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Negative and Positive Site-Site Interactions, and Their Proc. Nati. Acad. Sci. USA Vol. 85, pp. 8400-8404, November 1988 Biochemistry Negative and positive site-site interactions, and their modulation by pH, insulin analogs, and monoclonal antibodies, are preserved in the purified insulin receptor (receptor-affinity changes/negative cooperativity/insulin struct ction relationships) CHIH-CHEN WANG*, IRA D. GOLDFINEt, YOKo FUJITA-YAMAGUCHI4, HANS-GREGOR GATTNER§, DIETRICH BRANDENBURG§, AND PIERRE DE MEYTS*t *Department of Diabetes, Endocrinology and Metabolism, City of Hope National Medical Center, Duarte, CA 91010; tCell Biology Laboratory and Department of Medicine, Mount Zion Hospital and Medical Center, San Francisco, CA 94120; *Department of Molecular Genetics, Beckman Research Institute, City of Hope, Duarte, CA 91010; and §Deutsches Wollforschungsinstitut, D-5100 Aachen, Federal Republic of Germany Communicated by Rachmiel Levine, August 1, 1988 (receivedfor review April 25, 1988) ABSTRACT The kinetic properties of the insulin receptor Although arguments have been raised against the negative were studied in solution after its purification to homogeneity. cooperativity hypothesis (23-25), no alternative model has Dissociation of "I-labeled insulin at a 1:50 dilution was not been proposed that is as compatible as the three-state model first order; unlabeled insulin at physiological concentrations outlined above with a variety of experimental findings using accelerated the dissociation rate with a maximal effect at "17 either insulin analogs, monoclonal antibodies, or alterations nM. At higher concentrations, the unlabeled insulin slowed the ofreceptors in clinical states, and with more recent studies of dissociation rate. Maximal acceleration was seen at pH 8.0. The alterations in the dimeric structure ofthe receptor (see ref. 22 ability to accelerate the dissociation rate was diminished with for review). While we believe that the experimental evidence [LeuB24]insulin and suppressed with desoctapeptide, [LeuB21], favors the site-site interactions model, it is clear that ultimate [LeuB24,B2S], desalanine-desasparagine, and desheptapeptide proof will have to come from the actual demonstration by insulins, all of which slowed the dissociation at high concen- crystallographic or other physicochemical methods, of alter- trations. Monoclonal antibodies to the insulin receptor a native conformations of the purified receptor in various subunit (MA-5, MA-10, MA-20, and MA-51) all competed for liganded states, or by appropriate changes in receptor struc- insulin binding to the purified receptor. MA-10 and MA-51 ture and kinetics generated by site-directed mutagenesis. accelerated the dissociation of 12I-labeled insulin, while MA-5 As a first step in that direction, we have now characterized and MA-20 slowed the off rate. Thus, all the aspects of both in detail the kinetic behavior in solution of the insulin negatively and positively cooperative site-site interactions receptor purified to homogeneity from human placenta. One previously described in whole cells are present in solubilized of us previously demonstrated that Scatchard plots of 125I- purified receptors, demonstrating that these interactions rep- insulin binding to this purified preparation are curvilinear (1). resent intrinsic properties ofthe receptor molecule, most likely We now report that all the kinetic properties previously as a result of ligand-induced conformational changes. demonstrated with either whole cells or membrane prepara- tions have been preserved after extensive receptor purifica- The purification to homogeneity (1-3) and the cloning and tion, demonstrating that these properties are intrinsic to the expression (4, 5) of the insulin receptor, as well as the receptor. availability of monoclonal antibodies (6-12), have recently provided exciting means to investigate the exact mechanisms MATERIALS AND METHODS underlying the complex kinetics of insulin receptor binding. It is now well established that insulin binding departs mark- Porcine insulin (26.3 units/mg) was purchased from Sigma. edly from simple reversible mass action kinetics (13-22). [Tyr(125j)A14]insulin was prepared and purified by high- Scatchard plots are curvilinear in most systems studied, and performance liquid chromatography as described (26). Mono- the dissociation is not first order. Furthermore, dissociation clonal antibodies MA-5, MA-10, MA-20, and MA-51 have of 125I-labeled insulin (125I-insulin) at an "infinite" dilution is been described (6, 11-12, 22). Desalanine-desasparagine accelerated in the presence of unlabeled insulin. These basic insulin, desoctapeptide insulin, desheptapeptide insulin, findings, as well as the effects of numerous structural [Leun24]insulin, [LeuB25]insulin, and [LeuB24,B25]insulin modifications of the insulin molecule (16, 18-20) and more have been described (19, 27-29). Insulin receptors were recent studies with monoclonal antibodies (7, 8, 11, 12, 22), solubilized from human placental membranes and purified have led to the view (most explicitly developed in refs. 20 and 2400-fold by sequential affinity chromatography on wheat 22) that the insulin-receptor complex can shift reversibly germ agglutinin-Sepharose and insulin-Sepharose as de- between at least three interconvertible states by site-site scribed (1). This homogeneous preparation has full binding interactions, which depend on the nature of the ligand and kinase activity (1). occupying neighboring sites. We distinguished the initial, Association and dissociation of 125I-insulin were per- "empty" state with an apparent affinity denoted KAE; a lower formed in 50 mM Tris-HCl buffer containing 0.1% bovine affinity state, Kf, characterized by a faster dissociation rate serum albumin and 0.1% Triton X-100 (pH 7.4). Immuno- constant, induced by increased insulin occupancy-the phe- globulin and polyethylene glycol (PEG) were prepared in 50 nomenon known as "negative cooperativity"-and a higher mM Tris-HCl buffer (pH 7.4), except in the pH-dependence affinity state, which was denoted "Ksu.r" with a much determination in which 300 mM Tris HCl buffer was used to slower dissociation rate. ensure precipitation of the receptor in the pH range 7-8. Steady-state receptor binding was carried out as described The publication costs of this article were defrayed in part by page charge (1). Dissociation of 125I-insulin by dilution, with or without payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: l25l-insulin, l25l-labeled insulin. 8400 Downloaded by guest on October 2, 2021 Biochemistry: Wang et al. Proc. Natl. Acad. Sci. USA 85 (1988) 8401 S~PECIFIC BINDING unlabeled insulin present, was performed as follows (for more 100 detailed description, see figure legends). Briefly, purified DILUTION ONLY insulin receptor (1-2 gg/ml) was incubated with 0.1 nM 50 '25I-insulin at pH 7.4 and 40C overnight. The dissociation was 0 carried out by diluting 1:50 an aliquot of the equilibrium X 20-DILUTION' mixture in the absence or presence of the indicated insulin +COILD INSULIN concentrations at 15TC. Then, at intervals, the 125I-insulin- (o-)0 10 60 120 180 receptor complex was precipitated by 0.05% immunoglobulin and 10% PEG at pH 7.4 for 10 min in an ice bath. The C')Iq receptor-bound radioactivity was separated from dissociated (n radioactivity by centrifugation and then counted. The dilu- c tion study was carried out in two different ways. In most 0 20 experiments, 20-pl aliquots from the equilibrium mixture z_1)0(Q were distributed in multiple duplicate sets oftubes containing c 6:0 1 ml of buffer alone or buffer containing unlabeled insulin or I z 1 DILUTION + COLD INSULIN analogs; one set was precipitated immediately (time 0), while i WAJ 101 the other sets were precipitated at the indicated intervals. To Q obtain more precise initial time points, in subsequent exper- z iments studying time course of dissociation (Figs. 4 and 7) after determination of total binding, the remaining equilib- 51[ rium mixture was diluted 1:50 by the addition of buffer (time V) DILUTION ONLY 0), was thoroughly mixed, and 1-ml aliquots were precip- A £-". a A itated at various times (the first being 3 min after dilution). In en a _N all figures, the time indicated is the time when the insulin- DILUTION + COLD INSULIN receptor complex and PEG are put in contact (another 10 min elapses before centrifugation). In some dissociation experi- 60 120 180 ments, unlabeled insulin was replaced by various insulin MINUTES analogs or monoclonal antibodies. All experiments were performed at least three times with comparable results. FIG. 1. Dissociation of "25I-insulin from purified receptors in the absence and presence of unlabeled ("cold") insulin. Purified insulin RESULTS receptor (1-2 ,ug/ml) was incubated overnight at pH 7.4 and 4"C with 0.1 nM 125I-insulin. Three 20-sA aliquots were removed and precip- Kinetics of Dissociation of 125I-Insulin from Purified Recep- itated by adding 25 pA of 4% immunoglobulin and 2 ml of 10%o PEG. tors. Under our experimental conditions, 60-70% of the After 10 min at 4"C, the samples were centrifuged at 2000 x g for 20 125I-insulin tracer was bound to purified receptors after min. The counts in the receptor pellet represented 125I-insulin bound overnight equilibration. The dissociation of 125I-insulin was to receptors before dissociation [shown as T.B. (total binding) and not first order (Fig. 1). Unlabeled insulin markedly acceler- set as 100%1b]. Duplicate sets of 20 Al from the equilibration mixture ated the dissociation of 125I-insulin after a 50:1 dilution. After were then added to tubes containing 1 ml ofbuffer alone (e) or buffer 3 hr at 150C, 90% of bound tracer had dissociated in the containing unlabeled insulin (o) (100 ng/ml, 1.7 x 10-8 M) at 15'C. presence of 100 ng of insulin per ml (1.7 x 10-8 M) versus At the indicated intervals, contents of duplicate tubes were precip- itated by 25 Al of 4% immunoglobulin and 1 ml of ice-cold 20%o PEG only 60% in buffer only.
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