scholarly journals 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.

1988 ◽  
Vol 263 (15) ◽  
pp. 7068-7072
Author(s):  
P Maly ◽  
C Lüthi
Keyword(s):  
Monoclonal Antibody ◽  
Growth Factor ◽  
Chemical Modification ◽  
Binding Sites ◽  
Type I ◽  
Tyrosine Residues ◽  
Factor I ◽  
Type I Igf Receptor ◽  
Igf I ◽  
Igf Receptor

Growth hormone (GH) modulates insulin-like growth factor I (IGF-I) and type I IGF receptor mRNA levels in the ovary of prepubertal GH-deficient rats

1995 ◽  
Vol 132 (4) ◽  
pp. 497-501 ◽  
Author(s):  
Saul Malozowski ◽  
Toni G Parmer ◽  
Sabina Trojan ◽  
George R Merriam ◽  
Geula Gibori ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Growth Factor ◽  
Type I ◽  
Mrna Levels ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Receptor Mrna ◽  
Type I Igf Receptor ◽  
Igf I ◽  
Igf Receptor

Malozowski S, Parmer TG, Trojan S, Merriam GR, Gibori G, Roberts Jr CT, LeRoith D, Werner H, Zilberstein M. Growth hormone (GH) modulates insulin-like growth factor I (IGF-I) and type I IGF receptor mRNA levels in the ovary of prepubertal GH-deficient rats. Eur J Endocrinol 1995;132:497–501. ISSN 0804–4643 In order to explore the potential role of growth hormone (GH) in modulating insulin-like growth factor I (IGF-I) gene expression in the prepubertal rat ovary, female rats were rendered GH deficient by neonatal administration of monosodium glutamate (MSG). One group of rats received vehicle and served as the control. At 21 days of age, MSG-treated rats received either GH or vehicle for 2 weeks. On days 21, 24, 28 and 31 animals were weighed and subsets were sacrificed for liver RNA extraction. The remaining animals were sacrificed at day 35 when livers and ovaries were collected, and serum was obtained for GH determinations. The IGF-I mRNA levels were estimated by Northern blots and corroborated further by slot-blot analysis. The MSG-treated rats had lower body weights (p < 0.01) and GH levels (p < 0.05) than controls. Growth hormone replacement significantly accelerated the weight gain of MSG-treated rats. At day 24 and thereafter, three RNA IGF-I species (7.5, 1.8 and 0.8–1.2 kB) were seen in the liver. In the ovary, at age 35 days, two major IGF-I mRNA species (7.5 and 0.8–1.2kb) were seen. The MSG treatment consistently reduced the levels of both IGF-I mRNA species in the ovary. Growth hormone administration partially restored their expression, both in the liver and in the ovary. In addition, ovarian type I IGF receptor mRNA levels were increased in the MSG-treated rats when compared to controls. This trend was reversed by GH replacement. In summary, we have found that in prepubertal female rats rendered GH deficient with MSG, ovarian IGF-I gene expression is reduced while type I IGF receptor mRNA levels are increased. These findings are reversed with GH replacement. These results suggest a physiological role for GH in modulating IGF-I and type I IGF receptor genes in the ovary. Saul Malozowski, FDA, HFD-510, Rockville, MD 20897, USA

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.

Immunoreactive and receptor-active insulin-like growth factor-I (IGF-I) and IGF-binding protein in blood plasma from the freshwater fish Macquaria ambigua (golden perch)

1993 ◽  
Vol 136 (2) ◽  
pp. 191-198 ◽  
Author(s):  
T. A. Anderson ◽  
L. R. Bennett ◽  
M. A. Conlon ◽  
P. C. Owens
Keyword(s):  
Binding Protein ◽  
Size Exclusion ◽  
Type I ◽  
Factor I ◽  
Type I Igf Receptor ◽  
Receptor Assay ◽  
Igf I ◽  
Igf Binding ◽  
Igf Receptor ◽  
Igf Binding Protein

ABSTRACT The presence of insulin-like growth factor-I (IGF-I)-related molecules and IGF-binding factors in blood from golden perch, Macquaria ambigua, an Australian native freshwater fish, was investigated. Serum was acidified to dissociate IGF and IGF-binding protein complexes that might be present, and fractionated by size-exclusion high-performance liquid chromatography at pH 2·8. Fractions were neutralized and their activities assessed by (i) an immunoassay for mammalian IGF-I which also detects chicken IGF-I but in which all known forms of IGF-II react very poorly, (ii) a receptor assay for IGF-II in which all known forms of IGF-I react poorly, and (iii) a type-I IGF receptor assay in which mammalian IGF-I and IGF-II polypeptides are almost equivalent. No IGF-II-like activity was detected. Three peaks of IGF-I-like activity were detected by IGF-I immunoassay and type-I IGF receptor assay. The major peak of activity was similar in molecular size to human IGF-binding protein-3, 45–55 kDa ('large IGF'), and a minor peak of activity which was similar in size to mammalian IGFs, 7·5 kDa. A third peak of activity was observed eluting at a time which indicates that it is a smaller molecule than any previously described IGF. The large IGF was temperature-sensitive, but was not a binding protein for 125I-labelled human IGF-I (hIGF-I). This material therefore was able to bind to anti-hIGF-I antibodies and to human type-I IGF receptors, and may represent the fish equivalent of mammalian prepro-IGFs. The two smallest forms of IGF activity identified by IGF-I radioimmunoassay and type-I radioreceptor assay following acidic size-exclusion chromatography were able to stimulate protein synthesis by L-6 myoblasts in culture, although large IGF did not. When fresh (but not frozen and thawed) golden perch serum was incubated with 125I-labelled hIGF-I and then fractionated by size-exclusion liquid chromatography at pH 7·4 through Sephadex G-100, the radioactivity became associated with a complex, intermediate in size between free IGF-I and the major IGF-binding protein in human serum. The association of 125I-labelled hIGF-I with the complex was inhibited by the presence of unlabelled hIGF-I in the incubation. These studies show that receptor-active, immunoreactive and bioactive IGF-I-like activity is present in golden perch serum, and demonstrate the presence of an IGF-I-binding factor in this species. Journal of Endocrinology (1993) 136, 191–198

Differential binding of 125I-IGF-I preparations to human fibroblast monolayers

1988 ◽  
Vol 118 (4) ◽  
pp. 513-520 ◽  
Author(s):  
C. A. Conover ◽  
P. Misra ◽  
R. L. Hintz ◽  
R. G. Rosenfeld
Keyword(s):  
Binding Sites ◽  
Human Fibroblast ◽  
Type I ◽  
Type Ii ◽  
Low Concentrations ◽  
Type I Igf Receptor ◽  
Igf I ◽  
Differential Binding ◽  
Igf Receptor ◽  
Binding Curves

Abstract. Specific, high affinity binding of 125I-IGF-I to the type I IGF receptor on human fibroblast monolayers was not altered by varying feeding schedules, serum lots, washing procedures, or incubation times and temperatures. However, markedly different competitive binding curves were obtained when different iodinated IGF-I preparations were used. Five of six radioligands bound preferentially to the type I IGF receptor on human fibroblast monolayers, with 50% displacement at 4–8 μg/l unlabelled IGF-I; with one radioligand a paradoxical 20–200% increase in 125I-IGF-I binding was observed at low concentrations of unlabelled IGF-I, while concentrations as high as 100 μg/l IGF-I failed to displace this radioligand. The latter binding pattern cannot be accounted for by 125I-IGF-I binding to the type II IGF receptor. These data indicate that various radioligands may have preferential affinities for different IGF-I binding sites on human fibroblast monolayers.

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.

Receptor binding of insulin-like growth factor-I to mammary microsomes from non-pregnant, pregnant and lactating sheep

1993 ◽  
Vol 136 (2) ◽  
pp. 297-304 ◽  
Author(s):  
S. J. Winder ◽  
S. D. Wheatley ◽  
I. A. Forsyth
Keyword(s):  
Growth Factor ◽  
Membrane Protein ◽  
Binding Affinity ◽  
Binding Sites ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Pregnant Sheep ◽  
Igf I ◽  
Igf Receptor

ABSTRACT Sucrose density centrifugation was used to prepare a partially purified membrane fraction from the mammary glands of non-pregnant, pregnant and lactating sheep. The binding of125 I-labelled insulin-like growth factor-I (IGF-I) was dependent on membrane protein concentration, pH, time and temperature. The binding showed the characteristics of a type-1 IGF receptor, being displaced by IGF-I (median effective dose (ED50) 0·55 nmol/l), less effectively by IGF-II (ED50 8·8 nmol/l) and least effectively by insulin. Glucagon, ovine prolactin and ovine placental lactogen could not displace binding. A molecular weight of 135 000 was determined by affinity cross-linking using disuccinimidyl suberate; this was consistent with the reported size of the type-1 receptor α-subunit. Scatchard analysis was used to determine binding affinity and numbers of IGF-I-binding sites. A single class of high-affinity binding sites was found in all physiological states. In non-pregnant sheep and sheep at days 40, 75 and 110–120 of pregnancy and at term, the binding affinity was similar (apparent dissociation constant (Kd) 2·73 ±0·31 nmol/l, n = 22). In lactating sheep (weeks 1, 4 and 10), the binding affinity was significantly (P = 0·02) higher (Kd 0·77± 0·06 nmol/l n = 9). Binding capacity was similar in non-pregnant and pregnant sheep (1005 ± 113 fmol/mg, n = 19), but fell by parturition and remained low in lactation (570±52 fmol/mg membrane protein, n = 12). The results suggest that the mammary growth of pregnancy is not regulated at the level of the type-1 IGF receptor. Journal of Endocrinology (1993) 136, 297–304

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