The nonclassical insulin binding of insulin receptors from rat liver is due to the presence of two interacting α-subunits in the receptor complex

Deger, Arno; Krämer, Helmut; Rapp, Reinhard; Koch, R; Weber, Ulrich

doi:10.1016/0006-291x(86)90016-1

Cited by 26 publications

(11 citation statements)

References 17 publications

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“…These differences, however, do not appear to change the major characteristics of the receptor kinetics. Several groups have observed that the curvilinear Scatchard plot (33-35, 38, 39) and the accelerated dissociation (33,34) …”

Section: ) Antibodies On 125i-insulin Disso-mentioning

confidence: 99%

Negative and positive site-site interactions, and their modulation by pH, insulin analogs, and monoclonal antibodies, are preserved in the purified insulin receptor.

Wang

Goldfine

Fujita‐Yamaguchi

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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The kinetic properties of the insulin receptor were studied in solution after its purification to homogeneity.Dissociation of "I-labeled insulin at a 1:50 dilution was not first order; unlabeled insulin at physiological concentrations accelerated the dissociation rate with a maximal effect at "17 nM. At higher concentrations, the unlabeled insulin slowed the dissociation rate. Maximal acceleration was seen at pH 8. The purification to homogeneity (1-3) and the cloning and expression (4, 5) of the insulin receptor, as well as the availability of monoclonal antibodies (6-12), have recently provided exciting means to investigate the exact mechanisms underlying the complex kinetics of insulin receptor binding. It is now well established that insulin binding departs markedly from simple reversible mass action kinetics (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Scatchard plots are curvilinear in most systems studied, and the dissociation is not first order. Furthermore, dissociation of 125I-labeled insulin (125I-insulin) at an "infinite" dilution is accelerated in the presence of unlabeled insulin. These basic findings, as well as the effects of numerous structural modifications of the insulin molecule (16,(18)(19)(20) and more recent studies with monoclonal antibodies (7,8,11,12,22), have led to the view (most explicitly developed in refs. 20 and 22) that the insulin-receptor complex can shift reversibly between at least three interconvertible states by site-site interactions, which depend on the nature of the ligand occupying neighboring sites. We distinguished the initial, "empty" state with an apparent affinity denoted KAE; a lower affinity state, Kf, characterized by a faster dissociation rate constant, induced by increased insulin occupancy-the phenomenon known as "negative cooperativity"-and a higher affinity state, which was denoted "Ksu.r " with a much slower dissociation rate.Although arguments have been raised against the negative cooperativity hypothesis (23-25), no alternative model has been proposed that is as compatible as the three-state model outlined above with a variety of experimental findings using either insulin analogs, monoclonal antibodies, or alterations ofreceptors in clinical states, and with more recent studies of alterations in the dimeric structure of the receptor (see ref. 22 for review). While we believe that the experimental evidence favors the site-site interactions model, it is clear that ultimate proof will have to come from the actual demonstration by crystallographic or other physicochemical methods, of alternative conformations of the purified receptor in various liganded states, or by appropriate changes in receptor structure and kinetics generated by site-directed mutagenesis.As a first step in that direction, we have now characterized in detail the kinetic behavior in solution of the insulin receptor purified to homogeneity from human placenta. One of us previously demonstrated that Scatchard plots of 125I-insulin binding to this purified preparation are curvilinear (1...

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Section: ) Antibodies On 125i-insulin Disso-mentioning

confidence: 99%

Negative and positive site-site interactions, and their modulation by pH, insulin analogs, and monoclonal antibodies, are preserved in the purified insulin receptor.

Wang

Goldfine

Fujita‐Yamaguchi

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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“…The insulin receptor is a heterotetrameric structure composed of two a-and two fl-subunits held together by two classes of disulphide bonds [10]. The binding of insulin to its receptor often produces non-linear Scatchard analysis in most cell types considered: adipocytes [11,12], hepatocytes [13], muscle cells [14] and partially purified receptors [15]. This behaviour is often treated as two independent insulin-binding sites with different affinities.…”

Section: Introductionmentioning

confidence: 99%

Chloroquine augments the binding of insulin to its receptor

Bevan

Christensen

Tikerpae

et al. 1995

Biochemical Journal

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The effect of chloroquine on the interaction of insulin with its receptor has been investigated under both equilibrium and non-equilibrium conditions. Chloroquine was found to augment insulin binding in a pH-dependent manner between pH 6.0 and pH 8.5, with the maximum occurring at approximately pH 7.0. Analysis of the equilibrium binding data in terms of independent binding sites gave equivocal results but suggested an increase in the high-affinity component. Analysis using the negative co-operativity binding model of De Meyts, Bianco and Roth [J. Biol. Chem. (1976) 251, 1877-1888] suggested that the affinity at both high and low occupancy was increased equally. The kinetics of association of insulin with the plasma-membrane receptor indicated that, although the net rate of association increased in the presence of chloroquine, this was due to a reduction in the dissociation rate rather than an increase in the association rate. This was confirmed by direct measurement of the rates of dissociation. Dissociation was found to be distinctly biphasic, with fast and slow components. Curve fitting suggested that the decrease in dissociation rate in the presence of chloroquine was not due to a decrease in either of the two dissociation rate constants, but rather to an increase in the amount of insulin dissociating by the slow component. It was also found that the increase in dissociation rate in the presence of excess insulin, ascribed to negative co-operativity, could be accounted for by an increase in the amount of insulin dissociating by the faster pathway, rather than by an increase in the dissociation rate constant. Thus chloroquine appears to have the opposite effect to excess insulin, and evidence was found for the induction of positive co-operativity in the insulin-receptor interaction at high chloroquine concentrations. Evidence was also found for the presence of low-affinity chloroquine binding sites with binding parameters similar to the concentration dependence of the chloroquine-induced augmentation of insulin binding.

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“…Scatchard plots (2) of equilibrium binding data are concave and curvilinear, suggesting heterogeneity of ligand binding sites, negatively cooperative site-site interactions, or a combination of both (1). These properties and high affinity interactions with insulin are dependent on the dimeric structure of the receptor; insulin binds non-cooperatively to a single population of binding sites in the monomeric receptor (3)(4)(5). The stoichiometry of binding to the native receptor appears to be one insulin molecule to one receptor dimer (6).…”

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confidence: 99%

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Whittaker

Whittaker²

2005

Journal of Biological Chemistry

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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 ␣ subunit (amino acids [705][706][707][708][709][710][711][712][713][714][715] , and Phe 89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC 50 commensurate with their effect on the affinity of the receptor for insulin.The initiating event in the insulin-signaling cascade is the binding of insulin to a specific plasma membrane receptor. This interaction has been studied extensively and was found to be extremely complex (see Ref. 1 for review). Scatchard plots (2) of equilibrium binding data are concave and curvilinear, suggesting heterogeneity of ligand binding sites, negatively cooperative site-site interactions, or a combination of both (1). These properties and high affinity interactions with insulin are dependent on the dimeric structure of the receptor; insulin binds non-cooperatively to a single population of binding sites in the monomeric receptor (3-5). The stoichiometry of binding to the native receptor appears to be one insulin molecule to one receptor dimer (6). The secreted recombinant extracellular domain of the receptor exhibits properties similar to those of the receptor monomer and has a stoichiometry of two insulin molecules to one receptor dimer (6, 7).A number of hypothetical models of insulin-receptor interactions have been proposed to explain these findings (7-9). The model that best explains the experimental findings is that of De Meyts (1). This proposes that insulin has two topographically distinct receptor binding sites and that the receptor has two topographically distinct insulin binding sites/monomer. In this model, insulin binds asymmetrically to the insulin dimer, with each of its binding sites contacting its cognate receptor site on a separate monomer. Although the detailed structural basis of this model has yet to be elucidated, the structure-function relationships of the insulin molecule and the receptor have been studied extensively and provide support for the essentials of the model.Insulin (1, 10). However, hagfish insulin, in which these residues and the tertiary structure of the molecule are conserved (11), displays anomalous receptor binding behavior (11,12), suggesting that other residues outside this region might also be involved in receptor interactions. This is supported by the finding that two recombinant insulin analogues with substitutions in residues located on the opposite side of the molecule, Leu A13 to Ser and Leu B17 to Gln, exhibited similar receptor binding behavior to hagfish insulin (7,8).The structure and structure-function relationships of the insulin receptor are not as well documented. It is a dimeric transmembrane protein (see Ref. 1 for review). Each monomer consists of disulfide-linked ␣ and ␤ subunits. The ␣ subunits are wholly extracellular and c...

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The nonclassical insulin binding of insulin receptors from rat liver is due to the presence of two interacting α-subunits in the receptor complex

Cited by 26 publications

References 17 publications

Negative and positive site-site interactions, and their modulation by pH, insulin analogs, and monoclonal antibodies, are preserved in the purified insulin receptor.

Negative and positive site-site interactions, and their modulation by pH, insulin analogs, and monoclonal antibodies, are preserved in the purified insulin receptor.

Chloroquine augments the binding of insulin to its receptor

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

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