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Abstract
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.
... In MCF-7 breast cancer cells, we have identified an atypical IGR-IR that binds both insulin and IGF-I with high affinity [21]. In human placenta and IM-9 lymphoblasts, the presence of an atypical IR that binds IGF-II with high affinity has been suggested [13,14]. Thus, understanding the complex and multiple interactions of insulin and the IR and the IGFs and their receptors is necessary to understand breast cancer biology. ...
In order to prevent and cure breast cancer it is important to identify and understand the biochemical pathways that are relevant to the biology of this disease. There is evidence, both in vitro and in vivo, that receptor tyrosine kinases play a key role in the formation and progression of breast cancer. The insulin-like growth factors I and II (IGF-I and IGF-II), and their receptor (the IGR-IR) have been well documented in cell culture, animal studies, and humans to play a role in malignant transformation, progression, protection from apoptosis, and metastasis [46]. Also, the hormone insulin (which is very closely related to the IGFs) and its receptor (the IR which is very closely related to the IGR-IR) have been documented both in vitro and in vivo to also play a key role in breast cancer biology [4]. The contribution, however, of the IR to the regulation of breast cancer cell function has not been appreciated. Insulin signals breast cancer cells via its own receptor, and new data indicate that the fetal form of the IR (IR-A) is expressed in breast cancers. IR-A is activated not only by insulin, but also by IGF-II [9,38]. In addition, the IR contributes to formation of hybrid receptors with the IGR-IR (Hybrid-R) [31,32]. This chapter will review these latest developments in our understanding of the IR in breast cancer.
... high-affinity component of binding (8 x 10-10 M for IGF-I and .ft^* --94 2.5 x 10-10 M for insulin) were in agreement with those found in cells that have both insulin and IGF-I receptors, such as IM-9 -67 cells [23]. However, in oocytes, there were about 15 times more high-affinity binding sites for IGF-I than for insulin (per oocyte, 7 x 107 for insulin, and 12 x 108 for IGF-I). ...
Insulin and insulin-like-growth-factor-I (IGF-I) receptors were partially purified from full-grown (stages V-VI) Xenopus laevis oocytes by affinity chromatography on wheat-germ agglutinin-agarose. Competitive-binding assays revealed high-affinity binding sites for both insulin and IGF-I (Kd = 2.5 x 10(-10) M and 8 x 10(-10) M respectively). However, IGF-I receptors were about 15 times more abundant than insulin receptors (22.5 x 10(11) versus 1.5 x 10(11)/mg of protein). Moreover, comparison of intact and collagenase-treated oocytes showed that most of the insulin receptors were in the oocyte envelopes, whereas IGF-I receptors were essentially at the oocyte surface. Oocyte receptors were composed of alpha-subunits of approximately 130 kDa and a doublet of beta-subunits of 95 and 105 kDa, which both had ligand-induced phosphorylation patterns compatible with IGF-I receptor beta-subunits. Accordingly, the receptor tyrosine kinase was stimulated at low IGF-I concentrations [half-maximally effective concentration (EC50) approximately 0.5-1 nM], and at higher insulin concentrations (EC50 approximately 20-50 nM). Partially purified glycoproteins from Xenopus liver and muscle contained mainly receptors of the insulin-receptor type, with alpha-subunits of 140 kDa in liver and 125 kDa in muscle, and doublets of beta-subunits of 92-98 kDa in liver and 85-94 kDa in muscle. Immunoprecipitation of receptors from oocytes, liver and muscle by receptor-specific anti-peptide antibodies suggested that the beta-subunit heterogeneity resulted from the existence of two distinct IGF-I receptors in oocytes and of two distinct insulin receptors in both liver and muscle. In the different tissues, the two receptor subtypes differed at least by their beta-subunit C-terminal region.
... Hybrid forms of insulin and type I IGF receptors exist that have been described in established cultured cell lines [33] and human placenta tissue [34]. The hybrid receptors are believed to be composed of one type I IGF receptor a3 half and one insulin receptor a'p3' half in P-o--a'-3' form. ...
The primary function of the placenta is to ensure an optimal environment for fetal growth and development. In normal pregnancy, placental vascular tone regulation assures fetus well-being and normal development by maintaining adequate blood flow so as to ensure materno-fetal exchanges. In human placenta, synthesis of insulin-like growth factor (IGF)-II and specific binding sites have been previously characterized in the trophoblast; in contrast, no studies have dealt with this subject in the fetoplacental vascular system, particularly in stem villi vessels. We thus investigated whether membranes of the muscular layer of stem villi vessels contained 125I-IGF-II binding sites. Two complementary approaches were used: 125I-IGF-II binding and affinity cross-linking studies. 125I-IGF-II labeled, in a saturable and noncooperative manner, a single class of high-affinity binding sites characterized by a Kd of 1.24 +/- 0.26 nM (n = 6), a maximum binding capacity (Bmax) of 3.02 +/- 0.45 pmol/mg protein, and a Hill coefficient of 1.00 +/- 0.15. Competitors for 125I-IGF-II binding to membranes were in the order of potency IGF-II > IGF-I. Insulin was not a competitor. Affinity cross-linking of membranes with 125I-IGF-II, followed by SDS-PAGE and autoradiography, revealed two labeled bands: a protein complex of 250 kDa, which corresponds to the type II IGF receptor, and another of 135 kDa, corresponding to the type I IGF receptor. Only IGF-II could displace 125I-IGF-II binding from the major 250-kDa band, while 125I-IGF-II bound to the minor 135-kDa band was displaced by either IGF-I, IGF-II, or insulin. In conclusion, high levels of specific binding sites for 125I-IGF-II are present in the muscular layer of stem villi vessels, which are considered placenta resistance vessels. The involvement of both type I and type II IGF receptors in the growth-promoting action of IGF-II remains to be determined in the fetoplacental vascular system.
... Furthermore, the ␣IR-3 mAb will bind to an occupied IGF-IR, whereas radiolabeled IGF-I cannot, and IGF-II binds nearly as well as IGF-I to the IGF-IR (35). Although IGF-I and IGF-II usually bind to the insulin receptor with a low affinity, they bind to atypical insulin receptors on the human lymphoid cell line, IM-9, with a moderately high affinity (36). The proliferative response of promyeloid HL-60 and U-937 monocytic cells to both endogenous and exogenous IGF-I depends on the expression of IGF-IR on their cell surface. ...
Flow cytometry was used to examine the expression of type I insulin-like growth factor receptors (IGF-IR) on three types of human hematopoietic cells that represent different stages of myeloid lineage development. Both HL-60 (promyeloid) and U-937 (monocytic) cells express abundant IGF-IR protein (> 79% cells positive for the IGF-IR), whereas KG-1 myeloblasts express negligible levels of IGF-IR (< 1% IGF-IR-positive cells). Exogenous IGF-I, IGF-II, and an IGF-I analog that binds poorly to IGF-binding protein-3 (des-IGF-I) increased DNA synthesis of HL-60 and U-937 cells in a dose-dependent (1-25 ng/ml) fashion by 2- to 4-fold in serum-free medium, whereas KG-1 cells did not respond to any of these growth factors. The IGF-induced increase in proliferation of HL-60 promyeloid cells was inhibited by soluble IGF-binding protein-3 (500 ng/ml) when these cells were stimulated with 10 ng/ml of either IGF-I (53 +/- 8%) or IGF-II (59 +/- 8%), but not with des-IGF-I (3 +/- 1%). In contrast, the anti-IGF-IR monoclonal antibody (mAb; alpha IR-3) inhibited the DNA synthesis caused by 10 ng/ml exogenous IGF-I (67 +/- 6%), IGF-II (72 +/- 8%), and des-IGF-1 (82 +/- 9%). Proliferation of KG-1 myeloblasts, however, was neither stimulated by the IGFs nor inhibited by the anti-IGF-IR mAb. In the absence of exogenous IGF-I, the mAb directed against the IGF-IR significantly suppressed basal DNA synthesis of HL-60 promyeloid (72 +/- 5%) and U-937 monocytic (39 +/- 7%) cells, but did not affect DNA synthesis of KG-1 myeloblasts (8 +/- 1%) compared to an isotype-matched control mAb. Similarly, the alpha IR-3 mAb abrogated vitamin D3-induced differentiation of the HL-60 cells into macrophages in serum-free medium, as assessed by expression of the leucam surface protein, CD11b. As the alpha IR-3 mAb inhibits DNA synthesis in the presence and absence of exogenous IGF-I on receptor-bearing cells, but not IGF-IR-negative cells, these data demonstrate that both endocrine and autocrine IGF-I are potent growth factors in human myeloid cells where expression of the surface receptor, rather than the ligand, is the critical control element. More importantly, these data support the hypothesis that autocrine IGF-I may play a significant role in the differentiation of promyeloid cells into macrophages.
... Another mechanism involves direct IR interaction with IGF-II. Although in most cells IGF-II interacts with IR with relatively low affinity, atypical IRs that bind IGF-II with high affinity have been described in human placenta and IM-9 lymphoblasts (19,20). In addition, in IGF-I-R-deficient mouse fibroblasts that overexpress the IR, IGF-II stimulates cell proliferation via this receptor, which behaves, therefore, as an IGF-II-R (21). ...
The insulin receptor (IR) occurs in two isoforms (IR-A and IR-B) resulting from alternative splicing of exon 11 of the gene. The IR-A isoform is predominantly expressed in fetal tissues and malignant cells and binds IGF-II with high affinity. We previously observed that IRs are overexpressed in thyroid cancer cells; now we evaluated whether these cells preferentially express IR-A and produce IGF-II, which would activate a growth-promoting autocrine loop. The IR content ranged 6.0-52.6 ng/100 microg cell membrane protein in thyroid cancer primary cultures (n = 8) and permanent cell lines (n = 6) vs. 1.2-1.7 in normal thyroid cells (n = 11 primary cultures; P < 0.0001). IR-A isoform relative abundance ranged from 36-79% in cancer cells (with the highest values in undifferentiated cancers) vs. 27-39% in normal cells. Similar results were obtained in normal vs. cancer thyroid tissue specimens. IGF-II caused IR autophosphorylation with an ED(50) of 1.5-40.0 nM in cancer cells vs. more than 100 nM in normal cells; IGF-II affinity correlated with the relative abundance of IR-A (r = 0.628; P < 0.0001). IGF-II was expressed in all cancer cells, highly expressed in anaplastic cells, and less expressed in normal cells. In conclusion, malignant thyrocytes, especially when poorly differentiated, produce IGF-II and overexpress IR, predominantly as IGF-II-sensitive isoform A. A growth-promoting autocrine loop is activated, therefore, and may affect thyroid cancer biology.
A substantial number of reports over more than a decade have described variant forms of receptors for insulin-like growth factors (IGFs) and insulin, which differ in structure or ligand specificity from classic type I IGF receptors and insulin receptors as characterized in most mammalian tissues or by expression of their cloned cDNAs. As an introduction to receptor subtypes, we first describe briefly the structure and function of the insulin receptor (IR), type I IGF receptor (IGFR), and insulin-receptor-related receptor (IRR), as reflected in the products of their cloned genes. We then review the properties of known splice variants of these receptors, which represent an obvious potential source of functional heterogeneity. Next, we discuss “atypical” insulin and IGF receptors that apparently bind both insulin and IGFs with high affinity, and attempt to rationalize anomalous observations and assess the structural basis and functional significance of these receptors. Finally we consider the properties of insulin/IGF hybrid receptors, which combine the structures and functions of both receptors and may account for much of the reported heterogeneity in receptor subtypes.
Competitive binding experiments from a number of laboratories showed that IGF receptors are distinct from insulin receptors, and that there are two types of IGF receptors based on their relative affinities for IGF-I and IGF-II and whether or not they bind insulin (reviewed in Rechler and Nissley, 1985). Later, affinity crosslinking experiments provided a physical basis for the two IGF receptor subtypes. The receptor which preferred IGF-II over IGF-I and did not bind insulin, was a large 250 kDa species with no apparent subunit structure (Fig. 1). The receptor which prefered IGF-I over IGF-II and bound insulin with low affinity had a binding sub-unit of 130 kDa after reduction of disulfide bonds. Biosynthetic labeling experiments demonstrated a 95 kDa beta subunit addition to the 130 kDa alpha subunit (Fig. 1). The heterotetrameric IGF-I receptor (a,& was therefore very similar to the insulin receptor, and the case for similarity was increased when the beta subunit was found to be auto-phosphorylated in response to IGF-I. Molecular cloning of the IGF-I receptor confirmed that the IGF-I receptor and the insulin receptor areclosely related structures (Ullrich et al., 1986). © 1991 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
Competitive binding experiments from a number of laboratories showed that IGF receptors are distinct from insulin receptors, and that there are two types of IGF receptors based on their relative affinities for IGF-I and IGF-II and whether or not they bind insulin (reviewed in Rechler and Nissley, 1985). Later, affinity crosslinking experiments provided a physical basis for the two IGF receptor subtypes. The receptor which preferred IGF-II over IGF-I and did not bind insulin, was a large 250 kDa species with no apparent subunit structure (Fig. 1). The receptor which prefered IGF-I over IGF-II and bound insulin with low affinity had a binding sub-unit of 130 kDa after reduction of disulfide bonds. Biosynthetic labeling experiments demonstrated a 95 kDa beta subunit addition to the 130 kDa alpha subunit (Fig. 1). The heterotetrameric IGF-I receptor (a,& was therefore very similar to the insulin receptor, and the case for similarity was increased when the beta subunit was found to be auto-phosphorylated in response to IGF-I. Molecular cloning of the IGF-I receptor confirmed that the IGF-I receptor and the insulin receptor areclosely related structures (Ullrich et al., 1986).
The cells of the human IM-9 lymphocyte-derived line contain a sub-population of insulin binding sites which differ from classical insulin binding sites in their higher binding affinity for insulin-like growth factor II (IGF-II) and insulin-like growth factor I (IGF-I). These atypical insulin binding sites are identified on IM-9 cells by [125I]IGF-II binding.
To determine whether the atypical and classical insulin receptors of IM-9 cells were subject to different modes of in vivo regulation, we treated IM-9 cells with agents known to alter the surface expression of insulin receptors - insulin, dexamethasone and monensin. We then measured insulin and IGF-II binding to the surface of the washed cells.
Pretreatment of IM-9 cells with 1 μM insulin for 20 h at 37°C induced a 44–48% decrease in the number of high affinity insulin binding sites, but no change in the number of IGF-II binding sites. In contrast, the surface expression of both insulin and IGF-II binding sites (classical and atypical insulin receptors) increased 1.3 to 1.7-fold after treatment with dexamethasone (200 nM) and decreased 30 to 45% after monensin (1 μM). These results suggest that atypical and classical insulin receptors are differentially susceptible to down-regulation by insulin.
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 μM) 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 ( K d 0.8–1.2 nM). Affinity cross‐linking of [ ¹²⁵ I]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 α subunit of the IGF‐I receptor.
In permissive quiescent myoblasts, linear Scatchard plots suggested that [ ¹²⁵ I]IGF‐II bound to a single class of binding sites ( K d 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 [ ¹²⁵ I]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 ( K d 0.2 nM) as evidenced by cross‐linking experiments. The second was the binding site of highest affinity ( K d 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 [ ¹²⁵ I]IGF‐II binding to this atypical receptor.
It emerges from these experiments that C2 cells express a putative α2β2 IGF‐II receptor structurally related to the insulin/IGF‐I receptor family. It is present in myoblasts but not in myotubes. The possible relationship between expression of this receptor and expression of MyoD1 as well as myoblast differentiation is discussed.
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)
The expression of insulin-like growth factor I (IGF-I) receptors (IGR-IR) on human B-lineage and T-lineage acute lymphoblastic leukaemias (ALL) representing different maturational stages has been studied. Immature (stage I) and mature (stage II) T ALL as well as pre-B ALL cell lines expressed high numbers of IGF-IR with high affinity for IGF-I. In contrast, on T ALL, stage II and B ALL only low specific binding of 125I-IGF-I was detected. No binding of 125I-IGF-I to Burkitt lymphoma cells was found. Primary human T, pre-B and cALL cells also expressed IGF-IR with Kd for IGF-I and IGF-IR number per cell in the same range as the investigated cell lines. Crosslinking of 125I-IGF-I to T and pre-B ALL cells revealed IGF-IR alpha-subunits of 135 and 116 kD for HSB2. Gene expression of IGF-IR could be detected in all T ALL cell lines but was undetectable in SKW6, a B ALL cell line.
It is commonly believed that the insulin receptor mainly mediates the metabolic effects of insulin, whereas the closely related IGF-I receptor is considered a major factor for the regulation of cell proliferation. Experimental and epidemiological evidence indicates, however, that insulin and insulin receptors may play an important role in breast cancer. This article reviews evidence indicating that (a) insulin receptors are overexpressed in human breast cancer, (b) insulin stimulates growth in breast cancer cells, (c) cells transfected with human insulin receptor may acquire a ligand-dependent transformed phenotype, and (d) breast cancer is associated with insulin resistance and hyperinsulinemia. These findings may open new possibilities in breast cancer prevention, prognosis assessment, and therapy. (Trends Endocrinol Metab 1997; 8:306–312). © 1997, Elsevier Science Inc.
The insulin receptor (IR) and type I IGF receptor (IGFR) are widely distributed in mammalian tissues, though varying in concentration between different cell types. The receptors show considerable similarity in primary sequence within a common disulphide linked (αβ)2 subunit structure. The insulin receptor-related receptor (IRR), a third member of this receptor family for which a ligand has yet to be identified, apparently has a much more restricted tissue distribution (Shier and Watt, 1992). The IR and IGFR mediate overlapping biological responses for which the intrinsic tyrosine-specific kinase activity of the receptors appears to be essential. These tyrosine kinases are highly homologous and differences in signalling capacities of the IR and IGFR in a given cell background have not been clearly defined.
GH induces hepatic IGF-I synthesis by increasing transcription of its gene. IGF-I is synthesized, however, in many other tissues where the effect of GH on its gene expression is less well characterized. IGF-I and GH are produced by human lymphocytes and may function as autocrine regulators of lymphoproliferation. We have therefore used the human IM9 lymphocyte cell line to (A) define the IGF-I gene transcripts expressed and (B) investigate the effect of GH on early (protein tyrosine phosphorylation) and late (changes in IGF-I mRNA levels) events in intracellular signal transduction. Multiple IGF-I mRNA species, ranging in size from 0·9 to 5·8 kb, were detected by Northern hybridization of poly(A) ⁺ mRNA from IM9 cells. The human IGF-I gene contains at least six exons and alternative splicing produces a number of transcripts. Solution hybridization with exon-specific riboprobes and amplification by PCR using exon-specific primers revealed that multiple transcripts were expressed in IM9 cells, and that exon 2 was the dominant leader exon.
Treatment of IM9 cells with 200 ng recombinant human (rh)GH/ml led to the specific tyrosine phosphorylation of three intracellular proteins (93, 120 and 134 kDa), which are involved in the initial signalling of the GH transduction pathway. However a solution hybridization assay using the IGF-IA specific riboprobe on IM9 cell RNA from similar experiments revealed that GH treatment did not change IGF-I gene expression.
This study has demonstrated (A) that the IGF-I gene is expressed in human IM9 lymphocytes, (B) that in contrast to other human tissue, exon 2 is the major leader exon, and (C) that rhGH induces tyrosine phosphorylation of 93, 120 and 134 kDa proteins but does not alter IGF-I gene expression. The IM9 cell may form an important model to investigate a GH transduction pathway not coupled to the IGF-I gene.
The insulin receptor and type 1 insulin-like growth factor (IGF) receptor as classically described are each the product of a single gene. Various receptor subtypes have been described, however, with distinct structures or binding properties. Two of these subtypes have been studied, namely hybrid and atypical IGF-I receptors. Hybrid receptors contain alpha beta halves of both the insulin and the IGF receptor. They are identifiable as a high-affinity IGF-I-binding species reacting with both IGF-receptor-specific and insulin-receptor-specific monoclonal antibodies, and account for a substantial fraction of IGF receptor in many mammalian tissues. Hybrid receptors purified from human placenta bind IGF-I with approximately 25-fold higher affinity than insulin, the affinity for insulin being 10-fold less than that of the classical insulin receptor. It is therefore likely that hybrids will respond more readily to IGF-I than to insulin in vivo. Atypical IGF receptors are characterized by an ability to bind insulin as well as IGFs with relatively high affinity, but are immunologically indistinguishable from classical IGF receptor and do not react with insulin receptor-specific antibodies. The structural basis of atypical binding behaviour is unknown, though the effect is mimicked by binding of certain anti-IGF receptor monoclonal antibodies, which dramatically increase the affinity of the IGF receptor for insulin. Specific physiological roles have not been demonstrated for hybrid or atypical receptors, but the available information concerning their distribution and properties suggests that these receptor subtypes may have an important influence on the specificity of action of insulin and IGFs in vivo.
The insulin like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is a bifunc-tional binding protein that binds lysosomal enzymes bearing the M6P recognition marker and IGF-II at distinct binding sites (45, 52). In addition, transforming growth factor (TGF) beta precursor, thyroglobulin and proliferin, a protein which is expressed in rapidly proliferating cells are also recognized by this receptor (Table 1). In avian and amphibian cells the receptor lacks the binding site for IGF-II but serves as a binding protein for M6P bearing ligands (7,9,76). Almost all mammalian cells described until today express IGF-II/M6P receptors that bind both classes of ligands, namely M6P-containing glycoproteins and IGF-II (55–59).
Human IM-9 lymphoblasts bind growth hormone (hGH) and insulin-like growth factors (IGFs). We have systematically examined the IM-9 cells as a valuable model of the interaction of hGH and the IGFs at the cellular level. Cells were cultured in medium with 10% serum and for a subset of experiments cultured in serum-free medium. Binding of [125I]hGH and [125I]IGF-I and -II to intact IM-9 cells was measured: unlabeled hGH inhibited binding of [125I]hGH (half max. 20 ng/ml). Binding of [125I]IGF-I was inhibited by IGF-I (half max. 7.5 ng/ml), IGF-II (half max. 60 ng/ml), and insulin and anti IGF-I receptor antibody (alpha IR3). [125I]IGF-II was inhibited by IGF-II (half max. 15 ng/ml), IGF-I (half max. 500 ng/ml), insulin (half max. 250 ng/ml) but not by alpha IR3. Crosslinking experiments with [125I]IGF-II and DSS as the crosslinking agent and analysis of radioligand-receptor complexes by SDS-PAGE under reducing conditions revealed that [125I]IGF-II bound to a 250 kDa and a 135 kDa receptor species. The latter possibly represents an insulin-type receptor whereas the 250 kDa species had the characteristics of the IGF-II/M6P receptor. When IM-9 cell conditioned medium was analyzed in ligand blotting experiments with either [125I]IGF-I or -II a 30 kDa IGFBP species was detected on the autoradiographs. Also, IGF-II immunoreactivity (approx. 1 ng/ml medium) was measured in the cell conditioned medium using an IGF-BP blocked RIA employing [125I]IGF-II. In a subset of experiments IM-9 cells were homogenized in 4 M guanidinium-thiocyanate and RNA extracted in 5.7 M CsCl. Denatured RNA was electrophoresed on 0.8% agarose gels and transferred to a nylon membrane, fixed and the blots hybridized with cDNA probes. Probes were labeled with [32P]dCTP using a random prime labeling procedure: a Pst I 700 bp fragment of the human IGF-I cDNA, a 554 bp Pst I-Sal I fragment of the IGF-II cDNA, a 614 bp Pst I fragment of the IGF-I receptor cDNA and a 663 bp Pst I fragment of the IGF-II/M6P receptor. Autoradiographs of Northern blots showed specific hybridization with the IGF-I probe at 3.7 kb and with the IGF-II probe at 5.3 kb. No signal was detected with the IGF-I receptor cDNA probe. Hybridization with the IGF-II/M6P receptor probe yielded a 9 kb RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)
In addition to its activity as a metabolic hormone and a regulator of somatic growth, insulin-like growth factor-I (IGF-I) has cytokine-like activities on lymphoid cells. A 14-day infusion of recombinant human (rh)IGF-I increased lymphocyte numbers in all the peripheral lymphoid organs examined. This increase was apparent for up to 3 weeks following cessation of hormone treatment. A second administration of rhIGF-I, given when the lymphocyte numbers in the rhIGF-I-treated mice had returned to control values, resulted in similar increases in the peripheral T and B cell populations. This increase in lymphocyte numbers had functional significance, since rhIGF-I-treated mice produced elevated antibody titres following primary or secondary antigen challenge compared with controls. In addition, when rhIGF-I-treated mice were immunized with a suboptimal dose of antigen they produced antibody titres which were equivalent to those generated by immunization with optimal doses of antigen. When examined in vitro, addition of rhIGF-I alone to cultures of splenocytes from antigen-primed mice stimulated immunoglobulin synthesis. These studies suggest that IGF-I produced locally by thymic and bone marrow stromal cells may be a natural component of B and T cell lymphopoiesis.
Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.
IGF-II, produced by breast cancer epithelial and stromal cells, enhances tumor growth by activating the IGF-I receptor (IGF-I-R) via autocrine and paracrine mechanisms. Previously we found that the insulin receptor (IR), which is related to the IGF-I-R, is overexpressed in breast cancer cells. Herein, we find that, in breast cancer the IR is activated by IGF-II. In eight human breast cancer cell lines studied there was high affinity IGF-II binding to the IR, with subsequent IR activation. In these lines, IGF-II had a potency up to 63% that of insulin. In contrast, in non malignant human breast cells, IGF-II was less than 1% potent as insulin. Via activation of the IR tyrosine kinase IGF-II stimulated breast cancer cell growth. Moreover, IGF-II also activated the IR in breast cancer tissue specimens; IGF-II was 10-100% as potent as insulin. The IR occurs in two isoforms generated by alternative splicing of exon 11; these isoforms are IR-A (Ex11-) and IR-B (Ex11+). IR-A was predominantly expressed in breast cancer cells and specimens and the potency of IGF-II was correlated to the expression of this isoform (P<0.0001). These data indicate, therefore, that the IR-A, which binds IGF-II with high affinity, is predominantly expressed in breast cancer cells and represents a new autocrine/paracrine loop involved in tumor biology.
In recent years, much interest has been devoted to defining the role of the IGF system in the nervous system. The ubiquitous IGFs, their cell membrane receptors, and their carrier binding proteins, the IGFBPs, are expressed early in the development of the nervous system and are therefore considered to play a key role in these processes. In vitro studies have demonstrated that the IGF system promotes differentiation and proliferation and sustains survival, preventing apoptosis of neuronal and brain derived cells. Furthermore, studies of transgenic mice overexpressing components of the IGF system or mice with disruptions of the same genes have clearly shown that the IGF system plays a key role in vivo.
It is commonly believed that the insulin receptor mainly mediates the metabolic effects of insulin, whereas the closely related IGF-I receptor is considered a major factor for the regulation of cell proliferation. Experimental and epidemiological evidence indicates, however, that insulin and insulin receptors may play an important role in breast cancer. This article reviews evidence indicating that (a) insulin receptors are overexpressed in human breast cancer, (b) insulin stimulates growth in breast cancer cells, (c) cells transfected with human insulin receptor may acquire a ligand-dependent transformed phenotype, and (d) breast cancer is associated with insulin resistance and hyperinsulinemia. These findings may open new possibilities in breast cancer prevention, prognosis assessment, and therapy. (Trends Endocrinol Metab 1997; 8:306-312). (c) 1997, Elsevier Science Inc.
Insulin-like growth factor (IGF)-binding sites copurifying with human placental insulin receptors during insulin-affinity chromatography consist of two immunologically distinct populations. One reacts with monoclonal antibody alpha IR-3, but not with antibodies to the insulin receptor, and represents Type I IGF receptors; the other reacts only with antibodies to the insulin receptor and is precipitated with a polyclonal receptor antibody (B-10) after labelling with 125I-multiplication-stimulating activity (MSA, rat IGF-II). The latter is a unique sub-population of atypical insulin receptors which differ from classical insulin receptors by their unusually high affinity for MSA (Ka = 2 x 10(9) M-1 compared with 5 x 10(7) M-1) and relative potencies for insulin, MSA and IGF-I (40:5:1 compared with 150:4:1). They represent 10-20% of the total insulin receptor population and account for 25-50% of the 125I-MSA binding activity in Triton-solubilized placental membranes. Although atypical and classical insulin receptors are distinct, their immunological properties are very similar, as are their binding properties in response to dithiothreitol, storage at -20 degrees C and neuraminidase digestion. We conclude that atypical insulin receptors with moderately high affinity for IGFs co-exist with classical insulin receptors and Type I IGF receptors in human placenta. They provide an explanation for the unusual IGF-II binding properties of human placental membranes and may have a specific role in placental growth and/or function.
We have identified high and low affinity insulin-like growth factor I (IGF I)-binding sites with mean dissociation constants of 0.37 and 6.25 nM, respectively, in solubilized placental membranes. We have separated these sites and purified the high affinity IGF I receptor 1,300-fold, with an overall yield of 9.9%, using wheat germ agglutinin-Sepharose chromatography, insulin affinity chromatography, and IGF I affinity chromatography. The Scatchard plot of IGF I binding to the high affinity receptor is linear, suggesting the purification of a single homogeneous class of binding sites. Insulin is two orders of magnitude less effective than IGF I in competitively inhibiting IGF I binding to this receptor. The high affinity IGF I receptor is composed of alpha and beta subunits with apparent molecular weights of 135,500 and 96,200, respectively. IGF I at concentrations of greater than or equal to 50 ng/ml stimulates autophosphorylation of the beta subunit of the purified high affinity receptor 4.6-fold. Low affinity IGF I-binding sites run through the IGF I affinity column or are eluted from the insulin affinity column. The separation of IGF I receptors with different binding affinities by sequential affinity chromatography will make it possible to examine directly the determinants of receptor affinity.
Insulin receptors purified from human placenta by sequential affinity chromatography on wheat germ lectin-agarose and insulin coupled to 1,1'-carbonyldiimidazole-activated agarose (CDI-agarose) retained full binding activity but bound a greater than predicted amount of 125I-labeled insulin-like growth factor I (IGF-I). IGF-I and multiplication-stimulating activity (MSA; the rat homologue of IGF-II) were equipotent in displacing either 125I-labeled IGF-I or 125I-labeled MSA from the purified receptors; insulin was 5-15 times more potent. Competitive binding studies indicated that this IGF binding activity could not be explained by cross-reaction with classical insulin receptors or by coelution of IGF-I or IGF-II receptors. Instead, it was due to a minor population of discrete atypical insulin receptors (6-18% total insulin receptors) with moderately high affinity (Kd = 2-4 X 10(-9) M) for IGF-I and MSA. These receptors were not an artifact of insulin-CDI-agarose chromatography, since they were present in wheat germ lectin-agarose-purified preparations and could also be purified from insulin-succinyldiaminodipropylamino-agarose. Affinity labeling with 125I-labeled MSA revealed that these atypical receptors had the same binding subunit (Mr 140,000) as classical insulin and IGF-I receptors. They displayed intermediate reactivity with polyclonal and monoclonal antibodies to the insulin and IGF-I receptors. It is therefore likely that insulin receptors purified by immunoadsorption would also contain atypical insulin receptors. The finding of more than one type of insulin receptor might relate to the slight variations in the cDNA nucleotide sequences and the multiple mRNA species reported for the insulin receptor [Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J.-H., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A. & Rutter, W. J. (1985) Cell 40, 747-758].
Intact cells and membranes isolated from several human and rodent tissues have been affinity-cross-linked to human 125I-insulin-like growth factor I (IGF-I) and 125I-insulin-like growth factor II (IGF-II). Dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity-labeled material resolves two types (type I and type II) of labeled membrane components that fulfill the properties expected for high affinity growth factor receptors. Type I receptors consist of three disulfide-linked forms (M(r)= 350,000, 320,000 and 290,000) structurally similar to the insulin receptor forms in membrane preparations from various tissues (Massague, J., Pilch, P.F., and Czech, M.P. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7137-7141). The proposed subunit stoichiometries of these type I IGF receptor forms are (β-S-S-α)-S-S-(α-S-S-β), (β1-S-S-α)-S-S-(α-S-S-β), and (β1-S-S-α) -S-S-(α-S-S-β1), respectively, based on two-dimensional electrophoretic analysis. The α and β subunits migrate with apparent M(r)= 130,000 and 98,000, respectively, and the β1 subunit is a proteolytic fragment of the β subunit. The disulfide-linked M(r)= 350,000 receptor species probably represent the native receptor complex. The type I IGF receptors exhibit a higher affinity for IGF-I than for IGF-II, and a low affinity for insulin. The type II receptor consists of one single labeled species (M(r)= 258,000-268,000) that is not disulfide-linked to any other membrane component. This type II IGF receptor has a higher affinity for IGF-II than for IGF-I, and has no significant affinity for insulin. This receptor type apparently corresponds to the same structure previously identified as a membrane receptor for multiplication-stimulating activity. The ability of IGF-I and IGF-II to compete with 125I-insulin for affinity labeling the high affinity insulin receptor in all tissues examined is lower than that of insulin. In conclusion, we have identified a specific growth factor receptor with high affinity for IGF-I that consists of a heterotetrameric disulfide-linked subunit composition virtually identical with the insulin receptor structure. A second growth factor receptor with high affinity for IGF-II is structurally distinct from these disulfide-linked receptor types.
Three monoclonal antibodies, designated alpha IR-1, alpha IR-2, and alpha IR-3, were prepared by fusing FO myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of insulin receptors from human placenta. These antibodies were characterized by their ability to immunoprecipitate solubilized receptors labeled with 125I-insulin or 125I-somatomedin-C in the presence or absence of various concentrations of unlabeled insulin or somatomedin-C. alpha IR-1 preferentially immunoprecipitates insulin receptors and also less effectively immunoprecipitates somatomedin-C receptors, while alpha IR-2 and alph IR-3 preferentially immunoprecipitate somatomedin-C receptors, but may also weakly immunoprecipitate insulin receptors. These three monoclonal antibodies, as well as A410, a rabbit polyclonal antibody, were used to immunoprecipitate insulin and somatomedin-C receptors from solubilized human lymphoid (IM-9) cells and human placenta membranes that had been 125I-labeled with lactoperoxidase. Analysis of the immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both receptors are composed of alpha and beta subunits. The beta subunit of the insulin receptor (immunoprecipitated by alpha IR-1 and A410) has a slightly more rapid mobility than the corresponding subunit of the somatomedin-C receptor (immunoprecipitated by alpha IR-2 and alpha IR-3). Interestingly, the alpha subunit of the placenta somatomedin-C receptor has a slightly faster mobility than its counterpart from IM-9 cells. Immunoprecipitation of receptor that had been reduced and denatured to generate isolated subunits indicates that alpha IR-2 and alpha IR-3 interact with the alpha subunit of the somatomedin-C receptor while A410 interacts with both subunits of the insulin receptor. alpha IR-1 failed to react with reduced and denatured receptors.
alpha IR-3, a monoclonal antibody that interacts with the somatomedin-C receptor, inhibited the binding of somatomedin-C, but not of insulin, to human placental membranes and intact IM-9 cells. alpha IR-1, a monoclonal antibody that interacts with the insulin receptor, did not inhibit the binding of either hormone. Inhibition of somatomedin-C binding by alpha IR-3 was mainly due to a decrease in its affinity. 125I-Labeled alpha IR-3 bound specifically to placental membranes and intact IM-9 cells and was inhibited by concentrations of unlabeled alpha IR-3 that were lower than those required to inhibit somatomedin-C binding. [125I]alpha IR-3 binding was also inhibited by somatomedin-C and insulin, but only at very high concentrations. A410, a rabbit antiserum that reacts with both receptors for insulin and somatomedin-C, also inhibited labeled alpha IR-3 binding. alpha IR-I did not. These results help to define the epitopes with which these antibodies interact.
The receptors for insulin and insulin-like growth factor-I (IGF-I) are closely related in primary sequence and overall structure. We have examined the immunological relationships between these receptors by testing the reactivity of anti-(insulin receptor) monoclonal antibodies with IGF-I receptors in various tissues and cell lines. Antibodies for six distinct epitopes reacted with a subfraction of IGF-I receptors, as shown by inhibition of 125I-IGF-I binding, precipitation of 125I-IGF-I-receptor complexes or immunodepletion of receptor from tissue extracts before binding assays. Both immunoreactive and non-immunoreactive subfractions displayed the expected properties of 'classical' IGF-I receptors, in terms of relative affinities for IGF-I and insulin. The proportion of total IGF-I receptors which was immunoreactive varied in different cell types, being approx. 40% in Hep G2 cells, 35-40% in placental membranes and 75-85% in IM-9 cells. The immunoreactive fraction was somewhat higher in solubilized receptors than in the corresponding intact cells or membranes. A previously described monoclonal antibody, alpha-IR-3, specific for IGF-I receptors, inhibited IGF-I binding by more than 80% in all preparations. When solubilized placental receptors were pretreated with dithiothreitol (DTT) under conditions reported to reduce intramolecular (class I) disulphide bonds, the immunoreactivity of IGF-I receptors was abolished although total IGF-I binding was little affected. Under the same conditions insulin receptors remained fully immunoreactive. When solubilized receptor preparations were fractionated by gel filtration, both IGF-I and insulin receptors ran as symmetrical peaks of identical mobility. After DTT treatment, the IGF-I receptor was partially converted to a lower molecular mass form which was not immunoreactive. The insulin receptor peak showed a much less pronounced skewing and remained fully immunoreactive in all fractions. It is concluded that the anti- (insulin receptor) antibodies do not react directly with IGF-I receptor polypeptide, and that the apparent immunoreactivity of a subfraction of IGF-I receptors reflects their physical association with insulin receptors, both in cell extracts and in intact cells. The most likely basis for this association appears to be a 'hybrid' receptor containing one half (alpha beta) of insulin receptor polypeptide and the other (alpha' beta') of IGF-I receptor polypeptide within the native (alpha beta beta' alpha') heterotetrameric structure.
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.
To identify structural characteristics of the closely related cell surface receptors for insulin and IGF-I that define their distinct physiological roles, we determined the complete primary structure of the human IGF-I receptor from cloned cDNA. The deduced sequence predicts a 1367 amino acid receptor precursor, including a 30-residue signal peptide, which is removed during translocation of the nascent polypeptide chain. The 1337 residue, unmodified proreceptor polypeptide has a predicted Mr of 151,869, which compares with the 180,000 Mr IGF-I receptor precursor. In analogy with the 152,784 Mr insulin receptor precursor, cleavage of the Arg-Lys-Arg-Arg sequence at position 707 of the IGF-I receptor precursor will generate alpha (80,423 Mr) and beta (70,866 Mr) subunits, which compare with approximately 135,000 Mr (alpha) and 90,000 Mr (beta) fully glycosylated subunits.
The structural and immunological properties of the insulin-like growth factor II (IGF-II) receptor on IM-9 lymphoblasts were studied using a combination of competitive binding and affinity cross-linking techniques as well as with a panel of polyclonal and monoclonal antireceptor antibodies. Unlike IGF-II binding to the classical type II IGF receptor, [125I] IGF-II binding to IM-9 cells was potently inhibited not only by unlabeled IGF-II, but also by insulin (50% inhibition of binding at 2.5 and 1.5 nM, respectively). Affinity cross-linking of [125I] IGF-II to intact cells, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, demonstrated that the overwhelming majority of IGF-II binding was to a type I receptor (apparent mol wt, greater than 300,000 unreduced and 135,000 reduced), with minimal binding to a type II receptor (apparent mol wt, 220,000 unreduced and 240,000 reduced). After preincubation with five different antireceptor antibodies, inhibition of [125I] IGF-II binding was comparable to inhibition of [125I]insulin binding. These studies demonstrate that in IM-9 cells, the majority of IGF-II binding is to a type I receptor with high affinity for both insulin and IGF-II. Whether this is an atypical insulin receptor or a unique type I receptor remains to be established.
The primary structure of human insulin-like growth factor II receptor, predicted from the complementary DNA sequence, reveals a transmembrane receptor molecule with a large extracellular domain made up of fifteen repeat sequences and a small region homologous to the collagen-binding domain of fibronectin. The structural and biochemical features of the IGF-II receptor appear identical to those of the cation-independent mannose-6-phosphate receptor.
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.
Somatomedin-C/insulin-like growth factor-I (Sm-C/IGF-I) and insulin stimulate DNA synthesis and cell replication in cultured human fibroblasts. It has been postulated that the growth-promoting actions of both peptides are mediated through the type I Sm-C/IGF-I receptor. This study tests this hypothesis using two recently developed monoclonal antibodies. The antibody designated sm 1.2 is directed to Sm-C, whereas the antibody designated alpha IR-3 is directed against the type I receptor for Sm-C/IGF-I. Radiolabeled monoclonal antibody alpha IR-3 was bound to human foreskin fibroblasts in a reversible time-dependent fashion, with 90% of the specific binding complete after 6 h of incubation at 15 C. Binding of [125I]alpha IR-3 was completely inhibited by excess unlabeled antibody, but not by 50 nM Sm-C or 1000 nM insulin. Specific binding of [125I]Sm-C fell to 27% of the control value in the presence of 50 nM alpha IR-3, and this concentration of antibody significantly reduced the mitogenic response to both Sm-C and insulin. Antibody sm 1.2 blocked the mitogenic response to exogenous Sm-C, but did not block the response to insulin; indeed, in some experiments, sm 1.2 enhanced the response to insulin. We postulate that this enhancement is the result of neutralizing endogenously produced Sm-like substances. This study provides further evidence that the growth-promoting effects of insulin in this cell type are the result of interaction with the Sm-C/IGF-I receptor.
A monoclonal antibody was identified which equally inhibits 125I-labeled insulin and insulin-like growth factor I (IGF-I) binding to their respective receptors in human IM-9 lymphoid cells and solubilized placenta receptor preparations. In contrast, this monoclonal antibody inhibits insulin but not IGF-I binding to human hepatoma (HepG2) cells, fibroblasts and muscle cells. These results indicate that there are two distinct species of the type I insulin-like growth factor receptor (which we have named type IA and type IB) and suggest that this monoclonal antibody may be useful in determining whether different biological effects are mediated through these two receptors.
A novel affinity gel, consisting of insulin coupled to 1,1'-carbonyldiimidazole-activated agarose (CDI-agarose), was used to purify insulin receptors from human placenta to homogeneity. This affinity gel is reproducibly prepared and is reported to have a number of advantages over the standard cyanogen bromide activated supports, such as ease and simplicity of coupling and minimal ligand leakage and non-specific binding. Insulin receptors in Triton X-100-solubilized microsomal membranes were purified 2,000-fold by sequential affinity chromatography on wheat germ lectin-agarose and insulin-CDI-activated agarose. They have one of the highest specific insulin-binding capacities (6 nmol/mg protein) reported and can be calculated to have a binding valence of two on the basis that the molecular weight of the oligomeric receptor is 300-350,000.
A membrane receptor for somatomedin C (SM-C) on cultured IM-9 cells has recently been reported. The current studies were undertaken to further characterize this intact cellular receptor in terms of both kinetics and specificity and to investigate the ability of SM-C to induce homologous receptor loss. The binding of [125I]iodo-SM-C was rapid, achieving a steady state within 90 min and was greater than 95% reversible. Specific binding at 15C averaged 25-30% for 20 X 10(6) cells/ml. Competition for binding was observed at SM concentrations as low as 2 mU/ml, with half-maximal displacement of [125I]iodo-SM-C at SM concentrations of 33 mU/ml (3.33 X 10(-9) M). Insulin-like growth factor I (IGF-I) and a purified SM-C preparation were approximately equipotent in their ability to displace [125I]iodo-SM-C from the IM-9 receptor. The relative potencies of other growth-related peptides, in comparison with SM-C/IGF-I, were IGF-II (1:10), multiplication stimulating activity (1:10), insulin (1:100), and hGH (nonreactive). Preexposure of IM-9 cells to SM-C at 37 C resulted in a time- and concentration-dependent reduction in [125I]iodo-SM-C. A 10-15% decrease in binding was observed after preincubation with SM in concentrations of 7 mU/ml. Preincubation with 100 mU/ml SM resulted in 50% reduction in binding, but no further decreases were observed after preincubation with higher concentrations of SM (up to 1 U/ml). Scatchard analysis indicated that the reduction in binding was due to a loss of available specific receptors on the cell membrane. These data indicate that the IM-9 cell possesses a specific SM receptor and that SM-C and IGF-I compete equally for occupancy. Furthermore, the data provide a direct demonstration in cell culture that SM-C, like insulin and growth hormone, can modulate homologous receptor concentrations and potentially alter target-cell sensitivity.
Five sera containing autoantibodies to the insulin receptor were used to compare the immunological characteristics of the insulin and the somatomedin-C/insulin-like growth factor-1 receptors of Triton-solubilized human placental membranes. Complete immunoprecipitation of [125I] insulin-labelled receptors was achieved using all five antisera. Three antisera precipitated 90, 65 and 40% of [125I] insulin-like growth factor-1-labelled receptors while the other two caused less than 7% immunoprecipitation. These results are consistent with the view that insulin and insulin-like growth factor-1 receptors are separate molecules which although structurally similar, possess a significant degree of immunologic non-identity.
Preexposure of IM-9 lymphocytes to the somatomedin peptide insulin-like growth factor-I (IGF-I) results in a time- and concentration-dependent reduction in specific receptors for IGF-I. Since insulin and proinsulin are structurally homologous to IGF-I, we investigated the ability of insulin analogues to compete for occupancy and to directly modulate IGF-I receptor concentrations.
IGF-I binds rapidly and reversibly to IM-9 cells at 15°C, with half-maximal displacement of 125I-I-IGF-I at IGF-I concentrations of 3.6 × 10−9 M and insulin concentrations of 5 × 10−7 M. Preexposure of cells at 37°C to either IGF-I or insulin produced a concentration-dependent reduction in binding of 125I-IGF-I. A 50% decrease in binding was observed following preincubation of cells with IGF-I at 2.5 × 10−9 M and insulin at 2 × 10−7 M. At higher insulin concentrations (10−6–10−5 M), up to 70% reduction in 125I-IGF-I binding occurred. Bovine proinsulin and guinea pig insulin competed less potently than porcine insulin for the IGF-I receptor, and produced receptor loss in proportion to their ability to occupy the IGF-I receptor. Scatchard analysis indicated that at all insulin concentrations, the decrease in binding was secondary to loss of available IGF-I receptors, with no change in affinity. Receptor loss was evident following 1–2 h preexposure to insulin, with a t½ of 4 h and maximal receptor loss within 10 h. Similarly, IGF-I and IGF-II competed for occupancy of the IM-9 insulin receptor, with 50% displacement of 125I-insulin occurring at peptide concentrations of 3.5 × 10−9 M (insulin), 3.5 × 10−8 M (IGF-II), and 3 × 10−7 M (IGF-I). Preexposure of cells to these peptides at 37°C for 20 h resulted in a concentration-dependent reduction in binding of 125I-insulin, with the order of analogue effectiveness being insulin > IGF-II > IGF-I.
These data emphasize the structural and functional homology of insulin and the somatomedin peptides, IGF-I and II, as well as their respective receptors. Additionally, the data support the conclusion that the insulin and somatomedin peptides not only bind to both receptors, but downregulate each receptor in proportion to their ability to occupy that receptor.
We have studied the binding of 125I-GF-II to the IM-9 human lymphoid cell line, and to human placental membranes. All of IGF-II radioligand binding to IM-9 cells, and half of the binding to human placental membranes is to a previously unrecognized common (Type-III) high affinity receptor site for insulin-like peptides, in which IGF-I and IGF-II are equipotent and insulin only slightly less potent. This common receptor represents another mechanism by which insulin, and the somatomedins can exert biological action.
Insulin and such insulinlike growth factors as multiplication stimulating activity (MSA) are related polypeptides that have common biological activities. Both insulin and MSA produce acute metabolic responses (stimulation of glucose oxidation in isolated fat cells) as well as growth effects (stimulation of [3H]thymidine incorporation into DNA in cultured fibroblasts). In addition, most cells have separate receptors for insulin and insulinlike growth factors, and both peptides have weaker affinity for each other's specific receptors than for their own. To determine, therefore, whether these effects are mediated by receptors for insulin, insulinlike growth factors, or both, we have selectively blocked insulin receptors with a specific antagonist, namely Fab fragments derived from naturally occurring antibodies to the insulin receptor.In rat adipocytes, 10 μg/ml of antireceptor Fab inhibited insulin binding by 90%, whereas it inhibited MSA binding
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