Insulin and insulin-like growth factor II differentially regulate endocytic sorting and stability of insulin receptor isoform A

The insulin receptor isoform A (IR-A) binds both insulin and insulin-like growth factor (IGF)-II, although the affinity for IGF-II is 3-10-fold lower than insulin depending on a cell and tissue context. Notably, in mouse embryonic fibroblasts lacking the IGF-IR and expressing solely the IR-A (R-/IR-A), IGF-II is a more potent mitogen than insulin. As receptor endocytosis and degradation provide spatial and temporal regulation of signaling events, we hypothesized that insulin and IGF-II could affect IR-A biological responses by differentially regulating IR-A trafficking. Using R-/IR-A cells, we discovered that insulin evoked significant IR-A internalization, a process modestly affected by IGF-II. However, the differential internalization was not due to IR-A ubiquitination. Notably, prolonged stimulation of R-/IR-A cells with insulin, but not with IGF-II, targeted the receptor to a degradative pathway. Similarly, the docking protein insulin receptor substrate 1 (IRS-1) was down-regulated after prolonged insulin but not IGF-II exposure. Similar results were also obtained in experiments using [NMeTyr(B26)]-insulin, an insulin analog with IR-A binding affinity similar to IGF-II. Finally, we discovered that IR-A was internalized through clathrin-dependent and -independent pathways, which differentially regulated the activation of downstream effectors. Collectively, our results suggest that a lower affinity of IGF-II for the IR-A promotes lower IR-A phosphorylation and activation of early downstream effectors vis à vis insulin but may protect IR-A and IRS-1 from down-regulation thereby evoking sustained and robust mitogenic stimuli.     

Soluble insulin-like growth factor II/mannose 6-phosphate receptor inhibits DNA synthesis in insulin-like growth factor II sensitive cells

The soluble form of the insulin-like growth factor II (IGF-II)/mannose 6-P (IGF-II/M6P) receptor is released by cells in culture and circulates in the serum. It retains its ability to bind IGF-II and blocks IGF-II-stimulated DNA synthesis in isolated rat hepatocytes. Because these cells are not normally stimulated to divide by IGF-II in vivo, the effect of soluble IGF-II/M6P receptor on DNA synthesis has been further investigated in two cell lines sensitive to IGF-II; mouse 3T3(A31) fibroblasts, stimulated by low levels of IGF-II following priming by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and Buffalo rat liver (BRL) cells, which secrete IGF-II and proliferate in the absence of exogenous growth factors. Soluble IGF-II/M6P receptor (0.2-2.0 microgram/ml) purified from a rat hepatoma cell line inhibited DNA synthesis (determined by dThd incorporation) in both cell lines. Basal DNA synthesis was very low in serum-free 3T3 cells, but high in serum-free BRL cells, possibly as a result of autocrine IGF-II production. The inhibitory effect was reversible in cells preincubated with soluble receptor prior to incubation with growth factors and could also be overcome by excess IGF-II. Soluble receptor was more potent in IGF-II-stimulated 3T3 cells and serum-free BRL cells than in BRL cells incubated with serum. Mean inhibition by four preparations of soluble receptor (1 microgram/ml) was 34.7% +/- 4.4% in BRL cells stimulated with fetal calf serum (FCS) (5%) compared to 54.8% +/- 4.2% in serum-free BRL cells (P = 0.05) and 60.6% +/- 6.5% (P = 0.02) in 3T3 cells stimulated by PDGF, EGF, and IGF-II. Soluble receptor had no effect on DNA synthesis in 3T3 cells stimulated with IGF-I. These results demonstrate that soluble receptor, at physiological concentrations, can block proliferation of cells by IGF-II and could therefore play a role in blocking tumor growth mediated by IGF-II.     

Variable accumulation of insulin-like growth factor II in mouse tissues deficient in insulin-like growth factor II receptor

The insulin-like growth factor II receptor mediates endocytosis of insulin-like growth factor II, resulting in growth factor degradation in lysosomes. This degradation is an important regulator of growth factor activity in vivo, as shown by the phenotype of receptor deficient mice. Recent evidence suggests that the insulin-like growth factor II receptor functions as a tumour supressor in humans, and that loss of receptor function leads to increased levels of the growth factor in tumours. It is difficult to establish such a causal relationship in human tumours however, since most tumours have undergone several genetic changes by the time they are examined. Using mouse embryos deficient in receptor expression, and an insulin-like growth factor II-specific radioimmunoassay, we tested the hypothesis that lack of receptor function leads to local accumulation of insulin-like growth factor II. We found that mutant blood and skeletal muscle had excess insulin-like growth factor II, but that mutant lungs and liver had no accumulation. Mutant hearts had less growth factor than wild-type hearts, an unexpected observation, since the normal embryonic heart expresses very high levels of insulin-like growth factor II receptor, and mutant mice apparently die of congestive heart failure. The placentas of mutant mice were larger than those of wild-type, but this did not correlate with an excess of placental insulin-like growth factor II. These results indicate that lack of insulin-like growth factor II receptor can lead to local excess of the growth factor but that such excess is not a necessary consequence of receptor-deficiency.     

Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR

The insulin receptor (IR) lacking the alternatively spliced exon 11 (IR-A) is preferentially expressed in fetal and cancer cells. The IR-A has been identified as a high-affinity receptor for insulin and IGF-II but not IGF-I, which it binds with substantially lower affinity. Several cancer cell types that express the IR-A also overexpress IGF-II, suggesting a possible autocrine proliferative loop. To determine the regions of IGF-I and IGF-II responsible for this differential affinity, chimeras were made where the C and D domains were exchanged between IGF-I and IGF-II either singly or together. The abilities of these chimeras to bind to, and activate, the IR-A were investigated. We also investigated the ability of these chimeras to bind and activate the IR exon 11+ isoform (IR-B) and as a positive control, the IGF-I receptor (IGF-1R). We show that the C domain and, to a lesser extent, the D domains represent the principal determinants of the binding differences between IGF-I and IGF-II to IR-A. The C and D domains of IGF-II promote higher affinity binding to the IR-A than the equivalent domains of IGF-I, resulting in an affinity close to that of insulin for the IR-A. The C and D domains also regulate the IR-B binding specificity of the IGFs in a similar manner, although the level of binding for all IGF ligands to IR-B is lower than to IR-A. In contrast, the C and D domains of IGF-I allow higher affinity binding to the IGF-1R than the analogous domains of IGF-II. Activation of IGF-1R by the chimeras reflected their binding affinities whereas the phosphorylation of the two IR isoforms was more complex.     

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.     

Insulin/insulin-like growth factor I hybrid receptors have different biological characteristics depending on the insulin receptor isoform involved

The insulin receptor (IR) and the insulin-like growth factor I receptor (IGF-IR) have a highly homologous structure, but different biological effects. Insulin and IGF-I half-receptors can heterodimerize, leading to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I with high affinity. As the IR exists in two isoforms (IR-A and IR-B), we evaluated whether the assembly of the IGF-IR with either IR-A or IR-B moieties may differently affect Hybrid-R signaling and biological role. Three different models were studied: (a) 3T3-like mouse fibroblasts with a disrupted IGF-IR gene (R(-) cells) cotransfected with the human IGF-IR and with either the IR-A or IR-B cDNA; (b) a panel of human cell lines variably expressing the two IR isoforms; and (c) HepG2 human hepatoblastoma cells predominantly expressing either IR-A or IR-B, depending on their differentiation state. We found that Hybrid-Rs containing IR-A (Hybrid-Rs(A)) bound to and were activated by IGF-I, IGF-II, and insulin. By binding to Hybrid-Rs(A), insulin activated the IGF-I half-receptor beta-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-Rs(B) bound to and were activated with high affinity by IGF-I, with low affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs were more effectively stimulated in Hybrid-R(A)-containing cells than in Hybrid-R(B)-containing cells. The relative abundance of IR isoforms therefore affects IGF system activation through Hybrid-Rs, with important consequences for tissue-specific responses to both insulin and IGFs.     

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.     

The receptor for insulin-like growth factor II mediates an insulin-like response.

Insulin-like growth factor II (IGF-II) shares sequence homology and predicted three-dimensional structure with insulin and IGF-I. IGF-II can bind, therefore, to a limited extent with the receptors for these two other hormones, as well as to a distinct receptor for IGF-II. Previous studies have been unable to attribute a particular response of IGF-II through its own receptor. In the present studies, the IGF-II receptor is shown to mediate the stimulation of glycogen synthesis in human hepatoma cells since: (i) IGF-II is found to be capable of stimulating a response at concentrations in which it would primarily interact with its own receptor; (ii) the response to IGF-II was not blocked by monoclonal antibodies which inhibit the responses of cells through the insulin and IGF-I receptors; and (iii) polyclonal antibodies to the IGF-II receptor were found to mimic the ability of IGF-II to stimulate glycogen synthesis. These results indicate that the IGF-II receptor mediates a particular biological response--stimulation of glycogen synthesis in hepatoma cells. Furthermore, a monovalent Fab fragment of the polyclonal antibody to the IGF-II receptor was also shown to stimulate glycogen synthesis in these cells. These data indicate that clustering of the IGF-II receptor is not required to stimulate a biological response.     

A dominant negative type I insulin-like growth factor receptor inhibits metastasis of human cancer cells

We have previously shown that LCC6 wild-type (WT) cells, a metastatic variant of MDA-MB-435 cancer cells originally derived from a breast cancer patient, exhibit enhanced motility in response to IGF-I compared with the parent MDA-MB-435 cells. To further understand the role of the type I insulin-like growth factor (IGF) receptor (IGF1R) in cancer metastasis we inhibited signaling via IGF1R using a C-terminal-truncated IGF1R. The truncated receptor retains the ligand binding domain but lacks the autophosphorylated tyrosine residues in the carboxyl terminus. Cells stably transfected with this truncated receptor (LCC6-DN cells) overexpressed the truncated IGF1R messenger RNA nearly 50-fold over endogenous receptor. The truncated receptor in the LCC6-DN cells behaved in a dominant negative manner to inhibit endogenous IGF1R activation by IGF-I. Compared with the LCC6-WT cells, LCC6-DN cells failed to phosphorylate the adaptor proteins insulin receptor substrate-1 and -2 in response to IGF-I and did not activate Akt after exposure to IGF-I. Unlike LCC6-WT cells, LCC6-DN cells did not show enhanced motility in response to IGF-I. To assay for metastasis, LCC6-WT and LCC6-DN cells were injected into the mammary fat pads of mice, and the primary xenograft tumors were removed after 21 days. Mice sacrificed 5 weeks later showed multiple lung metastases derived from LCC-WT xenografts, whereas mice harboring LCC6-DN xenografts showed no lung metastases. Our data show that IGF1R can regulate several aspects of the malignant phenotype. In these cells, metastasis but not proliferation requires IGF1R function.     

Distinct biologically active receptors for insulin, insulin-like growth factor I, and insulin-like growth factor II in cultured skeletal muscle cells

The expression of insulin-like growth factor (IGF) receptors at the cell surface and the changes in IGF responsiveness during differentiation were studied in the L6 skeletal muscle cell line. Throughout the entire developmental sequence, distinct receptors for IGF I and IGF II that differed in structure and peptide specificity could be demonstrated. During differentiation, both 125I-IGF I and 125I-IGF II binding to the L6 cells decreased as a result of a 3-4-fold reduction in receptor number, whereas 125I-insulin binding increased. Under nonreducing conditions, disuccinimidyl suberate cross-linked 125I-IGF I and 125I-IGF II to two receptor complexes with apparent Mr greater than 300,000 (type I) and 220,000 (type II). Under reducing conditions, the apparent molecular weight of the type I receptor changed to Mr 130,000 (distinct from the 120,000 insulin receptor) and the type II receptor changed to 250,000. IGF I and IGF II both stimulated 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake in the L6 cells with a potency close to that of insulin, apparently through interaction with their own receptors. The stimulatory effects of IGF II correlated with its affinity for the type II but not the type I IGF receptor, as measured by inhibition of affinity labeling, whereas the effects of IGF I correlated with its ability to inhibit labeling of the type I receptor. In spite of the decrease in type I and type II receptor number, stimulation of 2-deoxy-glucose and alpha-aminoisobutyric acid uptake by the two IGFs increased during differentiation.     

Transforming growth factor-beta isoform expression in insulin-like growth factor stimulated myogenesis

Transforming growth factor betas (TGF-b?s) are the defining members of a superfamily of small proteins that are involved in the regulation of development and morphogenesis in a wide array of systems. Previous studies have demonstrated that TGF-b?s both inhibit and, under specialized conditions, induce the differentiation of myoblasts. TGF-b?s have been shown to be secreted by mouse C2C12 myoblast cultures undergoing differentiation. Insulin-like growth factors (IGFs) have also been shown to be secreted by myoblasts and to induce myogenesis. This study characterizes the effects of IGF treatment on the expression and secretion of TGF-b?s in the IGF-sensitive L6A1 myoblast line. IGF downregulated the expression of TGF-b?3 in a concentration-dependent manner at 24 and 48 hours; TGF-b?1 was not sensitive to IGF treatment at 24 hours but was downregulated by IGFs at 48 hours. This downregulation was mediated by the type I IGF receptor and modulated by IGF binding proteins secreted by the myoblasts. Some reexpression of TGF-b?1 and TGF-b?3 mRNAs was observed after extensive morphological differentiation had occurred. These results support the hypothesis that IGFs act through the IGF type I receptor as part of a concerted mechanism to modulate expression of the TGF-b? genes, as part of a coordinated set of changes associated with terminal myogenic differentiation. © 1995 Wiley-Liss, Inc.     

Biosynthesis and processing of the type II insulin-like growth factor receptor in H-35 hepatoma cells

The biosynthesis and post-translational processing of the insulin-like growth factor II (IGF-II) receptor has been studied in H-35 hepatoma cells using a specific polyclonal anti-receptor immunoglobulin preparation. Cells were pulse-labeled with [35S]methionine followed by incubation with excess unlabeled methionine (chase). Gel electrophoresis of the immunoadsorbed receptors shows that the receptor is first synthesized as a 245-kDa precursor which is transformed to the mature 250-kDa form with a half-time of about 2 h. The 245-kDa precursor could also be labeled biosynthetically with [3H]mannose, only one-half of which was ultimately found associated with the 250-kDa product. Neuraminidase converts the 250-kDa receptor species to a 245-kDa form. Whereas the 250-kDa receptor is insensitive to detectable cleavage by endoglycosidase H, digestion of the 245-kDa species with this enzyme produces a 232-kDa form. A similar 232-kDa receptor species accumulates in H-35 cells incubated with tunicamycin (2 micrograms/ml). This tunicamycin-induced aglyco-receptor is not further processed to the 250-kDa form. Monensin (50 nM) blocks receptor processing at the 245-kDa stage. Endoglycosidase H treatment of the monensin-induced 245-kDa species indicates that this is a mixture of partially processed precursors having equivalent Mr. No evidence was obtained for the presence of O-linked oligosaccharides on the IGF-II receptor. The IGF-II binding activity of the three different biosynthetic forms of the receptor was assessed by affinity cross-linking of 125I-IGF-II to the receptors using disuccinimidyl suberate. Both the mature 250-kDa receptor and the neuraminidase-digested 245-kDa form specifically bound 125-I-IGF-II. However, the 232-kDa aglyco-receptor had no detectable IGF-II binding activity using this method. In summary, these studies show: 1) that the H-35 cell IGF-II receptor is synthesized first as a 245-kDa precursor having 4-6 high-mannose oligosaccharide side chains, 2) processing of the receptor oligosaccharides by mannose removal and terminal sialylation converts the 245-kDa precursor to the 250-kDa mature product which has been previously identified as the functional receptor in the plasma membrane, 3) the apparent molecular mass of the receptor in the absence of N-glycosylation is 232-kDa, and 4) glycosylation of the IGF-II receptor is required for the acquisition of IGF-II binding activity.     

The insulin-like growth factor II/mannose-6-phosphate receptor

Recent evidence from molecular cloning, biochemical and immunological experiments has established that the cation-independent mannose-6-phosphate (Man-6-P) receptor and insulin-like growth factor-II (IGF-II) receptor are the same protein. Although the role of the IGF-II/Man-6-P receptor as a transporter of hydrolytic enzymes in the biogenesis of lysosomes is certain, elucidation of the receptor's structure has not yet provided major insights into the function of IGF-II binding. Mutually exclusive binding of IGF-II and naturally occurring phosphomannosyl ligands to distinct but proximal sites on the receptor suggests that the IGF-II/Man-6-P receptor cannot simultaneously fulfill the functional requirements of both IGF-II and lysosomal enzymes. Does the receptor transduce on intracellular signal in order to mediate the biological effects of IGF-II? If so, then the receptor must interact with an effector molecule, perhaps a G protein, in the mechanism of IGF-II action. Further information from ligand binding and especially mutagenesis experiments will be needed to elucidate the potentially multiple functions of the IGF-II/Man-6-P receptor.     

How insulin-like growth factor I binds to a hybrid insulin receptor type 1 insulin-like growth factor receptor

1. Download : Download high-res image (161KB)
2. Download : Download full-size image

     

Mannose 6-phosphate/insulin-like growth factor II receptor: distinct binding sites for mannose 6-phosphate and insulin-like growth factor II

Pentamannosyl phosphate substituted bovine serum albumin (PMP-BSA) and insulin like growth factor II (IGF II) bind specifically to immobilized mannose 6-phosphate/insulin like growth factor II receptor. An excess of IGF II inhibited binding of PMP-BSA by less than or equal to 20%, and an excess of PMP-BSA inhibited binding of IGF II by less than or equal to 10%. Polyclonal antibodies against the receptor purified from human liver inhibited preferentially the binding of PMP-BSA, and a monocloncal antibody 2C2 inhibited only the binding of IGF II to the receptor. Similar results were obtained for binding of PMP-BSA and IGF II to human skin fibroblasts. These results suggest that the binding sites for mannose 6-phosphate and IGF II reside in different portions of the receptor.     

Evidence for the phosphorylation of the type II insulin-like growth factor receptor in cultured cells

The ATP pools of monolayer cultures of rat embryo fibroblasts and rat liver cells (BRL-3A2) were labeled with [32P]H3PO4. The type II insulin-like growth factor (IGF) receptor was purified by affinity chromatography on wheat germ lectin-Sepharose and IGF-II-Sepharose columns. A phosphorylated species having the expected size of the type II receptor (Mr = 220,000 without reduction, Mr = 260,000 with reduction) was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. IGF-II stimulated phosphorylation of the type II receptor in BRL-3A2 rat liver cells. Lability of the receptor phosphate bonds to alkaline pH suggests that the bulk of phosphorylation was occurring on serine residues.     

Endocytosis of insulin-like growth factor II by a mini-receptor based on repeat 11 of the mannose 6-phosphate/insulin-like growth factor II receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) plays an important role in controlling the extracellular level of the insulin-like growth factor II (IGF-II) by mediating its binding at the cell surface and delivery to lysosomes. Loss of the receptor is associated with an accumulation of IGF-II, which can cause perinatal lethality if it is systemic, or local proliferation and tumorgenesis if it is spatially restricted. The extracytoplasmic domain of the receptor consists of 15 homologous repeats, of which repeat 11 carries the IGF-II-binding site of the multifunctional receptor. To investigate whether repeat 11 is sufficient to mediate binding and internalization of IGF-II, a construct consisting of repeat 11 fused to the transmembrane and cytoplasmic domain of the M6P/IGF-II receptor was transfected into mouse embryonic fibroblasts. The construct was expressed as a stable membrane protein which binds IGF-II with a 10-fold lower affinity as observed for the M6P/IGF-II receptor and is found at the cell surface and in endosomes. It mediates the internalization of IGF-II and its delivery to lysosomes, suggesting that it can function as a IGF-II mini-receptor controlling the extracellular IGF-II level.     

Role of insulin-like growth factors and the type I insulin-like growth factor receptor in the estrogen-stimulated proliferation of human breast cancer cells

Estrogen sensitizes the MCF-7 estrogen-responsive breast cancer cell line to the mitogenic effect of insulin and the insulin-like growth factors (IGFs). This sensitization is specific for estrogen and occurs at physiological concentrations of estradiol. Dose-response experiments with insulin, IGF-I, and IGF-II suggested that the sensitization is mediated through the type I IGF receptor. Binding experiments with 125I-IGF-I and hybridization of a type I IGF receptor probe to RNA showed that the levels of the type I IGF receptor and its mRNA are increased 7- and 6.5-fold, respectively, by estradiol. IGF-I and estradiol had similar synergistic effects on other estrogen-responsive breast cancer cell lines, but IGF-I alone increased the proliferation of the MDA MB-231 cell line which is not responsive to estrogens. These experiments suggest that an important mechanism by which estrogens stimulate the proliferation of hormone-dependent breast cancer cells involves sensitization to the proliferative effects of IGFs and that this may involve regulation of the type I IGF receptor.