scholarly journals A single-chain insulin-like growth factor I/insulin hybrid binds with high affinity to the insulin receptor

1995 ◽  
Vol 305 (3) ◽  
pp. 981-986 ◽  
Author(s):  
C Kristensen ◽  
A S Andersen ◽  
M Hach ◽  
F C Wiberg ◽  
L Schäffer ◽  
...  
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Insulin Receptors ◽  
Insulin Like Growth Factor ◽  
Single Chain ◽  
Factor I ◽  
High Affinity ◽  
Igf I ◽  
Membrane Bound ◽  
Single Chain Insulin

1. To investigate the structure/function relationship of the interaction between ligand and receptor in the insulin-like growth factor I (IGF-I) and insulin receptor systems we have prepared and characterized a single-chain insulin/IGF-I hybrid. The single-chain hybrid consists of the insulin molecule combined with the C domain of IGF-I. The single-chain hybrid was found to bind with high affinity to both truncated soluble insulin receptors and membrane-bound holoreceptors. The affinity for interacting with the soluble truncated insulin receptors was 55-94% relative to insulin, and affinity for membrane-bound insulin receptors was 113% of that of insulin. Furthermore we found that the affinity of the single-chain hybrid molecule for IGF-I receptors was 19-28% relative to IGF-I. 2. The affinity of the single-chain hybrid for chimeric insulin/IGF-I receptors exceeded that of either natural ligand. This indicates that coordinately changing domains of the receptors and the ligands can induce higher affinity of ligand for receptor, supporting the idea that these receptors have a common ligand-binding site [Kjeldsen, Andersen, Wiberg, Rasmussen, Schäffer, Balschmidt, Møller and Møller (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4404-4408]. 3. In contrast with what was generally assumed about the ligand structure required for binding to the insulin receptor we demonstrate the first single-chain insulin analogue that can bind with high affinity to the insulin receptor.

scholarly journals Receptors for insulin and insulin-like growth factor-I can form hybrid dimers. Characterisation of hybrid receptors in transfected cells

1990 ◽  
Vol 270 (2) ◽  
pp. 383-390 ◽  
Author(s):  
M A Soos ◽  
J Whittaker ◽  
R Lammers ◽  
A Ullrich ◽  
K Siddle
Keyword(s):  
Monoclonal Antibodies ◽  
Growth Factor ◽  
Insulin Receptors ◽  
Insulin Like Growth Factor ◽  
Transfected Cells ◽  
Factor I ◽  
High Affinity ◽  
Igf I ◽  
Growth Factor I ◽  
Nih 3T3

We have demonstrated the formation of hybrid insulin/insulin-like growth factor-I(IGF-I) receptors in transfected rodent fibroblasts, which overexpress human receptors, by examining reactivity with species- and receptor-specific monoclonal antibodies. In NIH 3T3 and Rat 1 fibroblasts, endogenous IGF-I receptors were unreactive with anti-(human insulin receptor)monoclonal antibodies (47-9, 25-49, 83-14, 83-7, 18-44). However, in transfected cells expressing high levels of insulin receptors, 60-80% of high-affinity IGF-I receptors reacted with these antibodies, as assessed either by inhibition of ligand binding in intact cells or by precipitation of solubilized receptors. Conversely, endogenous insulin receptors in NIH 3T3 cells were unreactive with anti-(IGF-I receptor) antibodies alpha IR-3 and 16-13. However, approx. 50% of high-affinity insulin receptors reacted with these antibodies in cells expressing high levels of human IGF-I receptors. The hybrid receptors in transfected cells bound insulin or IGF-I with high affinity. However, responses to these ligands were asymmetrical, in that binding of IGF-I inhibited subsequent binding of insulin, but prior binding of insulin did not affect the affinity for IGF-I. The existence of hybrid receptors in normal tissues could have important implications for metabolic regulation by insulin and IGF-I.

scholarly journals Immunological relationships between receptors for insulin and insulin-like growth factor I. Evidence for structural heterogeneity of insulin-like growth factor I receptors involving hybrids with insulin receptors

1989 ◽  
Vol 263 (2) ◽  
pp. 553-563 ◽  
Author(s):  
M A Soos ◽  
K Siddle
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Insulin Receptors ◽  
Insulin Like Growth Factor ◽  
Intact Cells ◽  
Receptor Complexes ◽  
Factor I ◽  
Igf I ◽  
Alpha Beta ◽  
Growth Factor I

The receptors for insulin and insulin-like growth factor-I (IGF-I) are closely related in primary sequence and overall structure. We have examined the immunological relationships between these receptors by testing the reactivity of anti-(insulin receptor) monoclonal antibodies with IGF-I receptors in various tissues and cell lines. Antibodies for six distinct epitopes reacted with a subfraction of IGF-I receptors, as shown by inhibition of 125I-IGF-I binding, precipitation of 125I-IGF-I-receptor complexes or immunodepletion of receptor from tissue extracts before binding assays. Both immunoreactive and non-immunoreactive subfractions displayed the expected properties of ‘classical’ IGF-I receptors, in terms of relative affinities for IGF-I and insulin. The proportion of total IGF-I receptors which was immunoreactive varied in different cell types, being approx. 40% in Hep G2 cells, 35-40% in placental membranes and 75-85% in IM-9 cells. The immunoreactive fraction was somewhat higher in solubilized receptors than in the corresponding intact cells or membranes. A previously described monoclonal antibody, alpha-IR-3, specific for IGF-I receptors, inhibited IGF-I binding by more than 80% in all preparations. When solubilized placental receptors were pretreated with dithiothreitol (DTT) under conditions reported to reduce intramolecular (class I) disulphide bonds, the immunoreactivity of IGF-I receptors was abolished although total IGF-I binding was little affected. Under the same conditions insulin receptors remained fully immunoreactive. When solubilized receptor preparations were fractionated by gel filtration, both IGF-I and insulin receptors ran as symmetrical peaks of identical mobility. After DTT treatment, the IGF-I receptor was partially converted to a lower molecular mass form which was not immunoreactive. The insulin receptor peak showed a much less pronounced skewing and remained fully immunoreactive in all fractions. It is concluded that the anti- (insulin receptor) antibodies do not react directly with IGF-I receptor polypeptide, and that the apparent immunoreactivity of a subfraction of IGF-I receptors reflects their physical association with insulin receptors, both in cell extracts and in intact cells. The most likely basis for this association appears to be a ‘hybrid’ receptor containing one half (alpha beta) of insulin receptor polypeptide and the other (alpha‘beta’) of IGF-I receptor polypeptide within the native (alpha beta beta‘alpha’) heterotetrameric structure.

scholarly journals Differential Activation of Insulin Receptor Substrates 1 and 2 by Insulin-Like Growth Factor-Activated Insulin Receptors

2007 ◽  
Vol 27 (10) ◽  
pp. 3569-3577 ◽  
Author(s):  
Adam Denley ◽  
Julie M. Carroll ◽  
Gemma V. Brierley ◽  
Leah Cosgrove ◽  
John Wallace ◽  
...  
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Splice Variants ◽  
Biological Effects ◽  
Insulin Receptors ◽  
Insulin Like Growth Factor ◽  
Extracellular Signal ◽  
Igf I ◽  
High Concentrations ◽  
Kinase Pathway

ABSTRACT The insulin-like growth factors (insulin-like growth factor I [IGF-I] and IGF-II) exert important effects on growth, development, and differentiation through the IGF-I receptor (IGF-IR) transmembrane tyrosine kinase. The insulin receptor (IR) is structurally related to the IGF-IR, and at high concentrations, the IGFs can also activate the IR, in spite of their generally low affinity for the latter. Two mechanisms that facilitate cross talk between the IGF ligands and the IR at physiological concentrations have been described. The first of these is the existence of an alternatively spliced IR variant that exhibits high affinity for IGF-II as well as for insulin. A second phenomenon is the ability of hybrid receptors comprised of IGF-IR and IR hemireceptors to bind IGFs, but not insulin. To date, however, direct activation of an IR holoreceptor by IGF-I at physiological levels has not been demonstrated. We have now found that IGF-I can function through both splice variants of the IR, in spite of low affinity, to specifically activate IRS-2 to levels similar to those seen with equivalent concentrations of insulin or IGF-II. The specific activation of IRS-2 by IGF-I through the IR does not result in activation of the extracellular signal-regulated kinase pathway but does induce delayed low-level activation of the phosphatidylinositol 3-kinase pathway and biological effects such as enhanced cell viability and protection from apoptosis. These findings suggest that IGF-I can function directly through the IR and that the observed effects of IGF-I on insulin sensitivity may be the result of direct facilitation of insulin action by IGF-I costimulation of the IR in insulin target tissues.

scholarly journals Purified hybrid insulin/insulin-like growth factor-I receptors bind insulin-like growth factor-I, but not insulin, with high affinity

1993 ◽  
Vol 290 (2) ◽  
pp. 419-426 ◽  
Author(s):  
M A Soos ◽  
C E Field ◽  
K Siddle
Keyword(s):  
Monoclonal Antibody ◽  
Growth Factor ◽  
Insulin Receptors ◽  
Beta Subunit ◽  
Type I ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Igf Receptors ◽  
Igf I ◽  
Growth Factor I

Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.

scholarly journals Role of the Insulin-Like Growth Factor I/Insulin Receptor Substrate 1 Axis in Rad51 Trafficking and DNA Repair by Homologous Recombination

2003 ◽  
Vol 23 (21) ◽  
pp. 7510-7524 ◽  
Author(s):  
Joanna Trojanek ◽  
Thu Ho ◽  
Luis Del Valle ◽  
Michal Nowicki ◽  
Jin Ying Wang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Growth Factor ◽  
Homologous Recombination ◽  
Insulin Receptor ◽  
Insulin Receptor Substrate ◽  
Insulin Like Growth Factor ◽  
Insulin Receptor Substrate 1 ◽  
Factor I ◽  
Igf I

ABSTRACT The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome.

scholarly journals Insulin receptor is phosphorylated in response to treatment of HepG2 cells with insulin-like growth factor I

1990 ◽  
Vol 270 (1) ◽  
pp. 27-32 ◽  
Author(s):  
V Duronio
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Hepg2 Cells ◽  
Insulin Binding ◽  
Response To Treatment ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I ◽  
In Cells

1. Binding of insulin and insulin-like growth factor I (IGF-I) to HepG2 cells was analysed with regard to competition by both insulin and IGF-I. At concentrations of insulin that caused maximal phosphorylation of the insulin receptor, virtually no displacement of IGF-I binding was observed. Similarly, at concentrations of IGF-I that caused maximal phosphorylation of the IGF-I receptor, no displacement of insulin binding was observed. 2. When the phosphorylation of both receptors was examined individually by using specific monoclonal antibodies to immunoprecipitate the receptors, phosphorylation of the insulin receptor was found to increase on both serine and tyrosine residues in cells treated with 100 ng of IGF-I/ml. In contrast, no increased phosphorylation of IGF-I receptor was observed in cells treated with 100 ng of insulin/ml. 3. The increase in phosphorylation of insulin receptor in response to IGF-I correlated with the dose-response of IGF-I-stimulated phosphorylation of the IGF-I receptor. 4. The IGF-I-stimulated phosphorylation of the insulin receptor could be blocked by preincubation with a monoclonal antibody that blocks IGF-I binding to the IGF-I receptor.

scholarly journals Delineation of atypical insulin receptors from classical insulin and type I insulin-like growth factor receptors in human placenta

1989 ◽  
Vol 257 (1) ◽  
pp. 101-107 ◽  
Author(s):  
H A Jonas ◽  
A J Cox ◽  
L C Harrison
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Human Placenta ◽  
Insulin Receptors ◽  
Type I ◽  
Insulin Like Growth Factor ◽  
Binding Properties ◽  
High Affinity ◽  
Igf Receptors ◽  
Placental Membranes

Insulin-like growth factor (IGF)-binding sites copurifying with human placental insulin receptors during insulin-affinity chromatography consist of two immunologically distinct populations. One reacts with monoclonal antibody alpha IR-3, but not with antibodies to the insulin receptor, and represents Type I IGF receptors; the other reacts only with antibodies to the insulin receptor and is precipitated with a polyclonal receptor antibody (B-10) after labelling with 125I-multiplication-stimulating activity (MSA, rat IGF-II). The latter is a unique sub-population of atypical insulin receptors which differ from classical insulin receptors by their unusually high affinity for MSA (Ka = 2 x 10(9) M-1 compared with 5 x 10(7) M-1) and relative potencies for insulin, MSA and IGF-I (40:5:1 compared with 150:4:1). They represent 10-20% of the total insulin receptor population and account for 25-50% of the 125I-MSA binding activity in Triton-solubilized placental membranes. Although atypical and classical insulin receptors are distinct, their immunological properties are very similar, as are their binding properties in response to dithiothreitol, storage at -20 degrees C and neuraminidase digestion. We conclude that atypical insulin receptors with moderately high affinity for IGFs co-exist with classical insulin receptors and Type I IGF receptors in human placenta. They provide an explanation for the unusual IGF-II binding properties of human placental membranes and may have a specific role in placental growth and/or function.

Characterization of insulin-like growth factor I receptors in human esophageal epithelial cells

1994 ◽  
Vol 267 (1) ◽  
pp. G105-G114 ◽  
Author(s):  
R. Vinayek ◽  
L. S. Pichney ◽  
U. Tantry ◽  
S. K. Dutta ◽  
J. Resau ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
High Capacity ◽  
Cross Linking ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
High Affinity ◽  
Igf I ◽  
Growth Factor I ◽  
Mediate Cell

In the present study we used 125I-labeled insulin-like growth factor I (125I-IGF-I) to identify and characterize IGF-I receptors in the well-characterized and propagable human esophageal epithelial (HEE) cell line and to characterize their role in cell growth. Binding of 125I-IGF-I was saturable, time and temperature dependent, reversible, and specific for IGF-I and related peptides. Scatchard analysis of binding data demonstrated that HEE cells possess two classes of IGF-I receptors: high affinity [dissociation constant (Kd) = 0.058 nM] and low capacity (13,870 receptors/cell), and low affinity (Kd = 2.2 nM) and high capacity (39,000 receptors/cell). Binding of 125I-IGF-I was inhibited with the following relative potencies (half-maximal inhibition): IGF-I (3.0 pM) > IGF-II (1.2 mM) >> insulin (1.0 microM). Affinity cross-linking of cell membranes using disuccinimidyl suberate as a cross-linking agent under reducing conditions revealed a single polypeptide band (relative mol wt 133,000) representing the alpha-subunit of the IGF-I receptor. IGF-I stimulated [3H]thymidine incorporation and cell proliferation in a dose-dependent manner with detectable effect observed with 0.5 nM IGF-I and maximal effect at 50 nM IGF-I. IGF-I occupation of low-affinity IGF-I receptors appears to mediate cell growth. The present results demonstrate that HEE cells possess two classes of IGF-I receptors: one class has a high affinity and low capacity and the other has a low affinity and high capacity for IGF-I. Occupation of low-affinity IGF-I receptors by IGF-I appears to mediate cell growth.

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