Binding properties and biological potencies of insulin-like growth factors in L6 myoblasts.

Protein synthesis in rat L6 myoblasts is stimulated and protein breakdown inhibited in a co-ordinate manner by insulin-like growth factors (IGF) or insulin. For both processes, bovine IGF-1 was somewhat more potent than human IGF-1, which was effective at a tenth the concentration of insulin, rat IGF-2 or human IGF-2. A similar order of potency is noted when DNA synthesis or protein accumulation is monitored over a 24 h period, but between 20- and 50-fold higher concentrations of each growth factor are required than those needed to produce effects in the 4 h protein-synthesis or -breakdown measurements. Binding experiments with labelled human or bovine IGF-1 as ligand demonstrated competition at concentrations of IGF-2, especially human IGF-2, lower than that of either IGF-1 preparation. This pattern was much more pronounced when the radioligand was either human IGF-2 or rat IGF-2. Insulin competed 10-15% for the binding of labelled IGF-1, but not at all with labelled IGF-2. Ligand-receptor cross-linking experiments showed that labelled bovine IGF-1 bound approximately equally to the type 1 IGF receptor (Mr 130000 after reduction) and to the type 2 IGF receptor (Mr 270000 after reduction), and that unlabelled IGF-1 competed equally with radioligand binding to both receptors. On the other hand, rat IGF-2 competed more effectively for binding to the type-2 receptor, and insulin competed only for binding to the type-1 receptor. Further cross-linking experiments with rat IGF-2 as radioligand demonstrated binding only to the type-2 receptor and to proteins with Mr values after reduction of 230000 and 200000. This binding was prevented by high rat IGF-2 concentrations, less effectively by bovine IGF-1 and not at all by insulin. The apparently conflicting biological potencies and receptor binding of the different growth factors can be explained if all the biological actions are mediated via the type-1 IGF receptor, rather than through the abundant type-2 receptor.     

Assembly of insulin/insulin-like growth factor-1 hybrid receptors in vitro

Insulin and Mn/MgATP treatment of immunoaffinity-purified alpha beta heterodimeric insulin receptors induced the formation of an alpha 2 beta 2 heterotetrameric insulin receptor complex. In contrast, insulin-like growth factor-1 (IGF-1) treatment was completely ineffective in inducing the association of alpha beta heterodimeric insulin receptors. Similarly, IGF-1 or Mn/MgATP, but not insulin, treatment of immunoaffinity-purified alpha beta heterodimeric IGF-1 receptors induced the formation of an alpha 2 beta 2 heterotetrameric IGF-1 receptor complex. A monoclonal antibody specific for the insulin receptor (MA5) completely immunoprecipitated all the insulin binding activity from both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes but did not immunoprecipitate IGF-1 receptors. Conversely, the IGF-1 receptor-specific monoclonal antibody (alpha IR-3) immunoprecipitated all the IGF-1 binding activity, but not insulin receptors. The simultaneous treatment of pooled equal amounts of alpha beta heterodimeric insulin and IGF-1 receptors with a combination of insulin and IGF-1 resulted in the formation of alpha 2 beta 2 heterotetrameric insulin and IGF-1 receptor complexes. However, in the mixed alpha 2 beta 2 heterotetrameric receptor fraction MA5 immunoprecipitated 94% of the insulin binding in addition to 27% of the IGF-1 binding activity whereas alpha IR-3 immunoprecipitated 97% of the IGF-1 binding in addition to 38% of the insulin binding activity. Treatment of the mixed alpha beta heterodimeric insulin and IGF-1 receptors with Mn/MgATP also resulted in the formation of cross-immunoreactive (42-46%) alpha 2 beta 2 heterotetrameric receptors. These data directly demonstrate the formation of insulin/IGF-1 hybrid receptors by both a combination of insulin plus IGF-1 or Mn/MgATP treatment of purified human placenta alpha beta heterodimeric insulin and IGF-1 half-receptors in vitro.     

Specific binding of insulin-like growth factors 1 and 2 to the type 1 and type 2 receptors respectively.

1. Competitive binding and receptor cross-linking experiments have been used to examine the receptor-ligand interactions between three bovine insulin-like growth factors (IGF) and monolayer cultures of myoblasts and fibroblasts. 2. Labelled IGF-2 bound predominantly to the type 2 receptor with negligible label cross-linked to the type 1 receptor, notwithstanding the ability of IGF-2 to compete effectively for the binding of IGF-1 to the type 1 receptor. Approx. 100-fold higher concentrations of IGF-1 or the N-terminal truncated (des-Gly-Pro-Glu) IGF-1 (-3N:IGF-1) were required to produce competition equivalent to IGF-2. 3. All IGF peptides, but especially IGF-1, enhanced the binding of labelled IGF-2 to the type 2 receptor of lung fibroblasts. This unusual effect was probably a consequence of the displacement of labelled IGF-2 otherwise bound to a medium protein, a conclusion supported by the demonstration of a 38 kDa membrane protein cross-linked to labelled IGF-2. 4. Both IGF-1 and -3N:IGF-1 bound only to the type 1 IGF receptor in L6 myoblasts, rat vascular smooth-muscle cells and human lung fibroblasts. The peptides competed for labelled IGF-1 binding with potencies in the order -3N:IGF-1 greater than IGF-1 greater than IGF-2 much greater than insulin. Since the IGF peptides were equipotent in skin fibroblasts, it was proposed that the apparently higher affinity of -3N:IGF-1 for receptors in the other cell types was instead a consequence of a low affinity of this peptide for the competing 38 kDa binding protein.     

The inhibition of a membrane-bound enzyme as a model for anaesthetic action and drug toxicity.

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.     

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

The cells of the IM-9 human lymphocyte-derived line contain a sub-population of insulin-binding sites whose immunological and hormone-binding characteristics closely resemble those of the atypical insulin-binding sites of human placenta. These binding sites, which have moderately high affinity for multiplication-stimulating activity [MSA, the rat homologue of insulin-like growth factor (IGF) II] and IGF-I, are identified on IM-9 cells by 125I-MSA binding. They account for approximately 30% of the total insulin-receptor population, and do not react with a monoclonal antibody to the type I IGF receptor (alpha IR-3). The relative concentrations of unlabelled insulin, MSA and IGF-I required to displace 50% of 125I-MSA from these binding sites (1:4.7:29 respectively) are maintained for cells, particulate membranes, Triton-solubilized membranes precipitated either by poly(ethylene glycol) or a polyclonal antibody (B-10) to the insulin receptor, and receptors purified by insulin affinity chromatography. Because the atypical insulin/MSA-binding sites outnumber the type I IGF receptors in IM-9 cells by approximately 10-fold, they also compete with the latter receptors for 125I-IGF-I binding. Thus 125I-IGF-I binding to IM-9 cells is inhibited by moderately low concentrations of insulin (relative potency ratios for insulin compared with IGF-I are approx. 1/14 to 1/4) and is partially displaced (65-80%) by alpha IR-3. When type I IGF receptors are blocked by alpha IR-3 or removed by B-10 immunoprecipitation or insulin affinity chromatography, the hormone-displacement patterns for 125I-IGF-I binding resemble those of the atypical insulin/MSA-binding sites.     

The specificity of the human IGF-2 receptor

The specificity of the type 2 insulinlike growth factor (IGF) receptor is evaluated in human placenta membranes and the human cell line K562. K562 cells have type 2 but not type 1 IGF receptors. Native IGF-2 isolated from human plasma and synthetic IGF-2 were equipotent in competing with labeled IGF-2 in both systems. Pure IGF-1 isolated from plasma, synthetic IGF-1 and recombinant IGF-1 could not crossreact with the type 2 IGF receptor in concentrations up to 1 microgram/ml in both systems. Studies on placenta membrane were done in the presence of 300 ug/ml insulin to block the type 1 IGF receptors. It is concluded that IGF-1, as well as insulin, cannot crossreact with the human type 2 IGF receptor.     

Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe-1,Val1,Asn2, Gln3,His4,Ser8, His9,Glu12,Tyr15,Leu16]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has greater than 1,000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln3,Ala4]IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr15,Leu16]IGF-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. This peptide is also equipotent to hIGF-I at the types 1 and 2 IGF receptors. The peptide in which these four-point mutations are combined, [Gln3,Ala4,Tyr15,Leu16]IGF-I, has 600-fold reduced affinity for the serum binding proteins. This peptide has 10-fold increased potency for the insulin receptor, but is equipotent to hIGF-I at the types 1 and 2 IGF receptors. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, these peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I.     

Functional properties of an isolated alpha beta heterodimeric human placenta insulin-like growth factor 1 receptor complex

Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, resulted in the formation of a functional alpha beta heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native alpha 2 beta 2 heterotetrameric disulfide-linked state. The membrane-bound alpha beta heterodimeric complex displayed similar curvilinear 125I-IGF-1 equilibrium binding compared to the alpha 2 beta 2 heterotetrameric complex. Triton X-100 solubilization of the alkaline pH and DTT-pretreated placenta membranes, followed by Bio-Gel A-1.5m gel filtration chromatography, was found to effectively separate the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric IGF-1 receptor species, 125I-IGF-1 binding to both the isolated alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. Similar to the membrane-bound IGF-1 receptor species, the 125I-IGF-1 binding properties between the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes were not significantly different. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of alpha beta heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent alpha 2 beta 2 heterotetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Insulin receptor interaction in human placental plasma membranes

Insulin-receptor interaction in partially purified preparations of human placental plasma membranes from normal mothers at term of pregnancy has been characterized. 125I-insulin became rapidly and reversibly bound to plasma membranes, being time and temperature dependent. The binding readily appeared at 1.0 ng/ml insulin concentration which falls within the physiological range of peripheral blood. Low levels of unlabeled insulin inhibited binding; 20 ng/ml insulin produced fifty per cent inhibition. Scatchard plots of data from competitive insulin binding proved to be curvilinear. The insulin greater ability for binding observed in this preparation can be explained by the purification degree achieved at the plasma membranes. 125I-insulin was less degraded by partially purified placental plasma membranes than by a microsomal-membrane preparation obtained without differential centrifugation in sucrose linear gradient. All these properties strongly suggest that the insulin-binding sites characterized in the plasma membrane fraction of the placenta represent biologically important receptors to hormone.     

Insulin receptor is phosphorylated in response to treatment of HepG2 cells with insulin-like growth factor I.

1. Binding of insulin and insulin-like growth factor I (IGF-I) to HepG2 cells was analysed with regard to competition by both insulin and IGF-I. At concentrations of insulin that caused maximal phosphorylation of the insulin receptor, virtually no displacement of IGF-I binding was observed. Similarly, at concentrations of IGF-I that caused maximal phosphorylation of the IGF-I receptor, no displacement of insulin binding was observed. 2. When the phosphorylation of both receptors was examined individually by using specific monoclonal antibodies to immunoprecipitate the receptors, phosphorylation of the insulin receptor was found to increase on both serine and tyrosine residues in cells treated with 100 ng of IGF-I/ml. In contrast, no increased phosphorylation of IGF-I receptor was observed in cells treated with 100 ng of insulin/ml. 3. The increase in phosphorylation of insulin receptor in response to IGF-I correlated with the dose-response of IGF-I-stimulated phosphorylation of the IGF-I receptor. 4. The IGF-I-stimulated phosphorylation of the insulin receptor could be blocked by preincubation with a monoclonal antibody that blocks IGF-I binding to the IGF-I receptor.     

Calcineurin-mediated dephosphorylation of the human placental membrane receptor for epidermal growth factor urogastrone

The findings of our work were 2-fold: (1) calcineurin (from bovine brain) can catalyze the complete dephosphorylation of the phosphotyrosine and phosphoserine residues in the human placental receptor for epidermal growth factor urogastrone (EGF-URO), and (2) the major calmodulin-binding protein of human placental membranes is a calcineurin-related protein. In terms of its metal ion dependence (Ni2+ greater than Mn2+ greater than Co2+), its calmodulin dependence, and its sensitivity to inhibitors (Zn2+, fluoride, orthovanadate), the phosphotyrosyl protein phosphatase activity of calcineurin, using the EGF-URO receptor as substrate, paralleled the enzyme activity measured with p-nitrophenyl phosphate (PNPP) as a substrate. These characteristics distinguish calcineurin from other classes of protein phosphotyrosyl phosphatases. Calcineurin purified from placental membranes was similar to, if not identical with, bovine brain calcineurin in terms of enzymatic specific activity toward PNPP, subunit electrophoretic mobilities, and immunological cross-reactivity. The enzymatic properties and comparative abundance of calcineurin in the placenta membranes suggest that this enzyme may play an important role in regulating the phosphorylation state of those receptors (e.g., for EGF-URO or insulin) also known to be present in the membranes.     

Expression, purification and characterization of recombinant human insulin-like growth factor I in yeast

Insulin-like growth factor I (IGF-I) is a 70 amino acid (aa) protein that is structurally similar and functionally related to insulin. We have inserted a synthetic gene coding for human IGF-I into a Saccharomyces cerevisiae expression vector utilizing the MF alpha 1 promoter and pre-pro leader peptide. This vector directs the expression and secretion of native, biologically active growth factor. Cleavage of the pre-pro alpha factor leader sequence in vivo results in the secretion of a 70-aa recombinant IGF-I molecule with the native N-terminal glycine residue. Human IGF-I purified from yeast culture supernatant is equipotent to serum-derived IGF-I in inhibiting [125I]IGF-I binding to type-I IGF receptors and crude human serum-binding proteins. Recombinant IGF-I is also equipotent to human IGF-I in the stimulation of DNA synthesis in rat aortic smooth-muscle cells. In contrast, yeast recombinant IGF-I is less potent than serum-derived IGF-I in binding to type-2 IGF receptors. The ability to produce native, biologically active IGF-I in yeast will allow the elucidation of binding domains through the expression and characterization of specific structural analogs.     

Ligand-receptor interactions involved in the stimulation of Swiss 3T3 fibroblasts by insulin-like growth factors and insulin.

1. The binding of 125I-labelled insulin-like growth factor 1 (125I-IGF-1) to Swiss mouse 3T3 fibroblasts was time- and concentration-dependent. Unlabelled IGF-1 had a slightly higher potency than multiplication-stimulating activity (MSA) in inhibiting the binding of 125I-IGF-1, and insulin gave a parallel inhibition curve at 300-1000-fold lower potency. Chemical cross-linking of bound 125I-IGF-1 to its receptors, followed by polyacrylamide-gel electrophoresis under reducing conditions, revealed a major band of Mr 130,000, the labelling of which was inhibited by IGF-1 or high concentrations of insulin. 2. The binding of 125I-IGF-1 was not affected by either co-incubation or preincubation of the cells with a range of heterologous growth factors and mitogens. However, IGF-1 and MSA each induced down-regulation of 125I-IGF-1 binding sites. 3. The maximal stimulations of DNA synthesis induced by IGF-1, MSA and insulin, in the presence of a synergizing mitogen, were similar. The dose-response curve for insulin was not parallel to those for IGF-1 and MSA; in particular, low concentrations of insulin induced a greater stimulation than expected on the basis of its potency in the inhibition or down-regulation of 125I-IGF-1 binding. 4. The preincubation of 125I-IGF-1 with Swiss 3T3 cells at 37 degrees C decreased its ability to bind to a second batch of cells. This inactivation did not occur when the preincubation was performed at 4 degrees C or in the presence of cycloheximide. Chemical cross-linking revealed that the cells released an IGF-binding protein, giving a complex of Mr about 48,000. 5. It is concluded that type I IGF receptors mediate the stimulation of Swiss 3T3 cells by insulin-like mitogens, but that insulin probably stimulates the cells through insulin receptors. The cells can modulate the amount of ligand binding, both by down-regulation of the receptors and by the secretion of an IGF-binding protein.     

Binding and action of insulin-like growth factors and insulin in bovine luteal tissue during the oestrous cycle.

The effect of insulin-like growth factors (IGFs) and insulin on the release of progesterone and oxytocin from bovine corpus luteum was investigated at early (days 5-7), mid- (days 8-12) and late (days 15-18) luteal phases of the oestrous cycle in an in vitro microdialysis system. The expression of specific receptors was evaluated in bovine corpora lutea of the respective luteal stages. A 30 min infusion of IGF-1, IGF-2 (1.3, 13 and 130 nmol l-1) or insulin (13, 130 and 1300 nmol l-1) caused a stimulation of the release of progesterone (P < 0.05). IGF-1 was most effective in releasing progesterone. Oxytocin release from corpora lutea was stimulated by insulin at all doses tested (13-1300 nmol l-1), whereas the IGFs were only effective at the highest dose (130 nmol l-1) applied. The high doses of IGFs (130 nmol l-1) and insulin (1300 nmol l-1) stimulated the release of progesterone and oxytocin throughout the luteal phase (P < 0.05). For all three peptides, greatest stimulation was seen during the late luteal phase (days 15-18 of the oestrous cycle) with the peak of progesterone release directly related to peptide infusion (P < 0.05). In addition, IGF-1 stimulated total release of progesterone (units in 4 h) after the beginning of the stimulation during this phase (P < 0.05). IGF-1 caused a gradual increase of progesterone even beyond the time of peptide perfusion, whereas IGF-2 and insulin stimulated progesterone release only during the peptide perfusion. Distinct receptors for IGF-1 and IGF-2 were present in corpora lutea membrane preparations at all stages investigated. Specific binding for insulin was also seen in all stages of the cycle without any cycle-dependent changes in the amount of binding. The displacement of labelled insulin by unlabelled IGF-1 and IGF-2 did not show the rank of order that has been described as typical for insulin receptors (i.e. insulin > IGF-1 > IGF-2), but comparable binding affinities were observed for the three unlabelled ligands. Specific binding of IGF-2 was markedly higher than that of IGF-1 or insulin throughout the cycle (1.9- and 4.9-fold higher compared with IGF-1 and insulin, respectively). Receptor specificity did not change during luteal development. Binding affinity and capacity of IGF-1 receptor was constant throughout the oestrous cycle. Specific IGF-2 binding increased and showed a positive co-operativity towards the end of the cycle. Specific binding of insulin was not significantly different in the three luteal stages examined.     

Coupling of insulin-responsive glucose transport to receptors for insulin-like growth factor 1 in primary human fibroblasts

We have recently described an insulin-resistant patient with leprechaunism (leprechaun G.) having a homozygous leucine----proline mutation at amino acid position 233 in the alpha-chain of the insulin receptor. The mutation results in a loss of insulin binding to cultured fibroblasts. Fibroblasts from the patient and control individuals were used to quantify the stimulation of 2-deoxyglucose uptake by insulin and insulin-like growth factor 1 (IGF-1). Insulin hardly stimulates basal 2-deoxyglucose uptake in the patient's fibroblasts whereas in control fibroblasts the uptake of 2-deoxyglucose is stimulated by insulin approximately 1.7 times. In contrast, IGF-1 stimulates hexose uptake in the patient's fibroblasts 1.8 times, a similar value to that obtained by stimulation of control fibroblasts with insulin or IGF-1. With both types of fibroblasts, maximal IGF-1 response is reached at about 10 nM IGF-1, the ED50 being approximately 4 nM. The results indicate that the insulin responsive glucose transport in primary fibroblasts is functionally linked to the receptor for IGF-1. Insulin binds with an approximately 200-fold lower affinity to IGF-1 receptors, compared to homologous IGF-1 binding. As an insulin concentration of 10 microM is unable to give maximal stimulation of glucose uptake in the patient's fibroblasts, which is already seen with 10 nM IGF-1, it seems that occupation of IGF-1 receptors by insulin on the patient's cells is less efficient at stimulating hexose uptake compared to homologous activation.     

Altered placental insulin and insulin-like growth factor-I receptors in diabetes

We have compared the characteristics of IGF-I and insulin receptors in placentas of normals and insulin dependent diabetic patients. Specific binding of both IGF-I and insulin in placental membranes from patients with good glycemic control (as reflected by blood hemoglobin content) was unaltered while that in the placental membranes from the patients with poor glycemic control was increased to approximately 20% of the normals. This observed small but significant (p less than 0.05) increase in binding of IGF-I and insulin to placental membranes from diabetic patients with poor glycemic control was further magnified, approximately twice (p less than 0.001) the normal, when the membrane receptors were purified by lectin chromatography. The kinetic analysis of IGF-I and insulin binding in both membranes and lectin purified receptors revealed that the increased binding of insulin and IGF-I to the placentas from diabetic patients with poor glycemic control was due to an approximately 2 fold increase (p less than 0.001-0.05) in the receptor numbers without any significant changes of the affinities. The molecular characteristics of the receptors in these diabetic patients, as revealed by the cross-linking studies, did not reveal any changes when compared to the normals. The parallel changes of IGF-I and insulin receptors, shown here, are in accordance with the homologous nature of these two receptors. The increased receptor numbers of these two interrelated hormones in placentas of diabetics with poor glycemic control may be relevant to the altered placental functions in diabetic pregnancy.     

Influence of placental mannose/N-acetyl glucosamine-binding proteins on the interaction of insulin and insulin-like growth factors with their receptors

Placenta is a source of carbohydrate-binding proteins that function as molecular scavengers, but they could also be involved in interactions that assist in metabolic control. Mannose/N-acetyl-glucosamine (Man/GlcNAc)-binding proteins from placenta were isolated and their reactivity towards placental insulin and insulin-like growth factor receptors (IR and IGF-Rs) was analyzed. The lectins reduced the binding of insulin and IGF-I in a dose-dependent manner, while almost no effect was observed on the binding of IGF-II. The shape of the inhibition curves changed, suggesting altered binding specificity. The presence of sugar could not reverse completely the effect of the lectins, implicating both lectin-sugar and protein-protein conformational recognition. Since biological molecules in our experimental system were those that are in close relation in vivo, placental Man/GlcNAc-specific lectins may be regarded as potential allosteric modulators of lig- and-receptor interactions in a system of homologous ligands, selectively affecting only binding to tyrosine kinase type receptors (IR and IGF-1R).     

Immunological relationships between receptors for insulin and insulin-like growth factor I. Evidence for structural heterogeneity of insulin-like growth factor I receptors involving hybrids with insulin receptors.

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.     

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.