Characterization of solubilized insulin receptors from rat liver microsomes. Existence of two receptor species with different binding properties

Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-100. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak II) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak II). Both peaks were glycoproteins. At 4 degrees C peak I showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak II had its binding optimum at pH 7.0 and low ionic strength, where peak I binding was minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak II an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4 degrees C the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak II). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of peak I revealed five receptor bands with Mr 400 000, 365 000, 320 000, 290 000, and 245 000 under non-reducing conditions. For peak II two major receptor bands with Mr 210 000 and 115 000 were found. The peak II receptor bands were also obtained after mild reduction of peak I. After complete reduction both peaks showed one major receptor band with Mr 130 000. The reductive generation of the peak II receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak II receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.     

The different receptor species of liver have similar complex insulin binding properties

Liver plasma membranes bind insulin in a complex fashion via three prominent disulfide-linked insulin receptor structures of 360K, 300K, and 260K molecular weight. To determine if the complex binding is explained by different binding affinities among the different structures, 125I-insulin was specifically cross-linked to the binding sites and the amount of radiolabeled insulin was determined after SDS-gel electrophoresis. The insulin binding characteristics of each structure were similar to the binding properties of the intact membrane. The Scatchard plot for each structure was curvilinear and the Kd values for the high and low affinity components were similar to the membrane values. Thus, the curvilinear Scatchard plot of insulin binding to liver membranes is also a feature of each receptor structure and is not a function of different receptors with different binding properties.     

Diacytosis of human asialotransferrin type 3 in the rat liver is due to the sequential engagement of two receptors

The possible role of transferrin receptors in the diacytosis of human asialotransferrin type 3 (HAsTf-3) by the rat liver was studied in vivo. A trace dose of the ligand was allowed to compete for hepatic binding sites against diferric transferrin, the concentration of which was varied between 5 400- and 18 000-fold. Binding of HAsTf-3 was insensitive to the presence of 2Fe-transferrin in this range, and the liver bound the ligand equally efficiently, regardless of whether it was presented in the holo or apo form. In contrast, pretreating the animals with desialylated bovine submaxillary mucin (2 mg/100 g, 2 min before the dose) prevented the asialotransferrin-liver interaction. These findings indicate that endocytosis of HAsTf-3 is mediated by the Gal/GalNAc-specific lectin and not by transferrin receptors. Although 2Fe-transferrin did not affect binding, it did reduce the half-life of the ligand in the liver, thus suggesting that transferrin receptors play an important role in the exocytic leg of the diacytic cycle. Based on our present and earlier data, a model is proposed in which the engagement of lectin and transferrin receptor in the diacytic cycle is envisaged sequentially so that HAsTf-3 switches receptors at an acidified subcellular site.     

Studies on the nuclear binding of steroid hormone-receptor complex; characteristics of binding of nuclei from fetal rat liver to 3H-dexamethasone-liver cytoplasmic receptor complex

Binding of 3H-dexamethasone (Dex)-rat liver cytoplasmic receptor complex to nuclei from fetal rat livers in vitro exhibited a high-affinity and saturable nature (Kd=1.5 X 10- M, maximal binding sites=470 fmole/mg DNA), and the binding was inhibited competitively by prior injection of Dex in vivo. While binding of 3H-Dex-receptor complex to nuclei from adult rat liver was in low affinity and unsaturable, and injection of Dex prior to the sacrifice of animals did not influence the nuclear binding to 3H-Dex-receptor complex in vitro. Differential salt-extraction with KCl solution of the nuclear bound 3H-Dex receptor complex revealed the presence of salt-extractable and residual forms of bound receptors. The amount of the fraction extracted with 0.3 M KCl reached its maximum at 10 min after the start of incubation, while the 1.0 M KCl-extractable and residual fractions reached their maximum plateaus after 30 min of the incubation. Scatchard analysis revealed that the binding of the receptor complex to the 0.3M and 1.0M KCl fractions was saturable, while the residual fraction did not show any tendency of saturation under the experimental conditions employed in the present study. The results obtained in this work were compared to those which have been reported by other investigators.     

Differential ligand binding by two subunits of the rat liver asialoglycoprotein receptor

The rat liver asialoglycoprotein receptor consists of two typesof subunits, a predominant polypeptide designated rat hepaticlectin 1 (RHL-1) and a minor polypeptide, RHL-2/3, that comesin two differentially glycosylated forms. The exact stoichiometryand arrangement of the subunits in the RHL oligomer are notknown. The carbohydrate-recognition domain of RHL-2/ has beenprepared by limited proteolysis of the liver receptor so thatits properties can be compared with those of the correspondingdomain of RHL-1 previously produced in a bacterial expressionsystem. Binding studies indicate that while RHL-1 binds N-acetylgalactosaminewith approximately 60-fold higher affinity than it binds galactose,RHL-2/ has only 2-fold selectivity for N-acetylgalactosamine.In general, the pattern of monosaccharide-binding specificityfor RHL-2/ is similar to RHL-1, but the discrimination of varioussugars relative to galactose is reduced substantially. Limitedproteolysis and crosslinking studies demonstrate that RHL- 2/is easily removed from the RHL oligomer in detergent solutionand that RHL-1 remains at least trimeric following removal ofRHL-2/. These studies suggest that RHL-1 forms a ligand-bindingcore while RHL-2/ acts more as an accessory subunit contributingto selective binding of certain oligosaccharide structures. asialoglycoprotein receptor binding carbohydrate recognition lectin proteolysis     

Increases of spinal kinin receptor binding sites in two rat models of insulin resistance

An autoradiographic study was conducted to determine whether kinin receptors are altered in the rat spinal cord in two experimental models of chronic hyperglycemia and insulin resistance. Sprague-Dawley rats were given 10% d-glucose in their drinking water alone or with insulin (9 mU/kg/min with osmotic pumps) for 4 weeks. Both groups and control rats were treated either with a normal chow diet or with an alpha-lipoic acid-supplemented diet as antioxidant therapy. After 4 weeks of treatment, glycemia, insulinemia, blood pressure, insulin resistance index, the production of superoxide anion in the aorta and the density of B2 receptor binding sites in the dorsal horn were significantly increased in the two models. These effects were prevented or attenuated by alpha-lipoic acid. In contrast, B2 receptor binding sites of most spinal cord laminae were increased in the glucose group only and were not affected by alpha-lipoic acid. Results show that chronic hyperglycemia associated with insulin resistance increases B1 and B2 receptor binding sites in the rat spinal cord through distinct mechanisms, including the oxidative stress for the B1 receptor.     

Characterization of human somatotropin binding to detergent-solubilized lactogenic receptors from rat liver.

Lactogenic receptors from rat liver microsomal fraction ('microsomes') were extracted by treatment with 1% (w/v) Triton X-100. Triton X-100 exerts an inhibitory effect on both the binding reaction and the separation of the free hormone from the complex. The association and dissociation of 125I-labelled human somatotropin are time- and temperature-dependent processes. The association rate constant, k1, is 6.7 x 10(6) mol . litre-1 . min-1 at 25 decrees C, and the dissociation rate constant, k-1, is 1.1 x 10(-3) min-1 at 25 degrees C. Scatchard analysis of saturation data reveals the existence of a single class of receptors and that solubilization leads to a slight decrease in affinity and a sharp increase in binding capacity. The dissociation constant, Kd, of the solubilized preparation is 0.22 nM and the binding capacity 2900 fmol/mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the solubilized receptors is specifically inhibited by hormones with lactogenic activity. Incubation of the solubilized preparation with trypsin resulted in an 80% decrease in binding activity. The solubilized form of the receptor has a slightly increased sensitivity to the inactivation by trypsin, heat and extremes of pH, with respect to the membrane-bound form.     

A thiol-sensitive degradative process of liver uncouples autophosphorylation of the insulin receptor from insulin binding.

Insulin receptors derived from highly purified rat liver plasma membranes and Golgi membranes showed differences in insulin-mediated receptor autophosphorylation, even though their insulin-binding characteristics were similar. This difference was related to the generation of a Mr-84,000 fragment of the Mr-90,000 beta subunit of the plasma-membrane receptor, a fragment that was not present in the receptor from Golgi membranes, in the absence of a change in the insulin-binding alpha subunit. When autophosphorylation activity was based on insulin binding, the activity of the plasma-membrane-derived insulin receptor was decreased to 25-30% that of the Golgi-derived receptor. Endoglycosidase F digestion produced changes in the Mr values for both species, but they were not converted into a single subunit, thereby suggesting differences in the protein component of the two subunits. Although the proteinase inhibitors phenylmethanesulphonyl fluoride, ovomucoid and aprotinin failed to block the formation of the Mr-84,000 fragment, the presence of iodoacetamide or EDTA during liver homogenization markedly inhibited fragment generation and allowed the plasma-membrane insulin receptor to retain an autophosphorylation activity comparable with that present in insulin receptors from Golgi membranes. Thus a thiol-sensitive, cation-dependent, degrading activity has been identified that can uncouple the insulin-binding activity of the plasma-membrane insulin receptor from its tyrosine kinase activity.     

Reoxidation of the class I disulfides of the rat adipocyte insulin receptor is dependent upon the presence of insulin: the class I disulfide of the insulin receptor is extracellular

Elements of the quaternary structure of the native and dithiothreitol- (DTT) reduced rat adipocyte insulin receptor have been elucidated by vectorial probing and subunit cross-linking. The charged reducing agents glutathione and beta-mercaptoethylamine were used to reduce the class I disulfides of the receptor in intact adipocytes, demonstrating the extracellular location of the disulfide directly. This interpretation was confirmed by use of DTT as a reducing agent and the nonpermeant sulfhydryl blocking reagent Thiolyte MQ to prevent the reoxidation of the class I sulfhydryl groups which occurred when they were not blocked. It was found that the above reoxidation of the receptor is dependent on the concentration of insulin in the nanomolar range, not occurring measurably at 4 degrees C in its absence. Cross-linking studies with ethylene glycol bis(succinimidyl succinate) demonstrated that the alpha subunits could not be cross-linked to each other after reduction of the class I disulfides, suggesting that the interaction between the receptor heterodimers may be due primarily to the disulfide bonds.     

The insulin receptor of rat brain is coupled to tyrosine kinase activity

Insulin receptors from rat brain were studied for receptor-associated tyrosine kinase activity. In solubilized, lectin-purified receptor preparations, insulin stimulated the phosphorylation of the beta subunit of its receptor as well as of exogenous substrates. Phosphoamino acid analysis of casein phosphorylated by these preparations revealed that 32P incorporation occurred predominantly on tyrosine residues. Receptor and casein phosphorylations were specific for insulin and analogues that also bind to the insulin receptor. The insulin dose response for phosphorylation of brain receptor resembled that reported for the purified insulin receptor from human placenta (Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D.L., and Kahn, C.R. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2137-2141), suggesting similar insulin sensitivity and coupling of the brain receptor kinase. Four polyclonal antisera to the insulin receptor were able to bind and immunoprecipitate the brain receptor; however, only two antisera activated the receptor-associated kinase. Thus, the brain insulin receptor, like the well studied non-neural receptor, is coupled to tyrosine kinase activity, making regulation of cellular events by insulin in neural tissue possible.     

All‐atom structural models of insulin binding to the insulin receptor in the presence of a tandem hormone‐binding element

Insulin regulates blood glucose levels in higher organisms by binding to and activating insulin receptor (IR), a constitutively homodimeric glycoprotein of the receptor tyrosine kinase (RTK) superfamily. Therapeutic efforts in treating diabetes have been significantly impeded by the absence of structural information on the activated form of the insulin/IR complex. Mutagenesis and photo‐crosslinking experiments and structural information on insulin and apo‐IR strongly suggest that the dual‐chain insulin molecule, unlike the related single‐chain insulin‐like growth factors, binds to IR in a very different conformation than what is displayed in storage forms of the hormone. In particular, hydrophobic residues buried in the core of the folded insulin molecule engage the receptor. There is also the possibility of plasticity in the receptor structure based on these data, which may in part be due to rearrangement of the so‐called CT‐peptide, a tandem hormone‐binding element of IR. These possibilities provide opportunity for large‐scale molecular modeling to contribute to our understanding of this system. Using various atomistic simulation approaches, we have constructed all‐atom structural models of hormone/receptor complexes in the presence of CT in its crystallographic position and a thermodynamically favorable displaced position. In the “displaced‐CT” complex, many more insulin–receptor contacts suggested by experiments are satisfied, and our simulations also suggest that R‐insulin potentially represents the receptor‐bound form of hormone. The results presented in this work have further implications for the design of receptor‐specific agonists/antagonists. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Stoichiometry for the binding of insulin to insulin receptors in adipocyte membranes

Insulin receptor molecules in rat adipocyte plasma membranes were shown to be monovalent with respect to their capacity to bind insulin. The 1:1 stoichiometry for insulin binding was determined by a "double-probe labeling" procedure, wherein 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess saturating concentration of an unlabeled biotinylated insulin derivative (probe 2). If the receptor were competent to bind more than one insulin molecule, any receptor molecule that was cross-linked to probe 1 also should have been cross-linked to probe 2 in the double probe labeling procedure. The monovalent character of the insulin receptor was indicated by the failure of the probe 1-linked receptor to be cross-linked to probe 2. This was indicated by the failure of succinylavidin to increase the molecular weight of the probe 1-linked receptor. Control experiments indicated that succinylavidin increased the molecular weight of receptor that had been cross-linked to probe 2. The 1:1 stoichiometry for insulin binding demonstrated here indicates that if insulin receptors contain more than one insulin binding subunit, the binding of insulin to its receptor must be a highly negatively cooperative process.     

Characterization of the binding of ferritin to the rat liver ferritin receptor

The binding characteristics and specificity of the rat hepatic ferritin receptor were investigated using ferritins prepared from rat liver, heart, spleen, kidney and serum, human liver and serum, guinea pig liver and horse spleen as well as ferritins enriched with respect to either H- or L-type subunit composition, prepared by chromatofocusing of rat liver ferritin on Mono-P or by reverse-phase chromatography of ferritin subunits on ProRPC 5/10. No significant difference was apparent in the binding of any of the tissue ferritins, or of ferritins of predominantly acidic or basic subunit composition. However, serum ferritin bound with a lower affinity. The effect of carbohydrate on the ferritin-receptor binding was examined by glycosidase treatment of tissue and serum ferritins. Tissue ferritin binding was unaffected, while serum ferritin binding affinity was increased to that of the tissue ferritins. Inhibition of ferritin binding by lactoferrin was not due to common carbohydrate moieties as previously suggested but was due to direct binding of lactoferrin to ferritin. Therefore, carbohydrate residues do not appear to facilitate receptor-ferritin binding, and sialic acid residues present on serum ferritin may in fact interfere with binding. The results indicate that the hepatic ferritin receptor acts preferentially to remove tissue ferritins from the circulation. The lower binding affinity of serum ferritin for the ferritin receptor explains its slower in vivo clearance relative to tissue ferritins.     

Decreased binding of insulin to liver plasma membrane receptors in hereditary diabetic mice.

The interaction of insulin with its receptors was studied in liver plasma membranes of the young non-obese hereditary diabetic mouse (KK strain). Under identical conditions of preparation and incubation, the membranes of the KK mouse bind only 55-70% as much insulin per mg of protein as those of the control mouse (Swiss albino). Scatchard analysis suggests that this decrease in binding is due to a decrease in the number of receptor sites in the membrane of the diabetic mouse. However, the membranes of diabetic and control mice do not exhibit significant differences in hexosamine and sialic acid contents, enzyme activities, and protein and glycoprotein analysis. The decrease in insulin receptors in the KK mouse seems to correlate with the insulin resistance which they exhibit.     

Characterization of the binding of ferritin to the rat liver ferritin receptor

The binding characteristics and specificity of the rat hepatic ferritin receptor were investigated using ferritins prepared from rat liver, heart, spleen, kidney and serum, human liver and serum, guinea pig liver and horse spleen as well as ferritins enriched with respect to either H- or L-type subunit composition, prepared by chromatofocusing of rat liver ferritin on Mono-P or by reverse-phase chromatography of ferritin subunits on ProRPC 5/10. No significant difference was apparent in the binding of any of the tissue ferritins, or of ferritins of predominantly acidic or basic subunit composition. However, serum ferritin bound with a lower affinity. The effect of carbohydrate on the ferritin-receptor binding was examined by glycosidase treatment of tissue and serum ferritins. Tissue ferritin binding was unaffected, while serum ferritin binding affinity was increased to that of the tissue ferritins. Inhibition of ferritin binding by lactoferrin was not due to common carbohydrate moieties as previously suggested but was due to direct binding of lactoferrin to ferritin. Therefore, carbohydrate residues do not appear to facilitate receptor-ferritin binding, and sialic acid residues present on serum ferritin may in fact interfere with binding. The results indicate that the hepatic ferritin receptor acts preferentially to remove tissue ferritins from the circulation. The lower binding affinity of serum ferritin for the ferritin receptor explains its slower in vivo clearance relative to tissue ferritins.     

Kinetics of dexamethasone binding to the glucocorticoid receptor of intact erythroid cells from rat fetal liver

The erythroid cells from the rat fetal liver have been shown to possess a receptor for glucocorticoids. In the present work, the characteristics of [3H]dexamethasone binding have been studied on intact cells, in order to minimize receptor degradation, and at 4 degrees C, in order to prevent the activation of the hormone-receptor complex. Dissociation kinetics were those of a first-order reaction and the value of the rate constant of dissociation was similar to the values available in the literature. When studied at low concentrations of the ligand and using short-term incubations, association kinetics were apparently those of a simple bimolecular reaction. But at high ligand concentrations and/or using long-term incubations, association kinetics indicated a more complex reaction. Our results were compatible with the model proposed by Pratt W.B., Kaine J.L. and Pratt V.D. (J. Biol. Chem. 250 (1975) 4584-4591) for cytosolic preparations. This model implies the rapid formation of a transient unstable form of the complex, further converted into a stable form with slower kinetics. Equilibrium dissociation constant of the first (rapid) reaction was 80 microM and the rate constant of 'stabilization' was of the order of 70 X 10(-3) min-1. These values agree with the results of Pratt et al. relative to a cytosolic preparation from rat thymocytes.