Insulin-like growth factor (IGF)/somatomedin receptor subtypes: structure, function, and relationships to insulin receptors and IGF carrier proteins

The insulin-like growth factors IGF-I and IGF-II are mitogenic polypeptides with a high degree of chemical homology. Two distinct subtypes of receptors for the IGFs have been identified on the basis of structure and binding specificity. Type I IGF receptors bind IGF-I with equal or greater affinity than IGF-II, and also bind insulin with a low but definite affinity. They are structurally homologous to insulin receptors, containing disulfide-linked a-subunits that bind the peptides and beta-subunits that have intrinsic tyrosine-specific kinase activity. Type II IGF receptors typically bind IGF-II with greater affinity than IGF-I, and do not interact with insulin. They consist of a single polypeptide and lack tyrosine kinase activity. Because of the extensive cross-reactivity of IGF-I and IGF-II with both type I and type II receptors, we believe that potentially either receptor may mediate the biological responses of either peptide. Type I IGF receptors have been shown to mediate the mitogenic effects of the IGFs in some cell types. Whether type II IGF receptors mediate the same or different functions remains to be elucidated.     

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.     

Tyrosine phosphorylation of common and specific sets of cellular proteins rapidly induced by insulin, insulin-like growth factor I, and epidermal growth factor in an intact cell

KB cells respond to insulin and insulin-like growth factor I (IGF-I) in a closely similar way (induction of membrane ruffling, stimulation of pinocytosis, and amino acid transport) but respond to epidermal growth factors (EGF) in a similar but distinct way. In the KB cells, using phosphotyrosine-specific antibody we have found that: the receptors for insulin (beta subunit), IGF-I (beta subunit), and EGF undergo tyrosine phosphorylation as early as 10 s after addition of their respective ligands; a 185-kDa protein is rapidly (less than 10 s) tyrosine phosphorylated by insulin and IGF-I through their respective receptor kinases but not EGF; tyrosine phosphorylation of a 190-kDa glycoprotein is rapidly (less than 10 s) induced by EGF through EGF receptor kinase; and tyrosine phosphorylation of a 240-kDa protein is stimulated within 30 s by all three growth factors. These patterns of tyrosine phosphorylation could be causally related to biological responses induced by the three growth factors.     

Madin-Darby canine kidney cells display type I and type II insulin-like growth factor (IGF) receptors

Using affinity cross-linking techniques, we report the presence of type I IGF and type II IGF receptors in Madin-Darby canine kidney cells, a line of cells lacking insulin receptors. The IGF receptors were further characterized by competition binding studies and found to be similar to IGF receptors in other tissue types. In Madin-Darby canine kidney cells, the type I IGF receptor binds IGF-I greater than IGF-II greater than insulin and the type II IGF receptor binds IGF-II and IGF-I with approximately the same affinity, but does not bind insulin.     

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.     

Rapid stimulation of fluid-phase endocytosis and exocytosis by insulin, insulin-like growth factor-I, and epidermal growth factor in KB cells

Effects of growth factors on fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells were examined by measuring horseradish peroxidase (HRP) as a marker. Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) promoted HRP accumulation. They also stimulated the efflux of the preloaded HRP from the cells. From these results it follows that these growth factors stimulate the influx as well as the efflux of HRP, because the accumulation rate is the sum of the influx rate and the efflux rate. The stimulation of both HRP accumulation and HRP efflux was rapidly induced within 2-4 min of the addition of growth factors and persisted for at least 60 min. The concentrations eliciting half-maximal stimulatory effects of insulin, IGF-I, and EGF were about 5 X 10(-7), 1 X 10(-9), and 5 X 10(-10) M, respectively. aIR-3 (anti-type I IGF receptor antibody) completely blocked the stimulation of HRP accumulation by IGF-I but very slightly inhibited the stimulation by insulin. The 528 IgG (anti-EGF receptor antibody) inhibited the stimulation of HRP accumulation by EGF. These results indicated that each of these growth factors stimulates the HRP accumulation mediated by the corresponding (homologous) growth factor receptors. The rapid stimulation of fluid-phase influx and efflux may constitute one of the common early cellular responses to growth factors.     

Insulin-like growth factor II binding and action in human fetal fibroblasts

To investigate the role of insulin-like growth factor II (IGF-II) in human prenatal growth, IGF-II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific binding of IGF-II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5-10 times the amount of IGF-I binding found in these cells. IGF-I and IGF-II caused dose-dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF-II was sevenfold less potent than IGF-I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF-II in stimulating [14C]AIB uptake approach that of IGF-I. Similar results were obtained when IGF-I and IGF-II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alpha IR-3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF-I and IGF-II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF-II is a potent metabolic and mitogenic stimulus for human fetal fibroblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF-II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.     

The type I insulin-like growth factor receptor is a motility receptor in human melanoma cells

Insulin-like growth factors I and II (IGF-I and II) and insulin are chemotactic agents for the human melanoma cell line A2058. As shown in this report, the motility receptor mediating this response is the heterodimeric type I IGF receptor. These three factors are able to compete with 125I-labeled IGF-I for binding to the cell surface with IC50 values equal to approximately 2 (IGF-I), approximately 150 (IGF-II), and approximately 300 nM (insulin). Cross-linking of 125I-IGF-I to the cell surface with disuccinimidyl suberate followed by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveals a 130-kDa protein (reduced) consistent with the alpha component of a type I receptor and a 38-kDa protein which does not bind insulin, and thus could be another IGF-I cell surface binding protein. The anti-IGF-I receptor monoclonal antibody (alpha IR-3) also competes with labeled IGF-I in binding experiments. In contrast, a control monoclonal antibody, matched to alpha IR-3 with respect to IgG subclass, has no significant effect on IGF-I binding. While alpha IR-3 inhibits the motility induced by IGF-I, IGF-II, and insulin, pertussis toxin (0.01-1.0 micrograms/ml) has no significant effect on the motility induced by the insulin-like growth factors or insulin on this cell line. Therefore, the type I IGF receptor appears to mediate a highly potent pertussis toxin-insensitive motility response to IGF-I, IGF-II, and insulin. In contrast, motility induced by the autocrine motility factor, a cytokine produced by the A2058 cells, is not affected by alpha IR-3 but is extremely sensitive to pertussis toxin. When mixtures of autocrine motility factor and IGF-I are employed to induce chemotaxis, the resulting motility is greater than that induced by either agent alone. These data indicate that motility in this melanoma cell line can be initiated through multiple receptors that stimulate the cells by separate transduction pathways. This capability to respond to multiple stimuli could enhance the metastatic potential.     

Presence of insulinlike growth factor receptors and lack of insulin receptors on fetal bovine smooth muscle cells

Summary Previous investigations have demonstrated specific receptors and associated mitogenic actions for insulin and insulinlike growth factors I and II (IGF-I and II) in postnatal bovine aortic smooth muscle. Using fetal tissue we have observed different patterns of binding and action for these peptides. Smooth muscle cells isolated from near-term fetal bovine aortae were studied in early passage. Specific receptors for both IGF-I and IGF-II were identified. Specific binding averaged 5.7%/2.5×10⁵ cells for IGF-I, and 16.2% for IGF-II, and 0.3% for insulin. High affinity K _d for both IGF receptors were nanomolar. IGF-II was fivefold less potent than IGF-I in displacing IGF-I binding. IGF-I showed no affinity for the IGF-II receptor. Insulin, at physiologic concentrations, was incapable of displacing either IGF-I or IGF-II binding. Cellular incorporation of [methyl-³H]thymidine was stimulated at the lowest dose of IGF-I tested, 0.5 ng/ml. IGF-II showed no effect up to 100 ng/ml, after which a sharp increase in incorporation was noted. Insulin had a similar effect only at concentrations >0.5 μg/ml, with a maximal response noted at 5 to 10 μg/ml. Our results indicate that fetal bovine aortic smooth muscle cells have an abundance of IGF receptors but lack specific insulin receptors. In addition, IGF-II binding levels are three times higher than for IGF-I. These results are consistent with observations in other species, in which a predominance of IGF over insulin receptors has been demonstrated in fetal tissue, and provide further evidence for a role for the IGFs in embryonic cellular metabolism. This project was supported by grants AM22190 (R. L. H.), AM28229 (R. G. R.) from the National Institutes of Health, Bethesda, MD, and Research Career Development Award AM01275 from the NIH (R. G. R.). Dr. Lee was the recipient of a fellowship award from the Juvenile Diabetes Foundation International and is currently supported by funds from the American Diabetes Association. Dr. Benitz is the recipient of a Clinician-Scientist Award from the American Heart Association, with funds contributed in part by the California Affiliate.     

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.     

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 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.     

Interactions between growth factor receptors and corresponding monoclonal antibodies in human tumors

Monoclonal antibodies (MAbs) to the human epidermal growth factor (EGF) receptor, the type I insulin-like growth factor (IGF) receptor, and the nerve growth factor (NGF) receptor were used to study the growth regulation of malignant cells. Anti-EGF receptor MAb 425 inhibited the growth of A 431 squamous carcinoma cells which express high numbers of EGF receptors on their surfaces. Growth inhibition induced by MAb 425 was accompanied by alterations of the cell-cycle distribution of these cells, indicating the ability of a monoclonal antibody to act as a biologically active ligand. Growth stimulation of melanoma cells by EGF was unrelated to EGF receptor expression on the cell surface. Insulin- and IGF-I-induced growth stimulation of melanoma cells was inhibited by MAb alpha IR-3 which reacts with the type I IGF receptor. This result indicates that the type I IGF receptor mediated growth stimulation not only by IGF-I but also by insulin. Normal melanocytes and cells of all stages of tumor progression expressed in tissue culture the receptor for NGF, but no effect on the growth of these cells has been observed.     

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.     

Protein kinase C-dependent and -independent pathways in the growth factor-induced cytoskeletal reorganization

Human epidermoid carcinoma KB cells exhibit rapid induction of membrane ruffling in response to epidermal growth factor (EGF), insulin, and insulin-like growth factor-I (IGF-I) (Kadowaki, T., Koyasu, S., Nishida, E., Sakai, H., Takaku, F., Yahara, I., and Kasuga, M. (1986) J. Biol. Chem. 261, 16141-16147). We have analyzed the role of protein kinase C (PKC) in this response. Treatment of KB cells with 4 beta-phorbol 12,13-dibutyrate (PDBu) (100 ng/ml) for 30 min caused translocation of PKC to the membrane. This treatment completely inhibited the induction of membrane ruffling by EGF, insulin, and IGF-I. Prolonged treatment with PDBu (200 ng/ml for 15 h) induced complete depletion of the PKC activity in the cells. Under these conditions, EGF binding to cells and autophosphorylation of the EGF receptor occurred normally, while EGF could not induce membrane ruffling. In contrast, insulin- or IGF-I-induced membrane ruffling occurred normally in the PKC-depleted cells. Moreover, H-7 (PKC inhibitor) inhibited only EGF-induced membrane ruffling in a dose-dependent manner. We further found that EGF, but not insulin/IGF-I, caused transient translocation of PKC to the membrane. All these results suggest that PKC is required for the membrane ruffling induced by EGF but not for that induced by insulin/IGF-I. Therefore, there are PKC-dependent and independent pathways in the growth factor-induced membrane ruffling. Furthermore, we propose dual roles of PKC in the EGF signaling, a signal transmitting role and a negative feedback role.     

Regulation of the genes for insulin-like growth factor (IGF) I and II and their receptors by steroids and gonadotropins in the ovary

Insulin-like growth factors (IGFs) I and II are two single-chain polypeptide hormones that are structurally related to each other and to proinsulin. Among the large number of growth factors involved in ovarian physiology, IGF-I and IGF-II are considered to be important progression factors for ovarian follicular development. To explore the ovarian expression of IGF-I, IGF-II and their receptor genes, a solution hybridization/RNase protection assay, was used. IGF-I mRNA was seen in the granulosa cells, and IGF-II mRNA in the theca-interstitial compartment. To study the hormonal regulation of the IGF-I and IGF-II gene, immature (21-day-old) hypohysectomized rats were treated with FSH (10 μg/day),GH (150 μg/day) and diethylstilbestrol (DES subcutaneous implant/5 days). Estrogen differentially regulated ovarian IGF-I and IGF-II gene expression. In concert with GH, estrogen up-regulated ovarian IGF-I mRNA, but significantly decreased hepatic IGF-I gene expression. Both IGF receptors (type I and type II) as well as the insulin receptor gene, were expressed in both ovarian cells. The expression of the type IIGF receptor gene (but not the type II IGF gene) was up-regulated by FSH and estrogen in vivo. In conclusion, these studies may serve to better understand the auto paracrine role of IGF, and their receptors in the pathophysiology of follicle recruitment, oocyte maturation and potentially embryo development.     

Insulin-like growth factor II binding to the type I somatomedin receptor. Evidence for two high affinity binding sites

We have previously shown that the antireceptor antibody alpha IR-3 inhibits binding of 125I-somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) to the 130-kDa alpha subunit of the type I receptor in human placental membranes, but does not block 125I-insulin-like growth factor II (IGF-II) binding to a similar 130-kDa complex in these membranes. To determine whether the 130-kDa 125I-IGF-II binding complex represents a homologous receptor or whether 125I-IGF-II binds to the type I receptor at a site that is not blocked by alpha IR-3, type I receptors were purified by affinity chromatography on Sepharose linked alpha IR-3. The purified receptors bound both 125I-Sm-C/IGF-I and 125I-IGF-II avidly (KD = 2.0 X 10(-10) M and 3.0 X 10(-10) M, respectively). The maximal inhibition of 125I-Sm-C/IGF-I binding by the antibody, however, was 62% while only 15% of 125I-IGF-II binding was inhibited by alpha IR-3. In the presence of 500 nM alpha IR-3, Sm-C/IGF-I bound with lower affinity (KD = 6.5 X 10(-10) M) than IGF-II (KD = 4.5 X 10(-10) M) and IGF-II was the more potent inhibitor of 125I-Sm-C/IGF-I binding. These findings suggest that the type I receptor contains two different binding sites. The site designated IA has highest affinity for Sm-C/IGF-I and is blocked by alpha IR-3. Site IB has higher affinity for IGF-II than for Sm-C/IGF-I and is not blocked by alpha IR-3.     

Expression and function of insulin-like growth factor receptors on anti-CD3-activated human T lymphocytes.

The pattern of expression of receptors for insulin-like growth factors (IGF-I and IGF-II) and insulin was studied on monocyte-depleted human peripheral blood T cells activated via anti-CD3. Binding assays demonstrated the sequential appearance of receptors for IGF-I, IGF-II, and insulin on activated T cells. IGF-IR appeared early, their expression reaching maximum levels at or before the peak of cellular proliferation. IGF-IIR expression generally followed that of the IGF-IR and was more transient, with increases and decreases in expression paralleling the rise and decline of cellular proliferation. Insulin receptor expression remained low throughout the activation time course. The identity of the IGFR on anti-CD3-activated T cells was confirmed in affinity cross-linking experiments. These data demonstrated a 135,000 Mr peptide that specifically binds radiolabeled IGF-I and corresponds to the alpha subunit of the type I IGF-IR, and a 260,000 Mr peptide that specifically binds radiolabeled IGF-II and corresponds to the type II IGFR. We have additionally found that IGF-I and IGF-II (in nanomolar concentrations) produce as much as a threefold enhancement of T cell proliferation early in the activation process, correlating with the early appearance of IGF-IR. The effect of both IGF appeared to be mediated through the type I receptor, since an antibody (alpha IR3), which blocks binding to the alpha subunit of this receptor, inhibited enhancement by up to 83%. Furthermore, we have found expression of IGF-IR on T cells after activation to be associated with both CD4+ and CD8+ T cell subpopulations. These observations provide a foundation for investigating the contribution of IGF in regulating T cell proliferation, differentiation, and effector function.     

Insulin regulation of insulin-like growth factor action in rat hepatoma cells

We have reported previously that insulin causes a complete but reversible desensitization to insulin action in rat hepatoma HTC cells in tissue culture, and that this insulin resistance is mediated by postbinding mechanisms rather than receptor down-regulation (Heaton, J. H., and Gelehrter, T. D. (1981) J. Biol. Chem. 256, 12257-12262). We report here that insulin causes a similar desensitization to the induction of tyrosine aminotransferase by the insulin-like growth factors IGF-I and IGF-II isolated from human plasma, and by multiplication-stimulating activity, the rat homologue of IGF-II. The results of both competition-binding studies and affinity cross-linking experiments indicate that insulin-like growth factors (IGFs) bind primarily to IGF receptors rather than to insulin receptors. The low concentrations at which these factors induce transaminase is consistent with their acting primarily via IGF receptors. This is confirmed by experiments utilizing anti-insulin receptor antibody which both inhibits 125I-insulin binding and shifts the concentration dependence of insulin induction of tyrosine aminotransferase to the right. This same immunoglobulin does not inhibit 125I-multiplication-stimulating activity binding and only minimally inhibits 125I-IGF-I binding. Anti-insulin receptor antibody also does not significantly shift the concentration dependence for the IGFs, suggesting that IGFs induce transaminase by acting via IGF receptors. Although insulin down regulates insulin receptors, it does not decrease IGF-I or IGF-II binding. We conclude that insulin causes desensitization of HTC cells to IGFs by affecting a postbinding step in IGF action, which may be common to the actions of both insulin and insulin-like growth factors.     

Heterogeneity of insulin-like growth factor-I affinity for the insulin-like growth factor-II receptor: comparison of natural, synthetic and recombinant DNA-derived insulin-like growth factor-I

Although insulin-like growth factors (IGF) I and II bind with high affinity to structurally discrete receptors, they bind with a lesser affinity to each other's receptor. We have evaluated the affinity of five different IGF-I preparations (three natural IGF-I preparations, one synthetic preparation, and one recombinant DNA-derived) for the IGF-II receptor in rat placental membranes, 18-54,SF cells and BRL-3A cells. In all tissues tested, the natural IGF-I preparations demonstrated an affinity for the IGF-II receptor which was 10-20% that of IGF-II. However, the recombinant and synthetic IGF-I preparations exhibited substantially lower affinities than natural IGF-I for this receptor, with only 10-25% reduction in (125-I)iodo IGF-II binding at peptide concentrations up to 400 ng/ml. Radioimmunoassay of the natural IGF-I preparations with an antibody directed against the unique C-peptide region of IGF-II demonstrated that contamination of IGF-I preparations with immunoreactive IGF-II could not exceed 5%. These results demonstrate that IGF-I purified from human plasma has a different affinity for the IGF-II receptor than does synthetic or recombinant IGF-I. Furthermore, these data are consistent with the hypothesis that IGF-I, itself, may be heterogeneous, and that subforms may vary in their affinities for the IGF receptors. Alternatively, IGF-I preparations which have been considered to be pure may be contaminated with small amounts of IGF-II, resulting in overestimation of the affinity of IGF-I for the type II IGF receptor.