting in the generation of biologically active protein fragments. Presenilin-dependant gamma-secretase activity is central to this event and includes amyloid precursor protein (APP), Notch and ErbB4 as substrates. Here we show that the insulin-like growth factor 1 receptor (IGF-IR) undergoes regulated intramembrane proteolysis. A metalloprotease-dependant ectodomain-shedding event generates a approximately 52 kDa IGF-IR-carboxyl terminal domain (CTD). The IGF-IR-CTD is consequentially a substrate for gamma-secretase cleavage, liberating a approximately 50 kDa intracellular domain (ICD) that can be inhibited by a specific gamma-secretase inhibitor. This study suggests that the IGF-IR is a substrate for gamma-secretase and may mediate a function independent of its role as a receptor tyrosine kinase.     

Insulin-like growth factor-I is internalized after binding to the type I insulin-like growth factor receptor

Receptor-mediated endocytosis may represent an important mechanism whereby peptide hormones exert their biological effects. The ability of recombinant insulin-like growth factor (IGF)-I to be internalized by cultured cells was evaluated in BRL-3A2 cells, a rat liver-derived cell line which lacks insulin receptors. Since recombinant IGF-I does not bind to the Type II IGF receptor, all specific binding of 125I-IGF-I in BRL-3A2 cells represents binding to the Type I receptor. Exposure of BRL-3A2 cells to IGF-I resulted in a rapid 50% downregulation of Type I IGF receptors. Only one-half of these binding sites were sensitive to treatment with trypsin, a phenomenon which indicates that the peptide and its receptor were internalized after the cells were exposed to IGF-I. In conclusion, these experiments demonstrate that IGF-I can be internalized by cultured cells via the Type I IGF receptor, and suggest that IGF hormone action may be exerted by receptor-mediated endocytosis.