scholarly journals Insulin-like growth factor binding to the atypical insulin receptors of a human lymphoid-derived cell line (IM-9)

1990 ◽  
Vol 266 (3) ◽  
pp. 737-742 ◽  
Author(s):  
H A Jonas ◽  
A J Cox
Keyword(s):  
Growth Factor ◽  
Affinity Chromatography ◽  
Insulin Receptor ◽  
Binding Sites ◽  
Insulin Binding ◽  
Type I ◽  
Insulin Like Growth Factor ◽  
Igf Receptors ◽  
Type I Igf Receptor ◽  
Igf I

The cells of the IM-9 human lymphocyte-derived line contain a sub-population of insulin-binding sites whose immunological and hormone-binding characteristics closely resemble those of the atypical insulin-binding sites of human placenta. These binding sites, which have moderately high affinity for multiplication-stimulating activity [MSA, the rat homologue of insulin-like growth factor (IGF) II] and IGF-I, are identified on IM-9 cells by 125I-MSA binding. They account for approximately 30% of the total insulin-receptor population, and do not react with a monoclonal antibody to the type I IGF receptor (alpha IR-3). The relative concentrations of unlabelled insulin, MSA and IGF-I required to displace 50% of 125I-MSA from these binding sites (1:4.7:29 respectively) are maintained for cells, particulate membranes, Triton-solubilized membranes precipitated either by poly(ethylene glycol) or a polyclonal antibody (B-10) to the insulin receptor, and receptors purified by insulin affinity chromatography. Because the atypical insulin/MSA-binding sites outnumber the type I IGF receptors in IM-9 cells by approximately 10-fold, they also compete with the latter receptors for 125I-IGF-I binding. Thus 125I-IGF-I binding to IM-9 cells is inhibited by moderately low concentrations of insulin (relative potency ratios for insulin compared with IGF-I are approx. 1/14 to 1/4) and is partially displaced (65-80%) by alpha IR-3. When type I IGF receptors are blocked by alpha IR-3 or removed by B-10 immunoprecipitation or insulin affinity chromatography, the hormone-displacement patterns for 125I-IGF-I binding resemble those of the atypical insulin/MSA-binding sites.

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 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.

An acid-stable insulin-like growth factor (IGF)-binding protein from pig serum inhibits binding of IGF-I and IGF-II to vascular endothelial cells

1989 ◽  
Vol 120 (2) ◽  
pp. 231-236 ◽  
Author(s):  
R. Gopinath ◽  
P. E. Walton ◽  
T. D. Etherton
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Binding Protein ◽  
Type I ◽  
Insulin Like Growth Factor ◽  
Igf Receptors ◽  
Igf I ◽  
Igf Binding ◽  
Igf Binding Protein ◽  
Acid Stable

ABSTRACT The effects of a porcine insulin-like growth factor (IGF)-binding protein on binding of IGF-I and IGF-II to porcine aortic endothelial cells (PAEC) were determined. Binding of 125I-labelled IGF-I and -II to IGF receptors was inhibited by IGF-binding protein. IGF-binding protein inhibited binding of IGF-I and -II in a dose-dependent manner with half-maximal inhibition occurring at 5·43 and 108 μg/l respectively. A125I-labelled IGF-I–IGF-binding protein complex, formed by incubating 125I-labelled IGF-I with IGF-binding protein overnight at 4 °C, did not effectively bind to endothelial IGF receptors. Addition of IGF-binding protein to PAEC previously incubated with IGF-I caused a marked dissociation of bound IGF-I (47% dissociation within 12 h). These results indicate that the acid-stable IGF-binding protein which appears to be a part of the 150 kDa GH-dependent binding protein, blocks binding of IGF-I and -II by the IGF receptors and appears to exhibit a higher affinity for IGF-I than the endothelial type-I IGF receptor. The ramifications of this latter point with respect to transfer of circulating IGFs (bound to their IGF-binding proteins) across the vascular endothelium are not clear. Journal of Endocrinology (1989) 120, 231–236

scholarly journals Insulin-like growth factor induces phosphorylation of immunoreactive insulin receptor substrate and its association with phosphatidylinositol-3 kinase in human thymocytes.

1995 ◽  
Vol 182 (2) ◽  
pp. 593-597 ◽  
Author(s):  
R Kooijman ◽  
J J Lauf ◽  
A C Kappers ◽  
G T Rijkers
Keyword(s):  
T Cells ◽  
Growth Factor ◽  
Insulin Receptor ◽  
Signaling Pathway ◽  
Insulin Receptor Substrate ◽  
Type I ◽  
Insulin Like Growth Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Igf I ◽  
Phosphatidylinositol 3

Insulin receptor substrate 1 (IRS-1) is the principle cellular substrate for insulin and insulin-like growth factor I (IGF-I) receptor signaling. After phosphorylation of tyrosine residues within the YMXM or YXXM motifs, IRS-1 associates with phosphatidylinositol-3 kinase (PI3K). This signaling pathway and the presence of an IRS-1-like molecule have been demonstrated in IRS-1-transfected and in nontransfected hematopoietic cell lines, respectively. IGF-I has been implicated in lymphocyte development and function, and recently, we showed that functional type-I IGF receptors are present on human thymocytes and peripheral T cells. In this study, we addressed IGF-I signal transduction in nontransformed, freshly isolated, human thymocytes, as well as in blood T cells. Using Western blot analysis, we found that IGF-I induced phosphorylation of a 160-180-kD protein (pp170) in human thymocytes and that phosphorylated pp170 becomes associated with PI3K and is recognized by anti-IRS-1. In blood T cells, this immunoreactive IRS-1 (irIRS-1) is less abundantly expressed than in thymocytes, as assessed with immunoblotting. As a consequence, phosphorylated pp170 was not or hardly detectable after stimulation with IGF-I, and irIRS-1 was not detected in PI3K immunoprecipitates from lysates of IGF-I-stimulated T cells. However, IGF-I induced the tyrosine phosphorylation of other cellular proteins, indicating that differential expression of irIRS-1 contributes to a distinct signaling pathway in T cells.

Type I insulin-like growth factor receptor expression in different developmental stages of human thymocytes

1995 ◽  
Vol 147 (2) ◽  
pp. 203-209 ◽  
Author(s):  
R Kooijman ◽  
L E Scholtens ◽  
G T Rijkers ◽  
B J M Zegers
Keyword(s):  
T Cell ◽  
Growth Factor ◽  
Dna Synthesis ◽  
T Cell Development ◽  
Cell Development ◽  
Receptor Expression ◽  
Type I ◽  
Insulin Like Growth Factor ◽  
Igf Receptors ◽  
Igf I

Abstract Insulin-like growth factor-I (IGF-I) has been implicated in playing a regulatory role in T cell development and in T cell function. We demonstrate the presence of type I IGF receptors on human thymocytes using radioligand binding assays and flowcytometric analysis. The relative potencies of IGF-I, IGF-II and insulin for competition with 125I-IGF-I indicate the presence of type I IGF receptors. Scatchard analysis revealed that the average number of receptors per thymocyte is 257 ± 28 with a Kd of 0·12 ±0·01 With multicolour flowcytometry using a type I IGF receptor specific monoclonal antibody (αIR3), we show that CD4− CD8− cells express 3-4 times more receptors per cell as compared with CD4+ CD8+, CD4+ CD8− and CD4− CD8+ cells. IGF-I directly stimulated DNA synthesis of thymocytes and potentiated DNA synthesis in mitogen-activated thymocytes. These results indicate that IGF-I can influence T cell development in humans at the level of the thymus. Journal of Endocrinology (1995) 147, 203–209

scholarly journals Expression and function of insulin/insulin-like growth factor I hybrid receptors during differentiation of 3T3-L1 preadipocytes

1998 ◽  
Vol 333 (3) ◽  
pp. 825-831 ◽  
Author(s):  
Dalit MODAN-MOSES ◽  
Michel JANICOT ◽  
John C. McLENITHAN ◽  
M. Daniel LANE ◽  
Samuel J. CASELLA
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Fold Increase ◽  
Insulin Binding ◽  
Cell Surface Expression ◽  
Insulin Like Growth Factor ◽  
Hybrid Form ◽  
Igf Receptors ◽  
Igf I ◽  
Igf Receptor

During the assembly of cell surface receptors, insulin proreceptors are sometimes joined to insulin-like growth factor (IGF) receptor precursors to form covalently linked hybrid receptors. To address the biological consequences of hybrid receptor formation, we studied 3T3-L1 cells known to undergo a 50–70-fold increase in insulin binding while maintaining nearly constant levels of IGF-I binding during differentiation from preadipocytes into adipocytes. The presence of insulin/IGF receptor hybrids in 3T3-L1 adipocytes was demonstrated by the immunoprecipitation of phosphorylated receptors and a novel enzyme-linked immunoassay. Hybrid receptor levels were very low in the early stages of differentiation and increased rapidly between days 4 and 6, reaching a level about 100-fold higher in the mature adipocyte. Coincident with the hybrid assembly, the formation of archetypal (α2,β2) IGF receptors decreased. In fully differentiated adipocytes, virtually all of the IGF receptors were in hybrid form. Stimulation by IGF-I of receptors isolated from mature adipocytes caused autophosphorylation of IGF receptor β subunits in hybrid complexes, whereas autophosphorylated IGF holoreceptors were not demonstrable. Insulin and IGF-I were equipotent in stimulating glucose uptake in the differentiated adipocytes, leading to the conclusion that hybrid insulin/IGF receptors can transduce a transmembrane signal when activated by IGF-I. We conclude that hybrid formation constitutes a novel post-translational mechanism whereby increased synthesis of insulin receptors limits the cell surface expression of the homologous IGF receptor. Furthermore, biological actions in 3T3-L1 adipocytes, previously attributed to archetypal IGF receptors, are in fact mediated through hybrid receptors.

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