Characterization of a Novel Receptor in Toad Retina with Dual Specificity for Insulin and Insulin-Like Growth Factor I

The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with 125I-insulin resulted in the specific labeling of insulin receptor alpha-subunits of approximately 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (alpha beta-heterodimer) receptors affinity-labeled with 125I-insulin indicated the presence of a disulfide-linked beta-subunit of approximately 95 kDa. Endoglycosidase F digestion of the affinity-labeled alpha-subunits increased their mobility by reducing their apparent mass to approximately 83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657-670 of the carboxy terminal of the human insulin receptor alpha-subunit, whereas this antibody did label insulin receptor alpha-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor beta-subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose alpha-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.     

Evidence for separate receptors for insulin and insulin-like growth factor-I in choroid plexus of rat brain by quantitative autoradiography

Binding of insulin and insulin-like growth factor-I (IGF-I) to the choroid plexus was quantitatively characterized using autoradiography and computer densitometry. Slide-mounted brain slices were incubated in 0.1 nM [125I]-insulin or [125I]-[Thr59]IGF-I. To determine specificity of the binding sites, the labeled peptides were mixed with unlabeled analogues. Autoradiography was done with LKB Ultrofilm and analyzed with a computer image analysis system and program for densitometry. Results showed that binding was time and temperature dependent and reversible. Binding of the iodinated insulin and IGF-I was inhibited by unlabeled peptides in a dose-dependent manner. The rank order of potency of these peptides in competing for the choroid plexus iodoinsulin binding sites was: chicken insulin greater than porcine insulin greater than desoctapeptide insulin greater than IGF-I. IGF-I was more potent than porcine insulin in competing for the choroid plexus iodolGF-I binding sites. Somatostatin was ineffective. Non-linear regression analysis revealed the presence of high- (Kd 1.3 +/- 0.2 nM) and low-affinity (Kd 36 +/- 1.4 nM) binding sites for insulin and a single high-affinity binding site (Kd 3.1 +/- 0.3 nM) for IGF-I in the choroid plexus. There were approximately 50 times more binding sites (Bmax) for IGF-I than for insulin high-affinity sites, whereas the number of low-affinity sites for insulin was about equal to the number of IGF-I high-affinity sites. The results of these binding studies with iodinated insulin and [Thr59]IGF-I support the conclusion that the rat choroid plexus has separate high-affinity receptors for insulin and IGF-I, and that the IGF-I receptors outnumber the insulin receptors.     

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

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

Insulin-like growth factor-mediated phosphorylation and protooncogene induction in Madin-Darby canine kidney cells.

We have characterized the role of tyrosine phosphorylation in protooncogene induction mediated by insulin-like growth factors I and II (IGF-I and IGF-II) in the Madin-Darby canine kidney (MDCK) cell line. These cells possess few, if any, insulin receptors, thus allowing determination of the effects of these growth factors in the absence of any secondary signal mediated through the insulin receptor. We found that IGF-I produced a specific stimulation of tyrosine kinase activity of the 97-kDa beta-subunit of the IGF-I receptor, resulting in autophosphorylation of the receptor and an increase in kinase activity toward a synthetic peptide substrate. This was associated with a gradual decrease in the level of phosphorylation of pp120, the major constitutive phosphotyrosine-containing protein of MDCK cells, and an increase in the ratio of serine to tyrosine phosphorylation. This was followed by a rapid, but transient, induction of c-fos gene expression, with no change in the levels of c-myc mRNA. Cycloheximide treatment resulted in a superinduction of both c-fos and c-myc and prevented any further stimulation by IGF-I. IGF-II did not stimulate tyrosine phosphorylation of its own receptor, but was 25% as active as IGF-I in stimulating phosphorylation of the IGF-I receptor. Despite this, IGF-II did not significantly enhance the expression of either nuclear protooncogene. Insulin also produced a delayed stimulation of IGF-I receptor phosphorylation, but was unable to stimulate biological effects in these cells. Under these conditions neither of the IGFs nor insulin produced any significant stimulation of thymidine incorporation into DNA. These data indicate that the IGF-I receptor can be activated upon binding of IGF-I, and to a lesser extent IGF-II, in intact cells to mediate cellular events. The nature of the signal generated by the IGF-I receptor appears to vary depending on the ligand that occupies it.     

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.     

Insulin-like growth factor I binding and receptor kinase in red and white muscle

IGF-I receptors were partially purified from red and white skeletal muscle by lectin-affinity chromatography and the resultant fraction was depleted of insulin receptors by insulin affinity chromatography. Equilibrium binding of 125I-IGF-I to receptor preparations from red and white muscle yielded identical Scatchard plots. The integrity of the IGF-I receptor preparation in the two fiber types was identical as determined by affinity cross-linking. The tyrosine kinase activity of the receptor from red muscle was 2-3-fold more active towards exogenous substrates in both the basal and ligand-activated states as compared to white muscle. These data show that there is IGF-I-dependent kinase activity intrinsic to IGF-I receptors from skeletal muscle, and suggest that identical cellular factors may regulate the kinase activity of insulin and IGF-I receptors in a parallel manner in vivo.     

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

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

Insulin Promotes the Hydrolysis of a Glycosyl Phosphatidylinositol in Cultured Rat Astroglial Cells

Abstract: Glycosyl phosphatidylinositols have been implicated in insulin signaling through their action as precursors of second messenger molecules in peripheral tissues. In the present study, cultured rat astrocytes were used to investigate whether glycosyl phosphatidylinositol might be involved in the mechanism of insulin signal transduction in neural cells. A glycosyl phosphatidylinositol sensitive to hydrolysis by both phosphatidylinositol-specific phospholipase C and glycosyl phosphatidylinositol-specific phospholipase D and to nitrous acid deamination was purified. When astrocytes were exposed to 10 n M insulin, a rapid and significant reduction in the content of glycosyl phosphatidylinositol was observed within 1–2 min. In addition, an inverse concentration-dependent relationship between glycosyl phosphatidylinositol and diacylglycerol levels was found, suggesting a phospholipase C-mediated hydrolysis of glycosyl phosphatidylinositol in response to insulin. The effects of insulin were mediated through its own receptors and not through insulin-like growth factor (IGF)-I and/or IGF-II receptors, as demonstrated by affinity cross-linking studies. Also, the effects of 5 n M IGF-I or 5 n M IGF-II on glycosyl phosphatidylinositol and diacylglycerol levels were different from those caused by insulin and were not essentially modified by pretreatment of the cells with either platelet-derived growth factor (PDGF) or epidermal growth factor (EGF). When cells were sequentially incubated with PDGF and EGF, a reduction in both glycosyl phosphatidylinositol and diacylglycerol contents was observed; the diacyl-glycerol but not the glycosyl phosphatidyl content was reversed after incubation with IGF-I, and especially with IGF-II, for 10 min. Despite the remarkable homology among insulin, IGF-I, and IGF-II, our results indicate that in astrocytes these compounds probably use different signal transduction pathways.     

Receptor Binding, Endocytosis, and Mitogenesis of Insulin-Like Growth Factors I and II in Fetal Rat Brain Neurons

Cell surface binding, internalization, and biological effects of insulin-like growth factors (IGFs) I and II have been studied in primary neuronal cultures from developing rat brain (embryonic day 15). Two types of IGF binding sites are present on the cell surface. The IGF-I receptor alpha-subunit (Mr 125,000) binds IGF-I with a KD of 1 nM and IGF-II with 10 times lower affinity. The mannose-6-phosphate (Man-6-P)/IGF-II receptor (Mr 250,000) binds IGF-II with a KD of 0.5 nM and IGF-I with 100 times lower affinity. Surface-bound IGF-I and IGF-II are internalized by their respective receptors binding and internalization of IGF-II but not those of IGF-I. Neuronal synthesis of RNA and DNA is increased twofold by IGF-I with 10 times higher potency than IGF-II. Antibody 3637, which blocks receptor binding of IGF-II, has no effect on the DNA response to IGF-I or IGF-II. Double immunocytochemical staining with antibodies to bromodeoxyuridine and neurofilament shows that greater than 80% of the bromodeoxyuridine-positive cells become neurofilament positive. It is concluded that IGF-I and IGF-II bind to two receptors on the surface of neuronal precursor cells that mediate endocytosis and degradation of IGF-I and IGF-II. Proliferation of neuronal precursor cells is stimulated by IGF-I and IGF-II via activation of the IGF-I receptor.     

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

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

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

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

Effects of a single cleavage in insulin-like growth factors I and II on binding to receptors, carrier proteins and antibodies.

Two somatomedin-like peptides were extracted from Cohn fraction IV of human plasma and brought to homogeneity: one focused at pH 7.8 and the other at pH less than 5.6. Each consisted of two peptide chains interlinked by disulphide bonds. The basic peptide was identical to insulin-like growth factor I (IGF-I) and had a single cleavage in the C-domain before Arg37 [IGF-I(Arg36cl)]. The acid peptide showed identity with IGF-II, with a cleavage in the B-domain before Arg30 [IGF-II(Ser29cl)]. The effects of these cleavages on the characteristics of binding to type I and type II receptor sites, to binding proteins and to antibodies was studied. Binding of IGF-I(Arg36cl) to antibodies directed against the B-domain or against the AD-domain of IGF-I was the same as IGF-I binding. Thus the cleavage does not influence these antigenic sites. In contrast, binding of IGF-I(Arg36cl) to the type I receptor on human and bovine placental cell membranes was markedly decreased compared with IGF-I binding. Binding to the insulin receptor on human placental cell membranes was slightly diminished, whereas the interaction with specific type II receptors on bovine placental cell membranes was unaffected. There was only a minor influence of the cleavage on the region involved in binding to binding proteins. The cleavage in IGF-II(Ser29cl) diminished binding to antibodies directed against the C-domain of IGF-II, compared with binding of IGF-II itself. Binding to receptors (type I and type II) was changed less profoundly. With 125I-labelled IGF-II(Ser29cl), less insulin was needed in order to obtain 50% displacement of the tracer compared with displacement of 125I-labelled IGF-II. The cleaved form of IGF-II probably has a greater affinity towards the common receptor population than does native IGF-II. Binding to binding proteins was not affected by the cleavage in IGF-II.     

Evidence for an Insulin-Like Growth Factor Autocrine-Paracrine System in the Retinal Photoreceptor-Pigment Epithelial Cell Complex

The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin-like growth factor I (IGF-I) immunoreactivity that co-elutes with authentic human IGF-I in HPLC analyses. Cultured human RPE cells synthesize and release IGF-I, raising the possibility that the RPE serves as a source of IPM IGF-I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF-I receptors with alpha-subunits of 120 and 138 kDa, respectively. They thus appear to be of the "brain" (in photoreceptors) and "peripheral" (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF-BP) that specifically binds IGF-I and IGF-II. The IPM-BP is visualized as a single radiographic band by both ligand blot and affinity cross-linking procedures. With enzymes specific for removing N- and O-linked oligosaccharides, the IPM-BP was found to contain O- but not N-linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM-BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF-I and IGF-BP in the IPM, together with the presence of IGF-I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine-paracrine system.     

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

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

TGF-beta 1 modulation of IGF-I signaling pathway in rat thyroid epithelial cells

Transforming growth factor beta 1 (TGF-beta 1) and insulin-like growth factor I (IGF-I) have contrasting effects on cell cycle regulation in thyroid cells and TGF-beta 1 induces a dramatic decrease in IGF-I-induced cell proliferation. The aim of the present study was to investigate the molecular mechanism of cross-talk between TGF-beta 1 and IGF-I in FRTL-5 cells. TGF-beta 1 affected IGF-I-stimulated insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with Grb2 protein. Moreover, TGF-beta 1 decreased the IGF-I-induced tyrosine phosphorylation of the adaptor protein CrkII and its association with the IGF-I receptor. These results were accompanied by TGF-beta 1 inhibition of IGF-I-stimulated mitogen-activated protein kinase phosphorylation and activation. Conversely, TGF-beta 1 did not alter IGF-I-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity, IGF-I-induced tyrosine phosphorylation of Shc, and its binding to Grb2. Taken together, these findings provide a molecular basis for the growth-inhibitory action of TGF-beta 1 on the IGF-I-induced mitogenic effect.     

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

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

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

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

Characterization of Insulin-Like Growth Factor-I Receptors in PC 12 Pheochromocytoma Cells and Bovine Adrenal Medulla

Competitive binding studies indicated that PC12 cells have receptors for insulin-like growth factor-I (IGF-I). There are approximately 11,000 +/- 1,500 IGF-I receptors/cell; these receptors have an apparent KD for IGF-I of 7.2 +/- 0.6 nM. Covalent cross-linking of 125I-IGF-I to PC12 cells labeled a 125,000-130,000-Mr protein, presumably the alpha-subunit of the IGF-I receptor. Although PC12 cells also have insulin receptors, the 125I-IGF-I appeared to be cross-linked to IGF-I receptors, because 100 nM IGF-I competed for labeling but 100 nM insulin did not. Bovine chromaffin cells also have IGF-I receptors. The protein tyrosyl kinase activity of IGF-I receptors from bovine adrenal medulla and PC12 cells was examined after purification of the receptors by wheat germ agglutinin-Sepharose chromatography. IGF-I (10 nM) stimulated autophosphorylation of the beta-subunits of the IGF-I receptors from both preparations; the beta-subunits from both sources had Mr values of approximately 97,000. IGF-I also stimulated phosphorylation of the synthetic substrate poly(Glu:Tyr)4:1 by both receptor preparations. IGF-I (IC50 of approximately 0.2 nM) was much more potent than insulin at stimulating phosphorylation of poly(Glu:Tyr) by the bovine adrenal medulla preparation. A maximal concentration of IGF-I (10 nM) increased phosphorylation approximately threefold. IGF-I was slightly more effective than insulin at stimulating the phosphorylation of poly(Glu:Tyr) by the PC12 cell receptor preparation, but neither ligand produced a maximal effect at concentrations up to 100 nM. This result probably reflects the presence of comparable numbers of IGF-I and insulin receptors on PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification of an IGF-II receptor/binding protein from the erythroleukemia cell line K562

We previously reported that insulin-like growth factor II (IGF-11) stimulated clonal growth of an erythroleukemia cell line, K562, in semi-solid agar, an effect not mimicked by insulin-like growth factor I (IGF-1), as IGF-I receptors are generally not expressed in this cell line. Affinity crosslinking of intact K562 cells with ¹²⁵I-IGF-II revealed that the labeled hormone predominantly bound to a protein with a molecular weight of approximately 75 K. We report here the partial purification of the 75 K IGF-II binding protein from K562 cells. Triton X-100-solubilized K562 cells were subjected to Sephacryl-400, followed by Sephacryl-200 chromatography. Fractions of interest were collected and applied to a Sepharose-IGF-II column or an immunoaffinity column. The immuno-affinity column was prepared using an antiserum against placental membrane-derived material eluted from the Sephacryl-400 column in the elution volume, corresponding to the IGF-II binding protein from K562 cells. An affi-gel 10 affinity column, prepared with a protein A purified IgG fraction of this antiserum (antibody-29), retarded proteins showing binding specificity for IGF-II, with apparent molecular weights of 76 K, 87 K, and 70 K under reducing conditions. These protein bands were similar to the proteins retarded in the IGF-II affinity column, when evaluated by affinity crosslinking and SDS-PAGE. Fractionation of the purified material from the antibody-29 affinity column on Superose 12 revealed 6 protein peaks. Affinity crosslinking of the peak fractions from FPLC resulted in single bands with a molecular weight of 75 K under reducing conditions with variable specificity for IGF-II.