Optimum conditions for the binding of insulin to its receptor.

The specific binding of insulin to the membranes from lactating mouse mammary gland was studied as a model of hormonereceptor type of binding. The basic ingredients of binding, the concentration of receptor protein and the concentration of labeled insulin were mainly studied. The characteristic changes in specific binding were followed, the adequate regression equation was draen and the optimum conditions of binding were established for further experiments. The expediency of applying shortened orthogonal plans, regression and analysis and graphic conture analysis were proved.     

Production of antibodies that inhibit the binding of insulin to its receptor

In this study, we report a procedure for producing antisera that block the binding of ¹²⁵I-insulin to its receptor. After 2 injections with intact IM-9 cultured human lymphocytes, the antisera from 8 of 17 $\text{Balb}\text{C}$ mice inhibited the binding of ¹²⁵I-insulin to its receptor on IM-9 cells by 50% or greater. One antiserum at dilutions of 1:200 and 1:50 inhibited the binding of ¹²⁵I-insulin by 50% and 80%, respectively. Four lines of evidence indicated that the inhibition of ¹²⁵I-insulin binding by this antiserum was due to a specific immunoglobulin directed against the insulin receptor. First, removal of the immunoglobulin fraction of the antiserum resulted in a complete loss of its inhibitory activity. Second, the antiserum inhibited the binding of ¹²⁵I-insulin to its receptor on both human cultured lymphocytes and human placenta particles. Third, the antisera bound solubilized insulin-receptor complexes. Finally, the antiserum did not inhibit the binding of ¹²⁵I-human growth hormone to its receptor on IM-9 lymphocytes. These studies demonstrate therefore, a simple method for producing antibodies that block the binding of ¹²⁵I-insulin to the human insulin receptor.     

Effects of beta-hydroxy butyric acid on insulin binding to its receptor and on autophosphorylation of the receptor

We examined the effects of beta-hydroxy butyric acid (B-OH-butyrate) on insulin binding to its receptor and on autophosphorylation of the receptor in vitro. The affinity of isolated rat adipocyte insulin receptors was significantly increased by B-OH-butyrate at neutral pH. Similar results were obtained with soluble insulin receptors prepared from hepatocytes. Autophosphorylation of insulin receptor, however, was not affected by OH-butyrate at neutral pH. These results suggested insulin resistance in ketoacidosis is not caused by an interaction between B-OH-butyrate and the insulin receptor.     

A radioimmunoassay for human insulin receptor correlation between insulin binding and receptor mass.

We have developed a radioimmunoassay for human insulin receptor. Serum from a patient with Type B severe insulin resistance was used as anti-insulin receptor antiserum. Pure human placental insulin receptor was used as reference preparation and 125I labeled pure insulin receptor as trace. The radioimmunoassay was sensitive (limit of detection less than 17 fmol), reproducible (inter and intra-assay coefficients of variation 12.5% and 1.6% respectively) and specific (no crossreactivity with pure placental IGF-1 receptor, insulin and glucagon). The anti-insulin receptor antibody was, however, able to differentiate between insulin receptor from human placenta and from rat liver. To determine the number of insulin binding sites per receptor, we measured insulin binding (by insulin binding assay) and insulin receptor mass (by radioimmunoassay) in solubilized aliquots from 5 human placentas. The molar ratio of insulin binding to receptor mass was 0.86 +/- 0.12 when binding was determined with monoiodinated 125I-Tyr A 14-insulin. It was 1.94 +/- 0.27 when randomly iodinated 125I-insulin was used. In conclusion, using a sensitive, reproducible and specific radioimmunoassay, we have measured insulin receptor mass independent of insulin binding. Our data are most compatible with binding of one insulin molecule per human placental insulin receptor.     

Serine phosphorylation proximal to its phosphotyrosine binding domain inhibits insulin receptor substrate 1 function and promotes insulin resistance

Ser/Thr phosphorylation of insulin receptor substrate (IRS) proteins negatively modulates insulin signaling. Therefore, the identification of serine sites whose phosphorylation inhibit IRS protein functions is of physiological importance. Here we mutated seven Ser sites located proximal to the phosphotyrosine binding domain of insulin receptor substrate 1 (IRS-1) (S265, S302, S325, S336, S358, S407, and S408) into Ala. When overexpressed in rat hepatoma Fao or CHO cells, the mutated IRS-1 protein in which the seven Ser sites were mutated to Ala (IRS-1(7A)), unlike wild-type IRS-1 (IRS-1(WT)), maintained its Tyr-phosphorylated active conformation after prolonged insulin treatment or when the cells were challenged with inducers of insulin resistance prior to acute insulin treatment. This was due to the ability of IRS-1(7A) to remain complexed with the insulin receptor (IR), unlike IRS-1(WT), which underwent Ser phosphorylation, resulting in its dissociation from IR. Studies of truncated forms of IRS-1 revealed that the region between amino acids 365 to 430 is a main insulin-stimulated Ser phosphorylation domain. Indeed, IRS-1 mutated only at S408, which undergoes phosphorylation in vivo, partially maintained the properties of IRS-1(7A) and conferred protection against selected inducers of insulin resistance. These findings suggest that S408 and additional Ser sites among the seven mutated Ser sites are targets for IRS-1 kinases that play a key negative regulatory role in IRS-1 function and insulin action. These sites presumably serve as points of convergence, where physiological feedback control mechanisms, which are triggered by insulin-stimulated IRS kinases, overlap with IRS kinases triggered by inducers of insulin resistance to terminate insulin signaling.     

The structure of the hepatic insulin receptor and insulin binding.

Hepatocytes or hepatic plasma membranes were photoaffinity-labelled with radioiodinated N epsilon B29-monoazidobenzoyl-insulin. Analysis of the samples by SDS/polyacrylamide-gel electrophoresis and autoradiography revealed the insulin receptor as a predominant band of 450 kDa. When hepatic plasma membranes were first treated with clostridial collagenase and then photolabelled, the insulin receptor appeared as a predominant band of 360 kDa. This effect of collagenase treatment on the insulin receptor was due to Ca2+-dependent heat-labile proteinases contaminating the preparation of collagenase, and it could be mimicked by elastase. The decrease in size of the insulin receptor to 360 kDa resulted from the loss of a receptor component that was inaccessible to photolabelling. In contrast, the size of the insulin receptor of intact cells was not affected by collagenase treatment. This suggests that the site sensitive to proteolysis was located on the cytoplasmic side of the plasma membrane. In hepatic plasma membranes that were treated with collagenase or elastase, and contained the 360 kDa form of the insulin receptor, the binding affinity for insulin was increased by up to 2-fold. These findings support the concept that a component which is either a part of, or closely associated with, the insulin receptor may regulate its affinity for insulin.     

Mechanism of transmembrane signaling: insulin binding and the insulin receptor

Transmembrane signaling via receptor tyrosine kinases generally requires oligomerization of receptor monomers, with the formation of ligand-induced dimers or higher multimers of the extracellular domains of the receptors. Such formations are expected to juxtapose the intracellular kinase domains at the correct distances and orientations for transphosphorylation. For receptors of the insulin receptor family that are constitutively dimeric, or those that form noncovalent dimers without ligands, the mechanism must be more complex. For these, the conformation must be changed by the ligand from one that prevents activation to one that is permissive for kinase phosphorylation. How the insulin ligand accomplishes this action has remained a puzzle since the discovery of the insulin receptor over 2 decades ago, primarily because membrane proteins in general have been refractory to structure determination by crystallography. However, high-resolution structural evidence on individual separate subdomains of the insulin receptor and of analogous proteins has been obtained. The recently solved quaternary structure of the complete dimeric insulin receptor in the presence of insulin has now served as the structural envelope into which such individual domains were fitted. The combined structure has provided answers on the details of insulin/receptor interactions in the binding site and on the mechanism of transmembrane signaling of this covalent dimer. The structure explains many observations on the behavior of the receptor, from greater or lesser binding of insulin and its variants, point and deletion mutants of the receptor, to antibody-binding patterns, and to the effects on basal and insulin-stimulated autophosphorylation under mild reducing conditions.     

Characterization of the functional insulin binding epitopes of the full-length insulin receptor

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg14, Phe64, Phe705, Glu706, Tyr708, Asn711, and Val715 inactivated the receptor. Alanine mutation of Asn15 resulted in a 20-fold decrease in affinity, whereas mutations of Asp12, Gln34, Leu36, Leu37, Leu87, Phe89, Tyr91, Lys121, Leu709, and Phe714 all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn15, Leu37, Asp707, Leu709, Tyr708, Asn711, Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn15, Gln34, and Phe89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.     

High-affinity insulin binding: insulin interacts with two receptor ligand binding sites

The interaction of insulin with its receptor is complex. Kinetic and equilibrium binding studies suggest coexistence of high- and low-affinity binding sites or negative cooperativity. These phenomena and high-affinity interactions are dependent on the dimeric structure of the receptor. Structure-function studies of insulin analogs suggest insulin has two receptor binding sites, implying a bivalent interaction with the receptor. Alanine scanning studies of the secreted recombinant receptor implicate the L1 domain and a C-terminal peptide of the receptor alpha subunit as components of one ligand binding site. Functional studies suggest that the first and second type III fibronectin repeats of the receptor contain a second ligand binding site. We have used structure-directed alanine scanning mutagenesis to identify determinants in these domains involved in ligand interactions. cDNAs encoding alanine mutants of the holo-receptor were transiently expressed in 293 cells, and the binding properties of the expressed receptor were determined. Alanine mutations of Lys(484), Leu(552), Asp(591), Ile(602), Lys(616), Asp(620), and Pro(621) compromised affinities for insulin 2-5-fold. With the exception of Asp(620), none of these mutations compromised the affinity of the recombinant secreted receptor for insulin, indicating that the perturbation of the interaction is at the site of mutation and not an indirect effect on the interaction with the binding site of the secreted receptor. These residues thus form part of a novel ligand binding site of the insulin receptor. Complementation experiments demonstrate that insulin interacts in trans with both receptor binding sites to generate high-affinity interactions.     

Structural domains of the insulin receptor and IGF receptor required for dimerisation and ligand binding

We investigated structural requirements for dimerisation and ligand binding of insulin/IGF receptors. Soluble receptor fragments consisting of N-terminal domains (L1/CYS/L2, L1/CYS/L2/F0) or fibronectin domains (F0/F1/F2, F1/F2) were expressed in CHO cells. Fragments containing F0 or F1 domains were secreted as disulphide-linked dimers, and those consisting of L1/CYS/L2 domains as monomers. None of these proteins bound ligand. However, when a peptide of 16 amino acids from the alpha-subunit C-terminus was fused to the C-terminus of L1/CYS/L2, the monomeric insulin and IGF receptor constructs bound their respective ligands with affinity only 10-fold lower than native receptors.     

Urea treatment allows dithiothreitol to release the binding subunit of the insulin receptor from the cell membrane: implications for the structural organization of the insulin receptor

The sequence of the human insulin receptor has only one identifiable transmembrane region which is located in the beta subunit. The structure predicts that the alpha subunit, which binds insulin, is attached to the cell only by disulfide bonds to the beta subunit. However, treatment of membranes with dithiothreitol is ineffective at releasing the alpha subunit. If the receptor structure is unfolded with urea, dithiothreitol is able to release the alpha subunit. These data provided confirmatory evidence that the alpha subunit is not a transmembrane protein.     

Polylysine increases the number of insulin binding sites in soluble insulin receptor preparations

The effects of cationic polyamino acids on insulin binding to soluble insulin receptor preparations were studied. Incubation of partially or fully purified receptor preparations with polylysine (pLys) increased by several-fold the amount of [125I]insulin that remained associated with the receptor, as determined both by precipitation of receptor-insulin complexes by polyethylene glycol or by separation of the complexes from the free hormone by gel filtration. This elevation in the amount of bound insulin resulted from increased number of insulin binding sites, and could not be attributed to an increased affinity of the receptors to insulin. In fact, pLys reduced 2-3-fold the affinity of insulin binding to its receptor as determined by equilibrium binding studies, and by monitoring the rate of exchange of bound [125I]insulin with unlabeled hormone. pLys induced specific interactions between insulin and its native receptor since other basic compounds such as histone, spermidine, polymixin B, compound 48/80, lysine, and arginine failed to reproduce its effects. pLys did not interact with the free ligand, nor did it promote interactions between insulin and denatured receptor forms. Furthermore, pLys did not induce binding of insulin to other proteins present in the partially purified receptor preparations. The effects of pLys were time and dose-dependent and were proportional to the pLys chain length. The longer the chain, the greater was the effect. Enhanced insulin binding and receptor beta-subunit autophosphorylation (in the presence of insulin) exhibited a similar dependency on the chain length of pLys. pLys effects on insulin binding were associated with formation of large protein aggregates that remained trapped at the top of Sephacryl S-300 columns. These aggregates contained substantial amounts of receptor-insulin complexes. Our results suggest that pLys induces formation of receptor clusters that create de novo insulin binding sites among adjacent receptor tetramers. Alternatively, formation of receptor aggregates might facilitate insulin binding to a soluble receptor subfraction that otherwise fails to bind the hormone.     

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.     

Inhibition of insulin receptor binding by dimethyl sulfoxide

Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured human lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of ¹²⁵I-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of ¹²⁵I-labeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor were affected to a much lesser extent.     

Inhibition of insulin receptor binding by phorbol esters

Phorbol esters inhibit the binding of insulin to its receptors on U-937 monocyte-like and HL-60 promyelocytic leukemia human cell lines. Within 20-30 min, exposure of these cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) at 37 degrees C results in a 50% reduction of the specific binding of 125I-insulin. Half-maximal inhibition occurs at 1 nM TPA. Other tumor-promoting phorbol esters also inhibit 125I-insulin binding in a dose-dependent manner which parallels their known promoting activity in vivo. TPA does not alter the degradation of the hormone nor does it induce any shedding of its receptors in the medium. The effect of phorbol esters is dependent on temperature and cell type. It is less prominent at 22 degrees C than at 37 degrees C. It is reversible within 2 h at 37 degrees C. TPA reduces the binding of insulin predominantly by increasing its dissociation rate. This effect results in an accelerated turnover of the hormone on its receptors.     

Purification of insulin receptor with full binding activity

Insulin receptor was purified 2400-fold with an overall yield of 40% from human placental membranes by affinity chromatography on wheat germ agglutinin-Sepharose and insulin-Sepharose. The receptor was eluted from insulin-Sepharose using mild conditions, eliminating urea, so that it was stable and retained full insulin-binding activity. Chromatofocusing and gel filtration analysis indicated that the receptor preparation was apparently pure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three high molecular weight protein bands with Mr = 320,000, 300,000, and 270,000 under nonreducing conditions and two major protein bands with Mr = 135,000 and 90,000 under reducing conditions. The purified receptor showed a curvilinear Scatchard plot with maximum insulin binding of 28.5 micrograms per mg of protein. In comparison, the receptor eluted from insulin-Sepharose with previously used conditions in the presence of urea resulted in maximum insulin binding of only 6 micrograms per mg of protein. This indicates that a 4-to 5-fold increase in specific activity can be obtained by using the new elution conditions.     

Decreased binding of insulin to its receptors in rats with hormone induced insulin resistance

Insulin and glucagon receptor binding was studied in purified liver membranes from rats made insulin resistant by implantation of an MtT pituitary tumor which secretes growth hormone, prolactin, and ACTH. Insulin binding to its receptors was decreased and correlated with the degree of insulin resistance. In contrast, binding of glucagon to its receptors was unchanged.     

Demonstration that the insulin receptor undergoes an early structural modification following insulin binding

Processing of the insulin receptor by hepatocytes was studied using a 125I-labelled photoreactive insulin derivative which could be covalently attached to the receptor and facilitate the analysis of receptor structure in isolated subcellular fractions by SDS-polyacrylamide gel electrophoresis. Following binding at the cell surface, the label was rapidly internalised and located in a low-density subcellular fraction ('endosomes'). The intact receptor (350 000 molecular weight) and binding (alpha) subunit (135 000), produced by in vitro disulphide reduction of the samples, were found in the plasma membrane fraction but not in endosomes. In endosomes, the label was concentrated in a band at 140 000 (non-reduced) which on reduction generated species of 100 000 and 68 000 predominantly. The insulin receptor therefore undergoes an early structural change during endocytosis. This modification does not involve complete disulphide reduction and may be due to a proteolytic event.     

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.