Characterization of the receptor for insulin-like growth factor II in bone cells

We have previously shown that insulin-like growth factor II (IGF-II) is produced by bone cells and that IGF-II stimulates cell proliferation and collagen synthesis in bone cells. We now extend these in vitro findings by demonstrating specific IGF-II binding to bone cells derived from newborn mouse calvaria and embryonic chick calvaria. The kinetics of [125I] IGF-II binding in embryonic chick calvaria cells showed time and temperature dependence. Scatchard analysis of [125I]IGF-II binding to chick calvaria cells showed an apparent Kd of 1.4 x 10(-10) M, with a calculated receptor site concentration of 40,000/cell. The specificity characteristics showed that IGF-II was significantly more potent than IGF-I or insulin in displacing IGF-II tracer. Competition for binding of [125I]IGF-II by unlabeled IGF-II showed a dose-dependent displacement between 0.5 and 25 ng/ml. Fifty percent displacement of [125I]IGF-II binding to chick and mouse calvarial cells was achieved at 1-2 ng/ml; 90% of specific binding of [125I]IGF-II was displaceable in the presence of 125 ng/ml of unlabeled IGF-II. IGF-I showed less than 5% cross reactivity for displacement of [125I]IGF-II binding to chick and mouse bone cells. Type II receptor inhibitory antibodies, R-II-PAB1 inhibited the binding of [125I]IGF-II to mouse bone cells and H-35 rat hepatoma cells (which contain type II but not type I receptors) in a dose-dependent manner. R-II-PAB1 also inhibited basal cell proliferation as well as IGF-II-, IGF-I-, and fibroblast growth factor (FGF)-induced cell proliferation in mouse bone cells. In chick calvaria bone cells and TE89 human osteosarcoma cells, R-II-PABI inhibited neither binding of [125I]IGF-II nor IGF-II-induced cell proliferation. These results together with our findings that IGF-II increased chick bone cell proliferation in the presence of maximal doses of IGF-I suggest that at least part of the mitogenic action of IGF-II is mediated through type II rather than type I receptors in bone cells.     

Specificity of insulin-like growth factor binding to type-II IGF receptors in rabbit mammary gland and hypophysectomized rat liver

We have reevaluated IGF binding specificity to membrane receptors in rabbit mammary gland (RMG) and hypophysectomized rat liver (HRL) using recombinant DNA-derived and synthetic analogues of human IGF-I and highly purified IGF-II. SDS-PAGE demonstrated that [125I]IGF-I bound to type-I IGF receptors in RMG; this binding was inhibited in a similar fashion by the IGF-I analogues (IC50 = 10 ng/ml) and to a lesser extent by IGF-II (IC50 = 60 ng/ml). [125I]IGF-II bound to type-II IGF receptors in both RMG and HRL. The IC50 for IGF-II was 9 and 3 ng/ml with RMG and HRL, respectively. At a dose as high as 1 microgram/ml, IGF-I analogues inhibited less than 20% of [125I]IGF-II binding. These results suggest that IGF-I has little or no affinity for type-II IGF receptors.     

Insulin-like growth factors in sheep thyroid cells: action, receptors and production

Sheep thyroid cells cultured in serum-free medium were used to study the biologic activity, binding, and production of the insulin-like growth factors (IGFs). IGF-I, IGF-II, and insulin stimulated thyroid cell division. Abundant, specific IGF receptors on sheep thyroid cell membranes were identified by binding displacement studies. Maximal specific binding of [125I]-labeled IGF-I and IGF-II to 25 micrograms of membrane protein averaged 21% and 27% respectively. The presence of type I and type II IGF receptors was confirmed by polyacrylamide gel electrophoresis of [125I]IGFs covalently cross-linked to cell membranes. Under reducing conditions, [125I]IGF-I bound to a moiety of approximate Mr = 135,000 and [125I]IGF-II to a moiety of approximate Mr = 260,000. Cross-linking of [125I]IGF-I to medium conditioned by thyroid cells indicated the presence of four IGF binding proteins with apparent Mr = 34,000, 26,000, 19,000 and 14,000. Thyroid cells also secreted IGF-I and II into the medium. IGF synthesis was enhanced consistently by recombinant growth hormone. These data indicate that sheep thyroid cells are a site for IGF action, binding, and production and provide further evidence that IGFs may modulate thyroid gland growth in an autocrine or paracrine manner.     

Receptors for insulin-like growth factor-I (IGF-I) in myocardial capillary endothelium of the intact perfused heart

Isolated intact, beating hearts were perfused with HPLC-pure [125]-IGF-I (1 ng/ml) alone or [125]-IGF-I (1 ng/ml) plus varying concentrations of unlabeled IGF-I (10-3,000 ng/ml) or unlabeled insulin (1,000-100,000 ng/ml). After 1 min of perfusion with peptides, the hearts were rapidly fixed, sectioned and analyzed for radioautographic [125I] grain counts. Greater than 90% of [125I] grains were shown to represent intact [125I]-IGF. Maximal grain counts over capillaries occurred after perfusion with [125I]-IGF-I alone and decreased in a dose-dependent manner when unlabeled IGF-I was coperfused. Coperfusion of [125I]-IGF-I with unlabeled insulin also decreased 125I grains over capillaries but less potently than unlabeled IGF-I. EM radioautography demonstrated that [125I]-IGF-I grains were localized over capillary endothelial cells. Thus, specific IGF-I receptors are present in the capillary endothelium of the intact heart and have properties similar to IGF-I receptors in cultured capillary endothelial cells.     

Production of insulin-like growth factor binding proteins by small-cell lung cancer cell lines

Conditioned serum-free media (CM) from small-cell lung cancer (SCLC) cell lines were examined for the presence of insulin-like growth-factor-binding proteins (IGF-BP). 6/9 SCLC cell lines secreted binding proteins with high affinity for IGFs. When [125I]IGF-I or [125I]IGF-II was incubated with the CMs, complexes of tracer with proteins could be demonstrated by gel filtration, by precipitation with polyethylenglycol, and after adsorption of unbound tracer with activated charcoal. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF-I and IGF-II. The dissociation constants were determined to be 0.106 nM for IGF-I and 0.209 nM for IGF-II binding. Cross-linking of [125I]IGF-I or [125I]IGF-II to the CMs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions revealed the presence of IGF-BPs with molecular masses in the range 24-32 kDa. The binding was competitively inhibited by addition of cold IGF-I and IGF-II but not by insulin. Northern blot hybridization with an IGF-BP cDNA probe encoding a low-molecular-weight IGF-BP from a human placenta cDNA library and Western blot analysis with a corresponding polyclonal antibody showed no expression of this gene. These data demonstrate that SCLC cell lines release IGF-BPs in culture supernatants, which differ from IGF-BPs detected in liver and placenta. These IGF-BPs might be important mediators in the autocrine/paracrine growth regulation of IGFs in SCLC.     

Insulin-like growth factor I (IGF-I) production and the presence of IGF-I receptors in rat medullary thyroid carcinoma cell line 6-23 (clone 6)

To clarify whether insulin-like growth factor I (IGF-I) is an autocrine growth factor of rat medullary thyroid carcinoma (MTC) cell line, 6-23 (clone 6), IGF-I binding to MTC cell membranes, IGF-I levels in the conditioned culture medium of MTC cells and the effects of IGF-I on methyl-[3H]thymidine incorporation to MTC cells were examined. Scatchard analysis of saturation binding studies revealed the association constant and the maximal binding capacity were 1.0 x 10(9) M-1 and 199 fmol/mg of membrane protein, respectively. The binding of [125I]IGF-I to MTC cell membranes was inhibited by unlabeled IGF-I, IGF-II and insulin; the relative potencies were IGF-I greater than IGF-II much greater than insulin, suggesting the presence of type I IGF receptors in MTC cells. IGF-I levels in the conditioned culture medium of MTC cells were 120 +/- 3 pM (mean + SE). IGF-I (10(-10) to 10(-8) M) dose-dependently stimulated methyl-[3H]thymidine incorporation to MTC cells. These findings suggest a possible role of IGF-I as an autocrine growth factor for MTC cells.     

Receptor autoradiographic localization of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands

We report here the first evidence of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands, obtained at autopsy. Sections of tissue were incubated with 0.1 nM [125I]IGF-I and analyzed using [3H]Ultrofilm autoradiography with image analysis coupled to computerized microdensitometry. Specific binding sites of [125I]IGF-I were found to be localized in the definitive zone, fetal zone, and fetal medulla of the fetal adrenal glands. In the adult adrenal glands, the entire cortex and medulla were specifically labeled with [125I]IGF-I. Specific binding obtained at a concentration of 0.1 nM [125I]IGF-I to areas in the fetal and adult human adrenal glands was competitively displaced by unlabeled IGF-I, with an IC50 value of 0.34-2.54 nM, and 0.38-0.73 nM, respectively, whereas insulin was much less potent in displacing the binding. Acquisition of this knowledge will aid in studies on cell growth and steroid-catecholamines biosynthesis of the human adrenal gland.     

IGF-I, IGF-II and insulin promote differentiation of spermatogonia to primary spermatocytes in organ culture of newt testes.

Recombinant human insulin-like growth factors (rhIGF-I and rhIGF-II) and human insulin promoted the differentiation of spermatogonia into primary spermatocytes in newt testes fragments cultured in a chemically defined medium. The biological potency for promoting differentiation was dose-dependent for all the ligands with the highest potency displayed by IGF-I, followed by IGF-II, and the least by insulin. The difference in potency was larger between IGF-II and insulin than that between IGF-I and IGF-II. This order of biological potency was in good accordance with the order of affinity in binding specificity of [125I]IGF-I to the testicular membrane fractions: IGF-II and insulin competed the binding of [125I]IGF-I only at concentrations 20-fold and 100-fold higher, respectively, than IGF-I. Specific binding was observed in both somatic cells (mostly Sertoli cells) and germ cells (spermatogonia and primary spermatocytes), though the binding to somatic cells was about 2.7 times higher than that to germ cells. These results indicate that (1) specific binding sites for IGF-I are present in the newt testes, (2) IGF-II and insulin also bind to these receptors but to a lesser degree, and (3) IGF-II and insulin as well as IGF-I promote spermatogonial differentiation into primary spermatocytes by binding to the IGF-I receptor.     

Preferential binding of insulin-like growth factor-II (IGF-II) to a putative alpha 2 beta 2 IGF-II receptor type in C2 myoblasts.

We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin-like growth factor (IGF) binding to human fibroblast and glioblastoma cells: the modulating effect of cell released IGF binding proteins (IGFBPs)

The cell surface of human fibroblasts contains not only type I IGF receptors but at least two forms of IGFBPs. Studies were undertaken to analyze the mechanisms by which these IGFBPs alter IGF-I-cell surface interactions. Human fetal fibroblasts (GM10) and a human glioblastoma cell line (1690) were chosen for analysis. During assays to quantify [125I]-IGF-I binding, both cell lines were shown to release IGFBPs into the binding assay buffer. Under equilibrium conditions, [125I]-IGF-I preferentially associates with IGFBPs in the assay buffer (up to 40% of the [125I]-IGF-I added) since they have a higher affinity than type I IGF receptors or IGFBPs associated with the cell surface. Likewise the addition of increasing concentrations of unlabeled IGF-I results in preferential competition for binding to assay buffer IGFBPs. This results in a repartitioning of the [125I]-IGF-I that is bound to assay buffer IGFBPs onto cell surface binding sites. The degree of repartitioning is quantitatively related to the amount of [125I]-IGF-I bound to released IGFBPs. When cultures are exposed to cycloheximide before the binding assay, both the amount of IGFBPs that are released into the assay buffer and the amount of [125I]-IGF-I that is repartitioned are decreased. In contrast when [Gln3, Ala4, Tyr15, Leu16]-IGF-I ([QAYL]-IGF-I, an IGF analog that has unaltered affinity for type I IGF receptors) is iodinated and tested, the competition curve with unlabeled IGF-I shows no repartitioning effect. This form of IGF can be used to quantify type I receptor number independent of the presence of IGFBPs. IGF-I and the [QAYL]-IGF-I compete equally with the [125I]-[QAYL]-IGF-I for binding to cell surfaces, whereas unlabeled [QAYL]-IGF-I is greater than 25-fold less potent compared to IGF-I in competing with [125I]-IGF-I for cell surface binding. Specific binding of [125I]-[QAYL]-IGF-I to GM10 and 1690 cell surfaces is less than 20% of [125I]-IGF-I binding. These findings suggest that IGFBPs that are present on human fibroblast surfaces represent a large portion of the IGF binding sites. We conclude that the amount of IGFBPs released into assay buffer is a major determinant of the repartitioning of [125I]-IGF-I to cell surface binding sites and that both cell surface and assay buffer IGFBPs modulate type I IGF receptor binding.     

IGF receptors in myocardial capillary endothelium: potential regulation of IGF-I transport to cardiac muscle

Beating rat hearts were perfused with 125I-IGF-II alone or 125I-IGF-II and unlabeled IGF-II or insulin, then prepared for radioautography. Maximal 125I-IGF-II grain counts over capillaries were decreased in a dose-dependent manner by unlabeled IGF-II but were unaffected by coperfusion with insulin. To determine a potential role for capillary receptors in the transfer of circulating IGF to cardiac muscle, the effects of sequential loss of capillary IGF binding sites was determined. For IGF-I, loss of capillary binding sites by trypsin perfusion was accompanied by proportional decreases in the subsequent appearance of IGF-I in cardiac muscle. In contrast, similar decrements of capillary IGF-II binding did not affect muscle levels of IGF-II. We conclude that capillary endothelium of the intact heart possesses distinct IGF-I and IGF-II binding sites, with the capillary IGF-I binding sites being of potential importance in the transfer of vascular IGF-I to subendothelial cardiac muscle.     

An antibody that blocks insulin-like growth factor (IGF) binding to the type II IGF receptor is neither an agonist nor an inhibitor of IGF-stimulated biologic responses in L6 myoblasts

To better define the biologic function of the type II insulin-like growth factor (IGF) receptor, we raised a blocking antiserum in a rabbit by immunizing with highly purified rat type II IGF receptor. On immunoblots of crude type II receptor preparations, only bands corresponding to the type II IGF receptor were seen with IgG 3637, indicating that the antiserum was specific for the type II receptor. Competitive binding and chemical cross-linking experiments showed that IgG 3637 blocked binding of 125I-IGF-II to the rat type II IGF receptor, but did not block binding of 125I-IGF-I to the type I IGF receptor, nor did IgG 3637 block binding of 125I-insulin to the insulin receptor. In addition, IgG 3637 did not inhibit the binding of 125I-IGF-II to partially purified 150- and 40-kDa IGF carrier proteins from adult and fetal rat serum. L6 myoblasts have both type I and type II IGF receptors. IGF-I was more potent than IGF-II in stimulating N-methyl-alpha-[14C]aminoisobutyric acid uptake, 2-[3H]deoxyglucose uptake, and [3H]leucine incorporation into cellular proteins. IgG 3637 did not stimulate either 2-[3H]deoxyglucose uptake, N-methyl-alpha-[14C]aminoisobutyric acid uptake, or [3H]leucine incorporation into protein when tested alone. Furthermore, IgG 3637 at concentrations sufficient to block type II receptors under conditions of the uptake and incorporation experiments did not cause a shift to the right of the dose-response curve for stimulation of these biologic functions by IGF-II. We conclude that the type II IGF receptor does not mediate IGF stimulation of N-methyl-alpha-[14C]aminoisobutyric acid and 2-[3H]deoxyglucose uptake and protein synthesis in L6 myoblasts; presumably, the type I receptor mediates these biologic responses. The anti-type II receptor antibody inhibited IGF-II degradation in the media by greater than 90%, suggesting that the major degradative pathway for IGF-II in L6 myoblasts utilizes the type II IGF receptor.     

Receptors for insulin-like growth factors and insulin on murine fetal cortical brain cells

Fetal murine neuronal cells bear somatomedin receptors which can be classified according to their affinities for IGF-I, IGF-II and insulin. Binding of 125I-IGF-I is half-maximally displaced by 7 ng/ml IGF-I while 15- and 700-fold higher concentrations are required for, respectively, IGF-II and insulin. Linear Scatchard plots of competitive-binding data with IGF-I suggest one single class of type I IGF receptors (Ka = 2.6 X 10(9) M-1; Ro = 4500 sites per cell). The occurrence of IGF-II receptors appears from the specific binding of 125I-IGF-II and competition by unlabeled IGF-II; the IGF-II binding sites display a low affinity for IGF-II and no affinity for insulin. IGF-II also interacts with insulin receptors although 50- to 100-fold less potent than insulin in competing for 125I-insulin binding. The presence of distinct receptors for IGF-I, IGF-II and insulin on fetal neuronal cells is consistent with a role of these peptides in neuronal development, although our data also indicate that IGF-I receptors could mediate the growth promoting effects of insulin.     

Decrease in IGF-I binding sites on human promyelocytic leukemia cell line (HL-60) with differentiation

Specific insulin-like growth factor I (IGF-I) receptors on human promyelocytic leukemia cell line (HL-60) were identified and characterized. [125I]IGF-I specifically bound to the cells, and [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose dependent manner. [125I]IGF-I binding to the cells were displaced by multiplication stimulating activity (MSA) and porcine insulin, with potencies that were 10 and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present on the HL-60 cells. After the cells were differentiated to the macrophage-like cells by 12-o-tetra-decanoyl-phorbol-13-acetate (TPA) and 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), [125I]IGF-I binding to the cells decreased significantly. By Scatchard analysis, it was found to be due to a decrease in the number of IGF-I receptors. Thus, the differentiation of HL-60 cells to the macrophage-like cells was accompanied by a decrease in IGF-I receptors.     

Distinct receptors for IGF-I, IGF-II, and insulin are present on bovine capillary endothelial cells and large vessel endothelial cells

Endothelial cells were cultured from bovine fat capillaries, aortae and pulmonary arteries and their interactions with 125I-IGF-I, 125I-MSA (an IGF-II), 125I-insulin and the corresponding unlabeled hormones were evaluated. Each endothelial culture showed similar binding parameters. With 125I-insulin, unlabeled insulin competed with high affinity while IGF-I and MSA were approximately 1% as potent. With 125I-MSA, MSA was greater than or equal to IGF-I in potency and insulin did not compete for binding. Using 125I-IGF-I, IGF-I was greater than or equal to MSA whereas insulin decreased 125I-IGF-I binding by up to 72%. Exposing cells to anti-insulin receptor antibodies inhibited 125I-insulin binding by greater than 90%, did not change 125I-MSA binding, while 125I-IGF-I binding was decreased by 30-44%, suggesting overlapping antigenic determinants between IGF-I and insulin receptors that were not present on MSA receptors. We conclude that cultured capillary and large vessel endothelial cells have distinct receptors for insulin, IGF-I and MSA (IGF-II).     

Characterization of pituitary IGF-I receptors: modulation of prolactin and growth hormone

There have been no studies in any vertebrate that have localized insulin-like growth factor (IGF)-I receptors in prolactin (PRL) cells or that have correlated pituitary binding to the potency of IGF-I in regulating both PRL and growth hormone (GH) secretion. We show that IGF-I binds with high affinity and specificity to the pituitary gland of hybrid striped bass (Morone saxatilis x M. chrysops). IGF-I and IGF-II were equipotent in inhibiting saturable (125)I-IGF-I binding, whereas insulin was ineffective. IGF-I binds with similar affinity to the rostral pars distalis (>95% PRL cells) as the whole pituitary gland and immunohistochemistry colocalizes IGF-I receptors and PRL in this same region. Des(1-3)IGF-I, a truncated analog of IGF-I that binds with high affinity to IGF-I receptors but weakly to IGF-I binding proteins (IGFBPs), showed a similar inhibition of saturable (125)I-IGF-I binding, but it was more potent than IGF-I in stimulating PRL and inhibiting GH release. These results are the first to localize IGF-I receptors to PRL cells, correlate IGF-I binding to its efficacy in regulating GH and PRL secretion, as well as demonstrate that IGFBPs may play a significant role in modulating the disparate actions of IGF-I on PRL and GH secretion.     

The insulin-like growth factors (IGFs) I and II bind to articular cartilage via the IGF-binding proteins

Bovine articular cartilage discs (3 mm diameter x 400 micrometer thick) were equilibrated in buffer containing (125)I-insulin-like growth factor (IGF)-I (4 degrees C) +/- unlabeled IGF-I or IGF-II. Competition for binding to cartilage discs by each unlabeled IGF was concentration-dependent, with ED(50) values for inhibition of (125)I-IGF-I binding of 11 and 10 nM for IGF-I and -II, respectively, and saturation by 50 nM. By contrast, an analog of IGF-I with very low affinity for the insulin-like growth factor-binding proteins (IGF-BPs), des-(1-3)-IGF-I, was not competitive with (125)I-IGF-I for cartilage binding even at 100-400 nM. Binding of the (125)I-labeled IGF-II isoform to cartilage was competed for by unlabeled IGF-I or -II, with ED(50)s of 160 and 8 nM, respectively. This probably reflected the differential affinities of the endogenous IGF-BPs (IGF-BP-6 and -2) for IGF-II/IGF-I. Transport of (125)I-IGF-I was also measured in an apparatus that allows diffusion only across the discs (400 micrometer), by addition to one side and continuous monitoring of efflux on the other side. The time lag for transport of (125)I-IGF was 266 min, an order of magnitude longer than the theoretical prediction for free diffusion in the matrix. (125)I-IGF-I transport then reached a steady state rate (% efflux of total added (125)I-IGF/unit time), which was subsequently accelerated approximately 2-fold by addition of an excess of unlabeled IGF-I. Taken together, these results indicate that IGF binding to cartilage, mostly through the IGF-BPs, regulates the transport of IGFs in articular cartilage, probably contributing to the control of their paracrine activities.     

Characterization of insulin-like growth factor I receptors on human erythroleukemia cell line (K-562 cells)

Specific insulin-like growth factor I (IGF-I) receptors on a human erythroleukemia cell line (K-562 cells) were identified and characterized. [125I]-IGF-I specifically bound to K-562 cells and the binding was displaced by unlabeled IGF-I in a dose dependent manner, and half maximal inhibition of the binding was observed at 7 ng/ml IGF-I. [125I]IGF-I binding to the cells was displaced by multiplication stimulating activity (MSA) and by porcine insulin, with potencies that were 10, and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present in the K-562 cells. When the cells were differentiated by hemin (40 microM), specific binding of [125I]IGF-I to the cells was decreased to 56.8 +/- 5.0% of that for undifferentiated cells. Furthermore, at physiological concentration of IGF-I stimulated thymidine incorporation into DNA and increased the number of cells. These data demonstrate that K-562 cells have specific receptors for IGF-I which may be functionally important for these cells, and that the IGF-I binding sites decrease with cell differentiation. This system might be useful in studying the interaction of IGF-I receptors.     

Epidermal growth factor enhancement of insulin-like growth factor-I-induced down-regulation of insulin-like growth factor I receptor in cultured placental trophoblastic cells.

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) synergistically stimulate placental lactogen (hPL) secretion by placental cells. To understand the mechanism of actions we have investigated a possible heterologous regulatory effect of EGF and IGF-I on each other's receptors. Pretreatment of the cells with IGF-I had no effect on [125I]-EGF binding or the down-regulation of EGF receptor. Pretreatment of the cells with EGF, concomitantly with IGF-I, had no effect on [125I]-IGF-I binding but it augmented the IGF-I down-regulation of IGF-I receptor. The time required to initiate the IGF-I-induced down-regulation of IGF-I receptor was reduced by 4 h in the presence of EGF. IGF-I-down-regulated decreased (P less than 0.05) receptor numbers were further decreased (p less than 0.05) in the presence of EGF. These results suggested that the synergistic effect of EGF and IGF-I seen in hPL secretion by placental cells is not due to direct heterologous hormone-receptor interactive effects. However, the effects seen may be due to a differentiating effect of EGF sensitizing the cells for responsiveness to IGF-I.     

Distribution of receptors for insulin and insulin-like growth factor I (somatomedin C) in the adrenal gland

Rat adrenal glands contain cell surface high-affinity receptors for several peptide hormones. Receptors for IGF-I were abundant in this tissue, but receptors for insulin were relatively scarce. The behavior of adrenal membrane IGF-I receptors in radioligand binding assays was similar to the behavior of IGF-I receptors from other tissues, with a KD congruent to 6.2 x 10(-9) M. Covalent cross-linking studies with [125I]IGF-I revealed an IGF-I receptor alpha-subunit with Mr congruent to 135,000 on dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions, as well as a smaller radiolabeled peptide, Mr = 116,000. In contrast, little binding of [125I]insulin to adrenal membranes was observed and no labeling occurred in cross-linking studies using [125I]insulin. These results contrast with the findings of whole-body autoradiographic studies that indicated substantial binding of [125I]insulin to adrenal glands and suggest that IGF-I, rather than insulin, may play a critical role in the growth and development of the adrenal gland.