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

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.     

Receptors for insulin-like growth factor-I and tumor necrosis factor-alpha are hormonally regulated in bovine granulosa and thecal cells.

Mastitis induces release of tumor necrosis factor-alpha (TNFalpha) and has been linked with reduced reproductive performance. To further elucidate the role and mechanism of action of TNFalpha on ovarian cells, the effect of TNFalpha on insulin-like growth factor-I (IGF-I)-induced steroidogenesis and IGF-I binding sites in granulosa and thecal cells as well as the hormonal regulation of TNFalpha receptors were evaluated. Granulosa and thecal cells were obtained from small (1-5mm) and large (> or =8mm) bovine ovarian follicles, respectively, and cultured for 3-4 days. During the last 2 days of culture, cells were treated with various hormones and steroid production and specific binding of 125I-IGF-I and 125I-TNFalpha was determined. Two-day treatment with 30 ng/ml of TNFalpha decreased (P<0.05) IGF-I-induced estradiol production by granulosa cells and IGF-I-induced androstenedione production by thecal cells. Two-day treatment with 10 and 30ng/ml of TNFalpha decreased (P<0.05) specific binding of 125I-IGF-I to thecal cells, but had no effect on specific binding of 125I-IGF-I to granulosa cells, or on specific binding of 125I-IGF-II to thecal cells. TNFalpha did not compete for 125I-IGF-I binding to granulosa or thecal cells whereas unlabeled IGF-I suppressed 125I-IGF-I binding. Insulin inhibited (P<0.10) whereas FSH had no effect on the number of specific 125I-TNFalpha binding sites in granulosa cells. In contrast, LH increased (P<0.10) whereas insulin had no effect on specific 125I-TNFalpha binding sites in thecal cells. These results suggest that IGF-I and TNFalpha receptors in granulosa and thecal cells are regulated by hormones differentially.     

Transport and binding of insulin-like growth factor I through articular cartilage

This study focused on the role of insulin-like growth factor (IGF) binding proteins (IGFBPs) in cartilage on the transport and binding of IGF-I within the tissue. We have developed experimental and theoretical modeling techniques to quantify and contrast the roles of diffusion, binding, fluid convection, and electrical migration on the transport of IGF-I within cartilage tissue. Bovine articular cartilage disks were equilibrated in buffer containing 125I-IGF-I and graded levels of unlabeled IGF-I. Equilibrium binding, as measured by the uptake ratio of 125I-IGF-I in the tissue (free plus bound) to the concentration of labeled species in the buffer, was found to be consistent with a first-order reversible binding model involving one dominant family of binding sites within the matrix. Western ligand blots revealed a major IGF binding doublet around 23 kDa, which has been previously shown to coincide with IGFBP-6. Diffusive transport of 125I-IGF-I through cartilage was measured and found to be consistent with a diffusion-limited reaction theoretical model incorporating first-order reversible binding. Addition of excess amounts of unlabeled IGF-I during steady state transport of 125I-IGF-I resulted in release of bound 125I-IGF-I from the tissue, as predicted by the diffusion-reaction model. In contrast, addition of the low-affinity Des(1-3)IGF-I analog did not result in release of bound 125I-IGF-I. Application of electric current was used to augment transport of IGF-I through cartilage via electroosmosis and electrophoresis. Taken together, our results suggest that a single dominant substrate family, the high-affinity IGFBPs, is responsible for much of the observed binding of IGF-I within cartilage. The data suggest that intratissue fluid flow, such as that induced by mechanical loading of cartilage in vivo may be expected to enhance IGF transport by an order of magnitude and that this increment may help to counterbalance the restrictions encountered by the immobilization of IGFs by the binding proteins.     

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).     

Insulin-like growth factor I/somatomedin C action on 2-deoxyglucose and alpha-amino isobutyrate uptake in chick embryo fibroblasts

Maximum 125I-IGF-I/Sm-C total binding to chick embryo fibroblasts was 3% at +37 degrees C and decreased to less than 1% in presence of 2.8 X 10(-9) M unlabelled IGF-I/Sm-C. Insulin did not compete with IGF-I/Sm-C for the binding to cells. Biological action of IGF-I/Sm-C was evaluated on 2-deoxyglucose and alpha-aminoisobutyrate uptake. Results are compared with those obtained with insulin. Maximal peptide effects on the two transport processes were obtained at a 0.65 X 10(-7) M concentration and for a 120 minute association time, whereas cells were markedly less sensitive to insulin and time response curves were different. These results suggest that insulin action on nutrient uptake in chick embryo fibroblasts is not mediated by the binding of the hormone to IGF-I/Sm-C receptors.     

Type I insulin-like growth factor receptors in human ovarian stroma

Hyperandrogenism observed in a variety of hyperinsulinemic states is thought to be due to an effect of insulin mediated through the type I insulin-like growth factor (IGF) receptors. These receptors, however, have not yet been demonstrated in normal human ovarian cells capable of androgen production. We now report the presence of type I IGF receptors in membrane preparations of human ovarian stroma. The ovarian stromal tissue was obtained from women undergoing indicated oophorectomy. Stromal plasma membranes were prepared. Specific 125I-IGF-I binding was 6.6 +/- 0.2%/100 micrograms protein. The affinity constant estimated by Scatchard analysis was 4.6 X 10(-9) M. 50% inhibition of 125I-IGF-1 binding was observed at 5 ng/ml of IGF-1. Specificity of the 125I-IGF-I-binding sites was confirmed by analogue specificity studies and in experiments utilizing monoclonal antibody to the IGF-I receptor, alpha-IR-3. IGF-II and insulin competed with 125I-IGF-I for the binding sites, but with an affinity significantly lower than that of IGF-I: 50% inhibition was observed at approximately 60 ng/ml of IGF-II or insulin. alpha-IR-3, a monoclonal antibody with high specificity for the type I IGF receptor, effectively inhibited 125I-IGF-I binding in a dose-dependent manner, confirming that the 125I-IGF-I binding was indeed to the type I IGF receptor. We conclude that type I IGF receptors are present in human ovarian stroma. These receptors may mediate effects of insulin on the ovary in hyperinsulinemic insulin-resistant states.     

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.     

Constitutive expression of uterine receptors for insulin-like growth factor-I during the peri-implantation period in the pig.

Insulin-like growth factor-I (IGF-I), synthesized by the uterine endometrium of cyclic and early pregnant gilts, accumulates in the uterine luminal fluid, where it comes in contact with the developing conceptus and the rapidly growing uterus. The uterus and the conceptus thus represent potential target sites for the biological effects of IGF-I, provided high-affinity Type I receptors are present. This study was undertaken to evaluate the expression of functional IGF-I receptors in the endometrium and myometrium of pregnant (Day 10, 12, and 15) gilts and in the endometrium of cyclic (Day 15) and pseudopregnant (Day 15) gilts and to correlate levels of these receptors with temporally regulated uterine production of IGF-I. Specific binding of 125I-IGF-I to endometrial membranes pretreated with MgCl2 (4 M) at 4 degrees C for 16 h, was saturable and membrane concentration-dependent. Competition of 125I-IGF-I binding to endometrial membranes was highest with unlabeled IGF-I greater than IGF-II much greater than insulin, whereas porcine relaxin was noncompetitive. Affinity cross-linking of endometrial membranes with 125I-IGF-I followed by SDS-PAGE and autoradiography revealed two labeled bands of Mr greater than 200,000 and Mr 135,000, with the major band being the Mr 135,000 species. Scatchard analysis of 125I-IGF-I binding to endometrial membranes from Day 12 pregnant gilts revealed a single class of binding sites with a dissociation constant (Kd) = 4.08 +/- 0.09 nM. Membranes prepared from endometrium of Day 10, 12, and 15 pregnant gilts exhibited comparable 125I-IGF-I binding (p greater than 0.05) that was higher (p less than 0.001) than that for the corresponding myometrial membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine Chromaffin Cells Have Insulin-Like Growth Factor-I (IGF-I) Receptors: IGF-I Enhances Catecholamine Secretion

The binding of 125I-insulin-like growth factor-I (125I-IGF-I) to bovine chromaffin cells was measured. Chromaffin cell cultures contained 111,000 +/- 40,000 IGF-I binding sites/cell. These sites bound IGF-I with a KD of 1.1 +/- 0.3 nM and had a much lower affinity for insulin. Cross-linking studies showed that 125I-IGF-I bound to a protein that had an Mr of approximately 125,000, similar to the Mr of the alpha subunit of the IGF-I receptor in other tissues. Cells cultured with IGF-I (10 nM) for 4 days exhibited an almost twofold increase in high K+-evoked catecholamine secretion. Insulin was much less potent than IGF-I in enhancing catecholamine secretion. These data indicate that binding of IGF-I to its receptors on chromaffin cells can modulate the function of these cells.     

Characterization of a neuronal subtype of insulin-like growth factor I receptor

Primary neuronal cultures from fetal rat brain were utilized to investigate the possible role of insulin-like growth factor I (IGF-I) in neuronal growth and differentiation. 125I-IGF-I binding to intact cultured neurons was specific and saturable with an apparent Kd of 7.0 +/- 1.2 nM and a Bmax of 1.8 +/- 0.3 pmol/mg protein. Binding of 125I-IGF-I to neurons was inhibited by IGF-I, followed by IGF-II and insulin. 7 S nerve growth factor, but not beta-nerve growth factor, also inhibited 125I-IGF-I binding. A similar binding site was detected on brain membranes. Affinity cross-linking of 125I-IGF-I to intact cultured neurons revealed, under reducing conditions, a major binding moiety with an Mr of 115,000 and a minor component at Mr 260,000. The former represents a neuronal type of the IGF-I receptor alpha subunit, whereas the latter probably represents an alpha dimer. The Mr = 115,000 binding component for 125I-IGF-I was also present in membranes prepared from postnatal whole brain. In contrast, the binding moiety in cultured glial cells was of Mr = 135,000, which was identical to the IGF-I receptor alpha subunit of placenta. Thus mature brain, despite its cellular heterogeneity, expresses a structural subtype of IGF-I receptor which appears to be unique to differentiated neurons. Moreover, glial and neuronal cultures secreted a polypeptide which specifically bound IGF-I; the apparent Mr of this binding protein was determined by affinity cross-linking to be approximately 35,000. The presence of neuronal IGF-I receptors and binding proteins suggested that IGF-I may exert neurotrophic effects on developing neurons. This possibility was supported by the observation that IGF-I markedly stimulated neuronal RNA synthesis.     

Up-regulation of IGF-I receptors on IM-9 cells by IGF-II peptides

To examine a possible role for IGF-II in the regulation of IGF-I receptors we measured 125I-IGF-I binding on IM-9 cells following pre-incubation with IGF-II/IGF-I mixtures, purified MSA (a rat IGF-II-like peptide), pure IGF-I, or insulin. Whereas all preparations tested induced down-regulation of IGF-I binding after 20 hours, distinct differences were noted after six hour pre-incubation: IGF-I (100 ng/ml) and insulin (1 microgram/ml) both induced down-regulation of IGF-I binding (15 +/- 2% and 19 +/- 2% respectively). However, a mixture of IGF-II and IGF-I (100 ng/ml each) induced consistent up-regulation of IGF-I binding (16 +/- 2%) (mean +/- SE, n = 14), and a preparation enriched in IGF-II (250 ng/ml IGF-II and 75 ng/ml IGF-I) induced 20 +/- 5% (n = 3) up-regulation at six hours. Purified MSA (200 ng/ml) induced 15% up-regulation of IGF-I binding at six hours. Scatchard analysis of displacement curves showed that increased binding was due to loss of low affinity binding, with enhancement of high affinity sites. The up-regulation of IGF-I binding was unaffected by treatment with 0.1 mM cycloheximide, but was blunted by 5 microM colchicine. It is concluded that 1. IGF-II induces up-regulation of IGF-I receptors on IM-9 cells following 6 hour pre-incubation; 2. This phenomenon is not mimicked by the structurally-related peptides IGF-I or insulin; The up-regulation is due to enhanced high affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factor I receptors in retinal rod outer segments

We have previously reported that the GDP-bound alpha-subunit of the GTP-binding protein transducin, present in outer segments of retinal rod cells (ROS), serves as a high affinity in vitro substrate (Km = 1 microM) for the insulin receptor kinase. The present study demonstrates that transducin also serves as in vitro substrate for an endogenous IGF-I receptor kinase isolated from ROS membranes. The presence of insulin-like growth factor I (IGF-I) receptors in ROS is evident from the high affinity and specific binding of 125I-IGF-I to ROS membranes (Kd = 3 nM) which contain 110 fmol of IGF-I binding sites/mg of membrane protein. Furthermore, cross-linking of 125I-IGF-I labels the 135-kDa alpha-subunit of this receptor. 125I-Insulin binding capacity to ROS membranes is less than 5% that of IGF-I. The IGF-I-stimulated tyrosine kinase activity in solubilized and partially purified receptors from ROS autophosphorylates its own 95-kDa beta-subunits as well as other substrates like transducin. Insulin, which is 200-fold less potent than IGF-I in competing for 125I-IGF-I binding, is only 5-fold less potent than IGF-I in stimulating the receptor kinase activity. This suggests that insulin is much more potent than IGF-I in coupling ligand binding with kinase activation. The previously reported presence of IGF-I in the vitreous, together with our present studies, strongly suggest that the IGF-I receptor kinase, through phosphorylation of endogenous proteins like transducin, could play a role in mediating transmembrane signal transduction in ROS.     

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.     

Expression of insulin-like growth factor-I and its receptor by SV40-transformed rat granulosa cells

Cellular proliferation is a dominant aspect of ovarian follicular development in the rat, and insulin-like growth factor I (IGF-I) has been proposed as a mediator of cellular growth and differentiation in the ovary. An SV40-transformed rat granulosa cell line (RGA-41S) has been established as a model for studies on dividing cells of granulosa origin. Granulosa cells from the ovaries of immature diethylstilbestrol-treated rats were infected with the tsA255 mutant of SV40, followed by cloning in serum-free medium to select transformed cell lines which were serum independent. At the permissive temperature (33 C), RGA-41S cells exhibited a transformed phenotype and rapidly formed high density multilayers of compact cells that readily overgrew nontransformed cells. At the nonpermissive temperature (40 C) cell replication declined and division ceased after 4 days. Furthermore, at 40 C the cells grew as a monolayer and assumed a tetrahedral shape with a high cytoplasm-to-nucleus ratio, and displayed reduced ability to overgrow nontransformed cells. The transformed ovarian cells did not express detectable gonadotropin receptors and steroidogenic activity but retained their epithelial phenotype as demonstrated by cytokeratin staining of the cytoskeleton, the presence of microvilli, and the formation of tight junctions between cells. In support of the proposed autocrine-paracrine actions of IGF-I in the ovary, assay of conditioned serum-free culture medium revealed secretion of IGF-I-immunoreactive material by RGA-41S cells. HPLC-purified IGF-I immunoreactivity from these cells eluted with the same retention time as recombinant human IGF-I. When hybridized with a 32P-labeled rat IGF-I cDNA probe, poly(A)+ mRNA prepared from RGA-41S cells grown at both temperatures showed the typical three size classes of IGF-I mRNA on Northern blots (7.5, 1.7, and 0.8-1.2 kilobase kb), although the levels were somewhat higher at 33 C. The presence of IGF-I receptors in transformed cells was demonstrated by specific 125I-IGF-I binding to intact cells. Scatchard analysis indicated a single class of high affinity receptors at a density of 10(5) binding sites per cell and a dissociation constant (Kd) = 0.52 x 10(-9) M. Furthermore, hybridization of a 32P-labeled IGF-I receptor probe to Northern blots of poly(A+) RNA prepared from cells grown at 33 C and 40 C revealed an 11-kilobase rat IGF-I receptor mRNA. Physiological concentrations of IGF-I increased [3H]aminoisobutyric acid uptake by RGA-41S cells grown at either temperature, attesting to the retention of responsiveness to IGF-I in these transformed granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Insulin-like growth factor-I is internalized after binding to the type I insulin-like growth factor receptor

Receptor-mediated endocytosis may represent an important mechanism whereby peptide hormones exert their biological effects. The ability of recombinant insulin-like growth factor (IGF)-I to be internalized by cultured cells was evaluated in BRL-3A2 cells, a rat liver-derived cell line which lacks insulin receptors. Since recombinant IGF-I does not bind to the Type II IGF receptor, all specific binding of 125I-IGF-I in BRL-3A2 cells represents binding to the Type I receptor. Exposure of BRL-3A2 cells to IGF-I resulted in a rapid 50% downregulation of Type I IGF receptors. Only one-half of these binding sites were sensitive to treatment with trypsin, a phenomenon which indicates that the peptide and its receptor were internalized after the cells were exposed to IGF-I. In conclusion, these experiments demonstrate that IGF-I can be internalized by cultured cells via the Type I IGF receptor, and suggest that IGF hormone action may be exerted by receptor-mediated endocytosis.     

Paracellular transport of insulin-like growth factor-I (IGF-I) across human umbilical vein endothelial cell monolayers

Insulin-like growth factors (IGFs) are well defined mitogens and growth promoters, which are found in blood associated with high affinity IGF binding proteins (IGFBPs). In vivo, the endothelium is potentially the primary site of uptake of IGFs or IGF-IGFBP complexes from blood for transport to the extravascular space. However, the pathway and mechanisms by which IGFs cross the endothelial cell barrier are not known. The presence of high affinity receptors for IGF-I and IGF-II on human umbilical vein endothelial (HUVE) cells was demonstrated by (i) radio-receptor assays using both IGF-I and IGF-II and (ii) affinity label cross-linking studies. In addition, Western ligand blotting and immunoblotting revealed that IGFBP-2, -3, and -4 are secreted into serum-free media conditioned by confluent HUVE cell monolayers. To study transendothelial migration of IGF-I, HUVE cells were grown on microporous membranes in a bichamber system. When compared with membranes without cells, HUVE monolayers restricted the passage of ¹²⁵I-IGF-I and [³H]inulin, whereas the control Madin Darby canine kidney (MDCK) cell line virtually excluded all passage of these molecules. Transport of ¹²⁵I-IGF-I across HUVE cell monolayers was not significantly different to that of [³H]inulin, a paracellular probe. Moreover, ¹²⁵I-IGF-I transport was not inhibited by either excess unlabelled IGF-I or a monoclonal antibody to the type I IGF receptor at a concentration shown to inhibit ¹²⁵I-IGF-I binding to HUVE cell monolayers. Our findings show that the movement of free IGF-I across HUVE cell monolayers occurs via a paracellular route and not by a receptor-mediated, transcellular pathway. J. Cell. Physiol. 170:290–298, 1997. © 1997 Wiley-Liss, Inc.     

Human recombinant insulin-like growth factor I. II. Binding characterization and radioreceptor assay development using BALB/c 3T3 mouse embryo fibroblasts

Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized, resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (¹²⁵I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding sites with K _d =59.6 pM and an estimated 1.57×10⁵ receptors/cell. Half-maximal displacement of bound¹²⁵I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for¹²⁵I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that¹²⁵I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Disulphide reduction alters the immunoreactivity and increases the affinity of insulin-like growth-factor-I receptors in human placenta.

We previously identified two forms of the insulin-like growth-factor-I (IGF-I) receptor in human placenta: a lower-affinity form reactive with an autoantiserum (B-2) to the insulin receptor and a higher-affinity non-immunoreactive form [Jonas & Harrison (1985) J. Biol. Chem. 260, 2288-2294]. Evidence is now presented that the lower-affinity immunoreactive forms are convertible into higher-affinity non-immunoreactive forms via reduction of receptor disulphide bonds. Treatment of placental membranes with increasing concentrations of dithiothreitol (DTT): (1) converted native Mr-290 000 heterotetrameric IGF-I receptors into Mr-180 000 dimers (determined by chemical cross-linking of 125I-IGF-I with disuccinimidyl suberate); (2) increased 125I-IGF-I binding, owing to an increase in receptor affinity; and (3) abolished the reactivity of Triton-solubilized IGF-I receptors with antiserum B-2 and transformed the curvilinear plot of IGF-I binding to a linear form. In isolated complexes between receptor and B-2 antibody, DTT increased 125I-IGF-I binding and released a single class of higher affinity IGF-I receptors of Mr 180,000. Thus DTT-treated IGF-I receptors have similar properties to the higher-affinity non-immunoreactive forms of the native receptor, except that reduced dimeric forms are not detected by cross-linking of 125I-IGF-I to native membranes. Cleavage of the inter-dimeric disulphide bonds is therefore not a prerequisite for higher-affinity binding or loss of immunoreactivity. These observations suggest that the thiol redox state of the IGF-I receptor in vivo is an important determinant of receptor conformation and therefore of the biological responses to IGF-I.