scholarly journals Insulin and insulin-like-growth-factor-I (IGF-I) receptors in Xenopus laevis oocytes. Comparison with insulin receptors from liver and muscle

1991 ◽  
Vol 273 (3) ◽  
pp. 673-678 ◽  
Author(s):  
P Hainaut ◽  
A Kowalski ◽  
S Giorgetti ◽  
V Baron ◽  
E Van Obberghen
Keyword(s):  
Growth Factor ◽  
Xenopus Laevis ◽  
Insulin Receptors ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Xenopus Laevis Oocytes ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Alpha Subunits ◽  
Igf I

Insulin and insulin-like-growth-factor-I (IGF-I) receptors were partially purified from full-grown (stages V-VI) Xenopus laevis oocytes by affinity chromatography on wheat-germ agglutinin-agarose. Competitive-binding assays revealed high-affinity binding sites for both insulin and IGF-I (Kd = 2.5 x 10(-10) M and 8 x 10(-10) M respectively). However, IGF-I receptors were about 15 times more abundant than insulin receptors (22.5 x 10(11) versus 1.5 x 10(11)/mg of protein). Moreover, comparison of intact and collagenase-treated oocytes showed that most of the insulin receptors were in the oocyte envelopes, whereas IGF-I receptors were essentially at the oocyte surface. Oocyte receptors were composed of alpha-subunits of approximately 130 kDa and a doublet of beta-subunits of 95 and 105 kDa, which both had ligand-induced phosphorylation patterns compatible with IGF-I receptor beta-subunits. Accordingly, the receptor tyrosine kinase was stimulated at low IGF-I concentrations [half-maximally effective concentration (EC50) approximately 0.5-1 nM], and at higher insulin concentrations (EC50 approximately 20-50 nM). Partially purified glycoproteins from Xenopus liver and muscle contained mainly receptors of the insulin-receptor type, with alpha-subunits of 140 kDa in liver and 125 kDa in muscle, and doublets of beta-subunits of 92-98 kDa in liver and 85-94 kDa in muscle. Immunoprecipitation of receptors from oocytes, liver and muscle by receptor-specific anti-peptide antibodies suggested that the beta-subunit heterogeneity resulted from the existence of two distinct IGF-I receptors in oocytes and of two distinct insulin receptors in both liver and muscle. In the different tissues, the two receptor subtypes differed at least by their beta-subunit C-terminal region.

Effects of insulin, insulin-like growth factor-I (IGF-I) and progesterone on glucose and amino acid uptake in Xenopus laevis oocytes

1991 ◽  
Vol 75 (2) ◽  
pp. 133-139 ◽  
Author(s):  
Pierre Hainaut ◽  
Aline Kowalski ◽  
Yannick Le Marchand-Brustel ◽  
Sophie Giorgetti ◽  
Nadine Gautier ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Xenopus Laevis ◽  
Amino Acid Uptake ◽  
Xenopus Laevis Oocytes ◽  
Acid Uptake ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I

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 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 Increased basal and induced tyrosine phosphorylation of the insulin- like growth factor I receptor beta subunit in circulating mononuclear cells of patients with polycythemia vera

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 877-882 ◽  
Author(s):  
AM Mirza ◽  
PN Correa ◽  
AA Axelrad
Keyword(s):  
Growth Factor ◽  
Polycythemia Vera ◽  
Tyrosine Phosphorylation ◽  
Mononuclear Cells ◽  
Beta Subunit ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I ◽  
Receptor Beta

We have previously shown that circulating progenitor cells in patients with polycythemia vera (PV) are hypersensitive to insulin-like growth factor I (IGF-I) with respect to erythroid burst formation in serum- free medium, and that this effect occurs through the IGF-I receptor. To investigate the molecular basis of this IGF-I hypersensitivity phenomenon, we examined tyrosine phosphorylation of the IGF-I receptor beta subunit in peripheral blood mononuclear cells (PBMNC) from eight PV patients and six normals. Cells were exposed to IGF-I at concentrations of 10(-8) and 10(-10) mol/L for 0, 1, 3, and 10 minutes, and then lysed. The IGF-I receptor beta subunit was immunoprecipitated, and the protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted with antiphosphotyrosine antibody (4G10). We found that, in the absence of exogenous IGF-I, there was a basal level of tyrosine phosphorylation of the IGF-I receptor beta subunit, and it was substantially greater in PV than in normal. At 10(-10) mol/L IGF-I in normals, no evidence of increased tyrosine phosphorylation was detected; however in PV, a pronounced increase in tyrosine phosphorylation was observed at both 10(-10) and 10(-8) mol/L IGF-I, and it occurred earlier and attained a higher level than in normal. In contrast, in PBMNC from three patients with erythrocytosis, no significant increase above normal was seen in either basal or induced tyrosine phosphorylation of the IGF-I receptor beta subunit. Thus, our findings show two distinctive features of the PV phenotype in PBMNC: (1) an increased basal tyrosine phosphorylation of the IGF-I receptor beta subunit, and (2) a hypersensitive and hyperresponsive receptor with respect to tyrosine phosphorylation. These features may influence the ability of the receptor to transmit a proliferative signal; thus, they may play a role in the pathogenesis of PV.

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 Increased basal and induced tyrosine phosphorylation of the insulin- like growth factor I receptor beta subunit in circulating mononuclear cells of patients with polycythemia vera

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 877-882 ◽  
Author(s):  
AM Mirza ◽  
PN Correa ◽  
AA Axelrad
Keyword(s):  
Growth Factor ◽  
Polycythemia Vera ◽  
Tyrosine Phosphorylation ◽  
Mononuclear Cells ◽  
Beta Subunit ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I ◽  
Receptor Beta

Abstract We have previously shown that circulating progenitor cells in patients with polycythemia vera (PV) are hypersensitive to insulin-like growth factor I (IGF-I) with respect to erythroid burst formation in serum- free medium, and that this effect occurs through the IGF-I receptor. To investigate the molecular basis of this IGF-I hypersensitivity phenomenon, we examined tyrosine phosphorylation of the IGF-I receptor beta subunit in peripheral blood mononuclear cells (PBMNC) from eight PV patients and six normals. Cells were exposed to IGF-I at concentrations of 10(-8) and 10(-10) mol/L for 0, 1, 3, and 10 minutes, and then lysed. The IGF-I receptor beta subunit was immunoprecipitated, and the protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotted with antiphosphotyrosine antibody (4G10). We found that, in the absence of exogenous IGF-I, there was a basal level of tyrosine phosphorylation of the IGF-I receptor beta subunit, and it was substantially greater in PV than in normal. At 10(-10) mol/L IGF-I in normals, no evidence of increased tyrosine phosphorylation was detected; however in PV, a pronounced increase in tyrosine phosphorylation was observed at both 10(-10) and 10(-8) mol/L IGF-I, and it occurred earlier and attained a higher level than in normal. In contrast, in PBMNC from three patients with erythrocytosis, no significant increase above normal was seen in either basal or induced tyrosine phosphorylation of the IGF-I receptor beta subunit. Thus, our findings show two distinctive features of the PV phenotype in PBMNC: (1) an increased basal tyrosine phosphorylation of the IGF-I receptor beta subunit, and (2) a hypersensitive and hyperresponsive receptor with respect to tyrosine phosphorylation. These features may influence the ability of the receptor to transmit a proliferative signal; thus, they may play a role in the pathogenesis of PV.

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 Distinct β-subunits are present in hybrid insulin-like-growth-factor-1 receptors in the central nervous system

1993 ◽  
Vol 294 (3) ◽  
pp. 685-692 ◽  
Author(s):  
A M Moss ◽  
J N Livingston
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Insulin Receptors ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Insulin Like Growth Factor ◽  
Binding Studies ◽  
Specific Sequence ◽  
Adult Rat ◽  
The Brain

Previous work suggests the existence of different isoforms of the insulin-like-growth-factor-1 (IGF-1) receptor in various tissues. In the present study we provide support for the concept that heterogeneous IGF-1 receptors exist in the brain and that part of the heterogeneity is derived from IGF-1 receptor hybrids formed from different beta-subunits. IGF-1 receptors were extracted from adult-rat forebrain synaptosomes and partially purified by wheat-germ agglutinin (WGA) chromatography. Hormone-binding studies in this preparation demonstrate the presence of receptors for IGF-1 and insulin. An antibody, a-RIR, specific for the rat insulin receptor was used to remove insulin receptors from the WGA extract. Studies with the immunodepleted material demonstrated two proteins of 92 and 99 kDa that are phosphorylated on tyrosine during incubation with low concentrations of IGF-1. Both proteins bound with high affinity and specificity to IGF-1 immobilized on agarose, and each underwent phosphorylation when the agarose beads were incubated with [gamma-32P]ATP and MnCl2. Two-dimensional phosphopeptide maps after exhaustive trypsin treatment of the two proteins showed significant differences in their structure as well as differences from the phosphopeptide map for the beta-subunit of the insulin receptor. The relationship of the two proteins to the IGF-1 receptor was further probed by an antibody (a-HF) raised against a specific sequence in the beta-subunit of the human IGF-1 receptor, and a polyclonal antibody raised against the liver insulin receptor (L1) which cross-reacts with the IGF-1 receptor. Both antibodies immunoprecipitated the two phosphorylated proteins. However, reduction of the receptors to form receptor dimers or monomers showed that a-HF precipitated only the 99 kDa protein, whereas L1 precipitated primarily the 92 kDa protein. In conclusion, the brain IGF-1 receptor apparently has two structurally different beta-subunits, one of 92 kDa and a second of 99 kDa. Interestingly, at least a portion of the IGF-1 receptor population has both isoforms in the same receptor.

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