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

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.

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

Biochemical Journal ◽

10.1042/bj2900419 ◽

1993 ◽

Vol 290 (2) ◽

pp. 419-426 ◽

Cited By ~ 178

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.

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

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.267.1.g105 ◽

1994 ◽

Vol 267 (1) ◽

pp. G105-G114 ◽

Cited By ~ 1

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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Functional Epitope Mapping of Insulin-Like Growth Factor I (IGF-I) by Anti-IGF-I Monoclonal Antibodies*

Endocrinology ◽

10.1210/endo.138.3.4965 ◽

1997 ◽

Vol 138 (3) ◽

pp. 905-915 ◽

Cited By ~ 9

Author(s):

Santos Mañes ◽

Leonor Kremer ◽

Juan Pablo Albar ◽

Catherine Mark ◽

Rafael Llopis ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Growth Factor ◽

Epitope Mapping ◽

Insulin Like Growth Factor ◽

Factor I ◽

Igf I ◽

Growth Factor I

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

Biochemical Journal ◽

10.1042/bj3050981 ◽

1995 ◽

Vol 305 (3) ◽

pp. 981-986 ◽

Cited By ~ 41

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.

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Physical mapping of human insulin-like growth factor-I using specific monoclonal antibodies

Journal of Endocrinology ◽

10.1677/joe.0.1540293 ◽

1997 ◽

Vol 154 (2) ◽

pp. 293-302 ◽

Cited By ~ 4

Author(s):

S Mañes ◽

L Kremer ◽

B Vangbo ◽

A López ◽

C Gómez-Mouton ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Growth Factor ◽

Growth Control ◽

Amino Acid Sequences ◽

Insulin Like Growth Factor ◽

Factor I ◽

Igf I ◽

Growth Factor I ◽

Α Helix ◽

Cell Growth Control

Abstract The primary structure of recombinant human (h) insulin-like growth factor-I (IGF-I) epitopes recognized by a panel of 28 monoclonal antibodies (mAbs) is characterized. Pairwise mAb epitope mapping defines eight 'epitopic clusters' (I–VIII) which cover nearly the entire solvent-exposed IGF-I surface. Monoclonal antibody reactivity with 32 overlapping synthetic peptides and with IGF-I mutants is used to associate these epitopic clusters with the probable primary IGF-I sequences recognized. Epitopic cluster I involves residues in the C-domain and the first α-helix of the A-domain; clusters II, V and VII involve principally the B-domain; clusters III and IV map to amino acid sequences (55–70) and (1–13) respectively; cluster VI includes the A- and B-domains; and cluster VIII involves mainly the C-terminal part of the B-domain. Data indicate that this mAb panel defines 14 distinct IGF-I epitopes. The specific inhibition of HEL 92.1.7 IGF-I-promoted proliferation by these mAbs was explored. Direct correlation between mAb affinity and inhibitory activity was observed except in the case of clusters III- and VII-specific mAbs. Finally, the combination of epitopic cluster I and II mAbs detect 0·5–10 ng/ml hIGF-I in a sandwich immunoassay, with no IGF-II crossreactivity. These anti-IGF-I mAbs are, therefore, useful for both the inhibition of IGF-I mitogenic activity and for the quantification of this growth factor. The potential use of this mAb panel in tumor cell growth control is discussed. Journal of Endocrinology (1997) 154, 293–302

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Dissection of the growth versus metabolic effects of insulin and insulin-like growth factor-I in transfected cells expressing kinase-defective human insulin receptors.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)40069-0 ◽

1990 ◽

Vol 265 (3) ◽

pp. 1678-1682

Author(s):

D A McClain ◽

H Maegawa ◽

R S Thies ◽

J M Olefsky

Keyword(s):

Growth Factor ◽

Human Insulin ◽

Insulin Receptors ◽

Metabolic Effects ◽

Insulin Like Growth Factor ◽

Transfected Cells ◽

Factor I ◽

Growth Factor I

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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

Biochemical Journal ◽

10.1042/bj2630553 ◽

1989 ◽

Vol 263 (2) ◽

pp. 553-563 ◽

Cited By ~ 124

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.

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Evidence for separate receptors for insulin and insulin-like growth factor-I in choroid plexus of rat brain by quantitative autoradiography.

Journal of Histochemistry & Cytochemistry ◽

10.1177/38.9.2167329 ◽

1990 ◽

Vol 38 (9) ◽

pp. 1289-1294 ◽

Cited By ~ 19

Author(s):

D A Davidson ◽

N J Bohannon ◽

E S Corp ◽

D P Lattemann ◽

S C Woods ◽

...

Keyword(s):

Growth Factor ◽

Choroid Plexus ◽

Binding Sites ◽

Porcine Insulin ◽

Image Analysis System ◽

Insulin Like Growth Factor ◽

Factor I ◽

High Affinity ◽

Igf I ◽

Growth Factor I

Binding of insulin and insulin-like growth factor-I (IGF-I) to the choroid plexus was quantitatively characterized using autoradiography and computer densitometry. Slide-mounted brain slices were incubated in 0.1 nM [125I]-insulin or [125I]-[Thr59]IGF-I. To determine specificity of the binding sites, the labeled peptides were mixed with unlabeled analogues. Autoradiography was done with LKB Ultrofilm and analyzed with a computer image analysis system and program for densitometry. Results showed that binding was time and temperature dependent and reversible. Binding of the iodinated insulin and IGF-I was inhibited by unlabeled peptides in a dose-dependent manner. The rank order of potency of these peptides in competing for the choroid plexus iodoinsulin binding sites was: chicken insulin greater than porcine insulin greater than desoctapeptide insulin greater than IGF-I. IGF-I was more potent than porcine insulin in competing for the choroid plexus iodolGF-I binding sites. Somatostatin was ineffective. Non-linear regression analysis revealed the presence of high- (Kd 1.3 +/- 0.2 nM) and low-affinity (Kd 36 +/- 1.4 nM) binding sites for insulin and a single high-affinity binding site (Kd 3.1 +/- 0.3 nM) for IGF-I in the choroid plexus. There were approximately 50 times more binding sites (Bmax) for IGF-I than for insulin high-affinity sites, whereas the number of low-affinity sites for insulin was about equal to the number of IGF-I high-affinity sites. The results of these binding studies with iodinated insulin and [Thr59]IGF-I support the conclusion that the rat choroid plexus has separate high-affinity receptors for insulin and IGF-I, and that the IGF-I receptors outnumber the insulin receptors.

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Specific immunoradiometric assay of insulin-like growth factor I with use of monoclonal antibodies.

Clinical Chemistry ◽

10.1093/clinchem/33.11.2019 ◽

1987 ◽

Vol 33 (11) ◽

pp. 2019-2023 ◽

Cited By ~ 3

Author(s):

M G Scott ◽

G C Cuca ◽

J R Petersen ◽

L R Lyle ◽

B D Burleigh ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Growth Factor ◽

Cross Reactivity ◽

Immunoradiometric Assay ◽

Insulin Like Growth Factor ◽

Factor I ◽

Igf I ◽

Growth Factor I ◽

Substantial Bias ◽

Growth Abnormalities

Abstract We identified two monoclonal antibodies that bind spatially distinct epitopes on insulin-like growth factor I (IGF-I). Using these two antibodies, we developed a simultaneous, two-site immunoradiometric assay (IRMA) specific for IGF-I. This IRMA has no detectable cross reactivity with insulin, proinsulin, prolactin, or somatotropin, and less than 2% crossreactivity with IGF-II. The assay response varies linearly with IGF-I concentrations of 0-800 micrograms/L in serum; the detection limit is about 10 micrograms/L. A comparison of 26 IGF-I serum values from the IRMA and from a previously reported IGF-I specific RIA gave a correlation coefficient of 0.96 with no substantial bias (slope = 1.10). IGF-I values for serum, as an aid in assessing growth abnormalities, are easily (only three pipetting steps) obtained in less than 4 h.

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