Site-site interactions among insulin receptors. Characterization of the negative cooperativity.

By studying the dissociation of 125I-instulin from its receptors in the absence and phe negatively cooperative type for the insulin receptors. In the present study we extend oy purified mouse and rat liver membranes as well as in human circulating monocytes and human cultured lymphocytes demonstrated negative cooperativity that was extraordinarily simn membranes more slowly than it does from its receptors on whole cells. The dissociaty a small percentage of the receptor sites (1 to 5%), are sufficient to accelerate dissociation of hormone from receptor. At these insulin concentrations insulin is entirely monomeric, and in fact at higher concentrations of insulin (greater than 10(-7) M) where insulin dimers predominate, the cooperativity effect is progressively lost. The dissociation rate of 125I-insulin alone (that is at very low fractional saturation of receptors) was markedly accelerated by dripping the pH from 8.0 to 5.0, whereas the dissociation of 125I-insulin at high receptor occupancy was only slightly accelerated by the fall in pH. The dissociation rate was directly related to temperature, but the dissociation rate of 125I-insulin at low receptor occupancy was much more affected by reduction in temperature and showed a sharp transition at 21 degrees. Urea at concentrations as low as 1 M produced a marked acceleration of 125I-insulin dissociation. Divalent cations (calcium and magnesium) appear to stabilize the insulin-receptor interaction, since higher degrees of receptor occupancy were required to achieve a given rate of dissociation of 125I-insulin. These data make it likely that the insulin receptors exist as oligomeric structures or clusters in the plasma membrane. Insulin receptor sites appear to switch from a "slow dissociating" state to a "fast dissociating" state when their occupancy increases; the proportion of sites in each state is a function of occupancy of the receptor sites by the insulin monomer as well as of the physiochemical environment. Other models which could explain apparent negative cooperativity besides site-site interactions, i.e. polymerization of the hormone, steric or electrostatic hindrance due to ligand-ligand interactions, or unstirred (Noyes-Whitney) layers are considered unlikely in the case of insulin receptors on both experimental and theoretical grounds.     

Reversible, positive cooperative interaction of 11 beta-chloromethyl-[3H]estradiol-17 beta with the calf uterine estrogen receptor

The binding of 11 beta-chloromethyl-[3H]estradiol-17 beta [3H]CME2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4-1.5. This positive cooperative interaction of [3H]CME2 was indistinguishable from the typical cooperative interaction of [3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics of [3H]CME2 from the receptor was biphasic, composed of a fast dissociating component (15%, t1/2 = 4 min at 0 degrees C; 9%, t1/2 = 4 min at 28 degrees C) and a slow dissociating component (85%, t1/2 greater than 50 h at 0 degrees C; 91%, t1/2 greater than 50 h at 28 degrees C). The dissociation kinetics of [3H]estradiol was also biphasic: the t1/2 of the fast dissociating component was 4 min at 0 and 28 degrees C and approximately 200 min for the slow dissociating component at both temperatures. The fraction of the slow [3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [3H]CME2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME2 are discussed.     

Regulation of formyl peptide receptor binding to rabbit neutrophil plasma membranes. Use of monovalent cations, guanine nucleotides, and bacterial toxins to discriminate among different states of the receptor

The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of [3H]FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM [3H]FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. [3H]FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of [3H]FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM [3H]FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state.     

Alteration by concanavalin A of the slow dissociable component in the human growth hormone-receptor interaction

In mice liver plasma membranes (PM), the binding affinity of receptors for [125I] human growth hormone (hGH) was dependent on the association time: after 18 hours, a high affinity receptor form with KA = 6.8 X 10(9) M-1 accumulated and, as compared to after 1 hour, an increase up to 88%, in a slow dissociating component was observed. Preincubation of PM with concanavalin A (Con A) or other lectins from Lens culinaris (LCA), Ricinus communis (RCA I), Wheat germ agglutinin (WGA) specifically inhibited the binding of hGH to receptors by 54, 28, 50 and 25%, respectively. Furthermore, PM pretreatment with Con A concomitantly increased the rate of dissociation of the hormone-receptor (H-R) complex to 92 or 65% after association for 1 or 18 hours. These Con A-induced alterations resulted from a reduced fraction of the slow dissociable component together with an increased rate constant. The treatment of PM with Con A subsequent to incubation with the hormone did not decrease hormone binding but caused the conversion of the class of hGH receptors exhibiting fast dissociation kinetics towards a form exhibiting slow ones. These data strongly suggest a role for glycoproteins of the N-acetyllactosaminic type in the affinity state of liver membrane hGH receptors.     

Alteration of binding properties and cytoskeletal attachment of nerve growth factor receptors in PC12 cells by wheat germ agglutinin

Incubation of PC12 cells preloaded with 125I-nerve growth factor (NGF) reveals rapidly and slowly dissociating binding components indicative of a heterogeneous population of receptors. If the cells are previously exposed to wheat germ agglutinin (WGA) for 30 min, NGF now binds to an apparently homogeneous receptor population which exhibit slow dissociation kinetics. Total binding is also reduced by 50%. If WGA is added subsequent to 125I-NGF, total binding is not diminished, but rapidly dissociating receptors occupied with NGF are all converted to the slowly dissociating form. This conversion of receptors occurs rapidly, reaching completion within 2 min at 37 degrees or 4 degrees C, and is unaffected by metabolic energy poisons, suggesting that WGA- induced slowly dissociating receptors are not the product of internalization. The effects of the lectin are blocked by the sugar N- acetyl-D-glucosamine, and the lectin-induced slowly dissociating receptors are converted back to rapidly dissociating receptors by addition of this same sugar. WGA also affects the association of the NGF receptor with the Triton X-100 cytoskeleton. Greater than 90% of bound 125I-NGF becomes associated with Triton X-100 insoluble cytoskeletons in the presence of the lectin, compared with less than 20% before lectin addition. Cytoskeleton association of the NGF receptor by WGA shows similar kinetics as the conversion of rapidly to slowly dissociating receptors. This interaction may be involved in the alteration of NGF-receptor binding properties produced by this lectin.     

Kinetic and Equilibrium Studies of (-)125Iodocyanopindolol Binding to β-Adrenoceptors on Human Lymphocytes: Evidence for the Existence of two Classes of Binding Sites

Abstract

Saturation experiments were performed on intact human peripheral mononuclear leucocytes (MNL) and MNL membranes with (-)¹²⁵Iodocyanopindolol (¹²⁵ICYP) over a large concentration range (1.5-600pmol/l). The corresponding Scatchard plots were curvilinear suggesting two saturable classes of binding sites: A high affinity binding site (B_max1=1000±400 sites/cell, K_d1= 2.1±0.9 pmol/l for intact MNL and B_max1=550±190 sites/cell, K_d1=4.1±0.9 pmol/l for MNL membranes)and a low affinity binding site (B_max2=9150±3590 binding sites/cell, K_d2=440±50 pmol/l for intact MNL and B_max2=11560±4690 sites/cell, K_d2=410±70 pmol/l for MNL membranes). Dissociation of (-)¹²⁵ICYP from MNL was biphasic consisting of a slow dissociating component (dissociation rate constant k_-1=(0.5±0.2)x10^?3 min^?1 for intact MNL and k_-1=(1.0±0.1)x10^?3min^?1 for MNL membranes) and a fast dissociating component (k_-2=(80±20)x10^?3min^?1 for intact MNL and k_-2=(60±10)x10^?3min^?1 for MNL membranes). In dissociation experiments started after equilibration with various (-)¹²⁵ICYP concentrations k_-1 and k_-2 were independent of the equilibrium concentration, whereas the percentual occupancy of the slow and the fast dissociating component varied and was similar to the estimated fractional occupancy of either binding site at the same (-)¹²⁵ICYP concentrations in saturation experiments. The association rate constant was in the same order of magnitude for both binding sites. These results suggest two independent classes of binding sites for (-)¹²⁵ICYP on MNL.     

Interaction of human monomeric C3b with its receptor (complement receptor type 1, CR1) on neutrophils. Evidence for negative cooperativity

The binding of highly purified monomeric 125I-C3b to its receptor (CR1) on resting human polymorphonuclear neutrophils (PMN) was analyzed under equilibrium conditions, at 4 degrees C and low ionic strength. Scatchard analysis of specific binding data yielded curvilinear concave upward plots, which resulted from the presence of site-site interactions of the negative type among PMN C3b-receptors (negative cooperativity), as shown by dissociation kinetic experiments. Indeed, the dissociation rate of 125I-C3b from PMN was markedly increased in the presence of an excess of unlabeled C3b in the dilution medium and was directly dependent on the degree of initial receptor occupancy with the radioligand. These interactions occurred when 2% of the receptors were occupied with 125I-C3b and resulted in a 4-fold decrease in CR1 affinity when the receptor went from its "empty" to its "filled" conformation. In a disease associated with a continuous production of C3b (factor I deficiency), CR1 on in vivo circulating PMN was found to be in a "low affinity" and "high dissociating" state similar to that of normal CR1 at high occupancy. Finally, negative cooperativity among CR1 sites disappeared after PMN activation with chemotactic peptides.     

Relationship among types of nerve growth factor receptors on PC12 cells

We analyzed the kinetics and thermodynamics of 125I-nerve growth factor (125I-NGF) binding to NGF-receptor on PC12 cells. We used conditions of pseudo-first order kinetics and techniques to quantitate internalized complexes, "slow" or high affinity binding complexes, and cell surface "fast" or low affinity complexes. Two possible models were examined: binding to two independent receptors at the cell surface (i.e. high and low affinity forms of NGF-receptor) and a model for consecutive formation of fast, low affinity binding followed by slow, high affinity binding or internalization. Our data are consistent with the consecutive model only. The rates of association and dissociation of NGF with slow, high affinity sites and internalized, acid wash-resistant sites are indistinguishable from each other. We also analyzed, in detail, the two assays primarily used to distinguish slow binding complexes from internalized complexes. Scatchard analysis of total binding and dissociation of pre-equilibrated 125I-NGF in the presence of unlabeled NGF at high concentration (cold wash). Neither of these assays shows any evidence that the slow, high affinity binding step is different from internalization of the 125I-NGF-receptor complex. Based on this analysis, there are only two detectable forms of NGF-receptor on PC12 cells: complexes on the surface of the cells with a binding affinity of 0.5 nM at 37 degrees C and complexes internalized by the cells. Furthermore, the data are consistent with a model in which NGF-receptor is internalized constitutively and independently of occupancy by NGF. We also examined the fate of internalized 125I-NGF. In the first 60 min after contact with PC12 cells, no degradation of 125I-NGF was observed. Moreover, a significant amount of 125I-NGF recirculates to the cell surface and is released as intact, Mr = 13,000 NGF. The cells were also stimulated by NGF in a primary neurite outgrowth assay with an ED50 of 2-16 pM under conditions of low initial cell numbers in a large extracellular volume of NGF-containing medium. Thus, low level occupancy of the cell surface receptors, Kd = 0.5 nM, for several days is sufficient to stimulate neurite outgrowth. This indicates the presence of spare NGF-receptors on the surface PC12 cells.     

Hormone-induced conformational changes in the hepatic insulin receptor

The insulin receptor can exist in either a lower or a higher affinity state. Hormone binding alters the equilibrium between the two states of the insulin receptor, favoring the formation of that of higher affinity (Corin, R.E., and Donner, D.B. (1982), J. Biol. Chem. 257, 104-110). After brief or extended incubations with hormone, during which the fraction of higher affinity receptors increased, 125I-insulin was covalently coupled to the alpha subunits of its receptor using disuccinimidyl suberate. Some 125I-insulin remained bound to higher affinity receptors after dissociation of hormone from lower affinity sites. This hormone could also be covalently coupled to the alpha subunit of the receptor. During extended incubations between 125I-insulin and liver plasma membranes, components of the receptor were cleaved to yield degradation products of 120,000 and 23,000 Da. The significance of this process remains undetermined. Unoccupied insulin receptors were cleaved by trypsin to produce fragments of 94,000 and 37,000 Da which remained membrane-bound and could be covalently coupled to 125I-insulin. Trypsin treatment after binding yielded an additional receptor fragment of 64,000 Da. As the incubation time between 125I-insulin and membranes was lengthened, components of the receptor became progressively less sensitive to trypsin. Higher affinity binding sites isolated after release of rapid dissociating insulin were less sensitive to trypsin than were mixtures of higher and lower affinity receptors. These observations suggest that hormone binding produces two conformational changes (alterations of tryptic lability) in the hepatic insulin receptor. The first change is rapid and exposes parts of the receptor to tryptic degradation. The second, slower conformational change renders the receptor less sensitive to trypsin and occurs with the same time course as the increase of receptor affinity mediated by site occupancy.     

The interaction of retinol-binding protein with its plasma-membrane receptor.

125I-labelled retinol-binding protein (RBP) bound to specific receptors in human placental brush-border membranes. Binding at 22 degrees C reached equilibrium within 15 min, but prolonged incubation caused a subsequent decline. Scatchard analysis of the equilibrium binding data at 22 degrees C and 15 min showed high-(3.0 +/- 2.7 x 10(-9) M) and low-(9.5 +/- 3.5 x 10(-8) M) affinity binding components. 125I-RBP, bound to membranes at 22 degrees C for 15 min and subsequently dissociated with excess unlabelled RBP, exhibited biphasic dissociation kinetics consisting of fast and slow components of release. In contrast, Scatchard analysis and dissociation kinetics of the binding that had taken place at 37 degrees C for 1 h showed the fast-dissociating/low-affinity binding component, but little of the slow-dissociating/higher-affinity binding component. When 125I-RBP, after incubation with membranes at 37 degrees C for 1 h, was re-isolated and subjected to dissociation kinetic analysis using a fresh batch of membranes, the fast-dissociating phase was unchanged, but the slow phase was almost absent. The complex kinetics were interpreted in terms of a heterogeneity in RBP consisting of high- and low-affinity binding forms. The higher-affinity-binding form is thought to be converted into the lower-affinity state on binding to the receptor. Transthyretin inhibited 125I-RBP binding to the membrane, suggesting that free, rather than transthyretin-associated, RBP bound to the receptor. The RBP receptor was trypsin-, heat- and thiol-group-specific-reagent sensitive and was highly specific for RBP.     

Characteristics of Partially Purified Nerve Growth Factor Receptor

Receptors for the nerve growth factor protein (NGF) have been isolated from three cell types [embryonic chicken sensory neurons (dorsal root sensory ganglia; DRG), rat pheochromocytoma (PC12) and human neuroblastoma (LAN-1) cells] and have been shown to be similar with respect to equilibrium dissociation constants. The present results demonstrate that there are multiple molecular weight species for NGF receptors from DRG neurons and PC12 cells. NGF receptors can be isolated from DRG as four different molecular species of 228, 187, 125, and 112 kilodaltons, and PC12 cells as three molecular species of 203, 118, and 107 kilodaltons. The NGF receptors isolated from DRG show different pH-binding profiles for high- and low-affinity binding. High-affinity binding displays a bell-shaped pH profile with maximum binding between pH 7.0 and 7.9, whereas low-affinity binding is constant between pH 5.0 and 9.1, with a twofold greater binding at pH 3.6. At 22 degrees C, the association rate constant was found to be 9.5 +/- 1.0 X 10(6) M-1 s-1. Two dissociation rate constants were observed. The fast dissociating receptor has a dissociation rate constant of 3.0 +/- 1.5 X 10(-2) s-1, whereas the slow dissociating receptor constant was 2.4 +/- 1.0 X 10(-4) s-1. The equilibrium dissociation constants calculated from the ratio of dissociation to association rate constants are 2.5 X 109-11) M for the high-affinity receptor (type I) and 3.2 X 10(-9) M for the low-affinity receptor (type II). These values are the same as those determined by equilibrium experiments on the isolated receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for isomerization during binding of apolipoprotein-B100 to low density lipoprotein receptors.

To determine the kinetics of human low density lipoproteins (LDL) interacting with LDL receptors, 125I-LDL binding to cultured human fibroblasts at 4 degrees C was studied. Apparent association rate constants did not increase linearly as 125I-LDL concentrations were increased. Instead, they began to plateau which suggested that formation of initial receptor-ligand complexes is followed by slower rearrangement or isomerization to complexes with higher affinity. To test this, 125I-LDL were allowed to associate for 2, 15, or 120 min, then dissociation was followed. The dissociation was biphasic with the initial phase being 64-110-fold faster than the terminal phase. After binding for 2 min, a greater percentage of 125I-LDL dissociated rapidly (36%) than after association for 15 min (24%) or 120 min (11%). Neither the rate constants nor the relative amplitudes of the two phases were dependent on the degree of receptor occupancy. Thus, the duration of association, but not the degree of receptor occupancy affected 125I-LDL dissociation. To determine if binding by large LDL, which is predominantly via apolipoprotein (apo) E, also occurs by an isomerization mechanism, the d = 1.006-1.05 g/ml lipoproteins were fractionated by ultracentrifugation. In contrast to small LDL which bound via apoB-100 and whose dissociation was similar to that of unfractionated LDL, large LDL dissociation after 2, 15, or 120 min of binding did not show isomerization to a higher affinity. This suggests that large and small LDL bind by different mechanisms as a result of different modes of interaction of apoE and apoB-100 with LDL receptors.     

Beta-adrenergic receptors: evidence for negative cooperativity.

The specific binding of [³H] (?)alprenolol to sites in frog erythrocyte membranes provides a tool for directly assessing ligand binding to adenylatecyclase coupled β-adrenergic receptors. Hill Plots of such binding data yield slopes (n_H=“Hill Coefficients”) less than 1.0, suggesting that negatively cooperative interactions among the β-adrenergic receptors may occur. The existence of such negative cooperativity was confirmed by a direct kinetic method. The dissociation of receptor bound [³H] (?)alprenolol was studied under two conditions: 1) with dilution of the ligand-receptor complex sufficient to prevent rebinding of the dissociated tracer and 2) with this same dilution in the presence of excess unlabeled (?)alprenolol. If the sites are independent, the dissociation rates must be the same in both cases. However, the presence of (?)alprenolol increases the rate of [³H] (?)alprenolol dissociation, indicating that negatively cooperative interactions among the β-adrenergic receptor binding sites do occur.     

The oligopeptide chemotactic factor receptor on human polymorphonuclear leukocyte membranes exists in two affinity states

The binding of the chemoattractant N-formyl-methionylleucyl-[³H]phenylalanine to intact polymorphonuclear leukocytes and membrane preparations was analyzed by computer methods. Whole viable cells bind the chemoattractant with a single dissociation constant (K_D) of 22.3 ± 2.4 nM and contain an average of 55,000 receptors percell. In contrast, the binding data using membrane preparations were consistent with the presence of two classes of binding sites with average K_Ds of 0.53 ± 0.01 nM and 24.4 ± 1.2 nM. The high affinity receptors accounted for ca. 25% of the binding sites. Increasing the receptor occupancy did not affect the rate of dissociation of the ligand-receptor complex thus negative cooperativity is not a likely explanation for the complex binding isotherms. On the other hand, the dissociation kinetics did agree with the two affinity receptor model.     

Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells

Steady state and kinetic studies on the binding of 125I-beta nerve growth factor (NGF) to single cells from sensory ganglia of 8-day-old chick embryos show two distinct, saturable binding sites with dissociation constants of Kd(I) = 2.3 X 10(-11) M and Kd(II) = 1.7 X 10(-9) M. The difference in the affinities is due to different rate constants of dissociation (k-1(I) = 10(-3) s-1, k-1(II) = 2 X 10(-1 s-1). The association to both sites is apparently diffusion controlled (k+1(I) = 4.8 X 10(7) M-1s-1, k+2(II) = 10(7) to 10(8) M-1s-1). The binding of betaNGF to both sites is specific, since none of a number of hormones or proteins tested compete for the binding of 125I-betaNGF to either of those two sites. The heterogeneity of the binding of 125I-betaNGF is not due to heterogeneity of the 125I-betaNGF preparation nor to a negatively cooperative binding. In experiments where the dissociation of 125I-betaNGF is induced by the addition of saturating amounts of unlabeled betaNGF, the ratio of the 125I-betaNGF released with either of the two dissociation rate constants is solely dependent on the occupancy of the two sites before dissociation is started and is independent of the total occupancy of the sites during dissociation. The rate of dissociation of 125I-betaNGF from the higher affinity binding site I is accelerated by unlabeled betaNGF under conditions where the occupancy is both increased and decreased. Although the dissociation characteristics of 125I-beta NGF change with increasing times of exposure of the cells to the ligand, and 125I-beta NGF is degraded after it binds to the cells, these secondary processes do not interfere with the analysis of the binding data. At the lowest concentration of 125I-beta NGF used for the analysis less than 10% of the 125I-beta NGF is degraded. Both kinetic and steady state binding data reveal the two NGF binding sites at 2 degrees C as well as at 37 degrees C.     

Formyl peptide chemotaxis receptors on the rat neutrophil: experimental evidence for negative cooperativity

To examine the existence of negative cooperativity among formyl peptide chemotaxis receptors, steady-state binding of f Met-Leu-[3H]Phe to viable rat neutrophils and their purified plasma membranes was measured and the data were subjected to statistical analysis and to computer curve fitting using the NONLIN computer program. Curvilinear, concave upward Scatchard plots were obtained. NONLIN and statistical analysis of the binding data indicated that a two-saturable-sites model was preferable to a one-saturable-site model and statistically valid by the F-test (P less than .010). In addition, Hill coefficients of 0.80 +/- 0.02 were obtained. Kinetic dissociation experiments using purified plasma membranes showed evidence of site-site interactions of the destabilizing type (negative cooperativity). Thus, unlabeled f Met-Leu-Phe accelerated the dissociation of f Met-Leu-[3H]Phe under conditions where no rebinding of radioligand occurred. The rate of dissociation of f Met-Leu-[3H]Phe from the plasma membranes was dependent on the fold excess of unlabeled f Met-Leu-Phe used in the dilution medium; at the highest concentration tested (10,000-fold excess), the dissociation rate was more than double the dissociation rate seen with dilution alone. In addition, occupancy-dependent affinity was ascertained directly by studying the effect of increasing fractional receptor saturation with labeled ligand on the dissociation rate of the receptor-bound labeled ligand. These data showed that the f Met-Leu-[3H]Phe dissociation rate was dependent on the degree of binding site occupancy over the entire biologically relevant range of formyl peptide concentrations. Furthermore, monitoring of the time course of dissociation of the receptor/f Met-Leu-[3H]Phe receptor/f Met-Leu-[3H]Phe complex as a function of receptor occupancy revealed that receptor affinity for f Met-Leu-Phe remained occupancy-dependent during the entire time of dissociation examined (up to 10 min). Finally, the average affinity profile of the equilibrium binding data demonstrated a 60% decrease in receptor affinity in changing from the high affinity to the low affinity conformation.     

Kinetic identification of a two-state glucagon receptor system in isolated hepatocytes. Interconversion of homogeneous receptors

A detailed kinetic study was performed to investigate the interaction of glucagon with receptors on freshly isolated hepatocytes. Competition binding assay results fit a mathematical expression for a single site noncooperative model of binding. Glucagon was shown to bind with first-order kinetics at six-hormone concentrations (0.02-0.50 nM) at 0 and 37 degrees C. The observed pseudo-first-order rate constants are directly proportional to the hormone concentration at 0 degree C, but display a downward deviation from linearity at 37 degrees C. Dissociation of glucagon exhibited biexponential character at 37 degrees C which was not seen at 0 degree C. The biphasic dissociation at 37 degrees C was resolved into rapid (t1/2 = 1.9 min) and slow (t1/2 = 27.7 min) components. The distribution of the total bound hormone between the rapidly and slowly dissociating complexes was not dependent upon the extent of receptor occupancy. The absolute quantity of rapidly dissociating hormone-receptor complexes was constant at all times examined; however, the fraction of slowly dissociating hormone-receptor complexes was found to increase with increasing incubation time. The results indicate that a homogeneous population of hepatic receptors undergoes a time-dependent, temperature-dependent conversion from one state to another in a two-stage sequential manner.     

Receptor-binding properties of gonadotropin-releasing hormone derivatives. Prolonged receptor occupancy and cell-surface localization of a potent antagonist analog

The receptor-binding properties and in vitro biological effects of a highly active gonadotropin-releasing hormone (GnRH) antagonist, [N-acetyl-D-p-chloro-Phe1,2D-Trp3,D-Lys6,D-Ala10]GnRH, were compared with those of the GnRH superagonist analog, [D-Ala6] des-Gly10-GnRH-N-ethylamide. In rat pituitary particles and isolated pituitary cells, the 125I-labeled GnRH antagonist showed saturable high-affinity binding (Ka v 8.4 +/- 1.4 X 10(9) M-1) to the same receptor sites which bound the GnRH agonist. The rate of dissociation of the receptor-bound antagonist from pituitary particles and cells was extremely slow in comparison with that of the agonist ligand. Also, dissociation of the antagonist analog was incomplete, with a residual fraction of tightly bound ligand that was proportional to the duration of preincubation. The [D-Lys6]GnRH antagonist prevented GnRH-induced luteinizing hormone release during static incubation and superfusion of cultured pituitary cells, but in contrast to the agonist did not cause desensitization of the gonadotroph. Although the antagonist caused a prolonged reduction in available GnRH receptor sites, this was attributable to persistent occupancy by the slowly dissociating ligand rather than to receptor loss. Autoradiographic analysis of [D-Lys6]GnRH-antagonist uptake by cultured pituitary cells revealed that the peptide remained bound at the cell membrane for up to 2 h, in contrast with the rapid endocytosis of GnRH agonists. The slow dissociation of receptor-bound antagonist was consistent with its ability to cause sustained blockade of GnRH actions, and its prolonged cell-surface location suggests that receptor activation is necessary to initiate the rapid internalization of hormone-receptor complexes that is a feature of the agonist-stimulated gonadotroph.     

Ligand-receptor interactions: detailed evaluation of occupancy-dependent affinity

The exact nature of the curvilinearity of Scatchard plots derived from hormonal and nonhormonal binding systems has not been definitively resolved. Such plots are compatible with heterogeneous receptors with different but fixed affinities and with negatively interacting binding sites resulting in occupancy-dependent affinity. In the current study we examined in detail the effect of receptor occupancy by the ligand on receptor affinity under a variety of experimental conditions. We chose the human lymphocyte-leukoagglutinin (LPHA) system, which closely mimics the IM9-insulin model. Reliable estimates of total binding capacity (728 ng/10(6) cells) essential to our report were calculated from a wide database by the least-squares model. At occupancies greater than or equal to 0.085, receptors are associated with low and fixed affinity (1.5 X 10(6) M-1), whereas at occupancies less than or equal to 0.085, affinity is high and fixed (1.8 X 10(8) M-1) or high but variable (1 X 10(7) M-1 to 1.5 X 10(6) M-1) depending on whether the binding is assumed to be noncooperative or cooperative, respectively. Calculation of receptor-ligand complex dissociation velocity over a wide range of occupancies (0.01-0.40) suggested that occupancy exerts an inversely proportional effect on affinity that is rapid and sustained. Cell activation (DNA synthesis) is initiated at receptor occupancy of approximately equal to 0.004 and is magnified as ligand binding to high affinity receptors increases up to approximately equal to 0.07 occupancy (functional sites), beyond which point further binding (to low affinity sites) becomes increasingly ineffective and cytotoxic (redundant sites). These findings suggest that occupancy influences affinity as postulated by the hypothesis of negative cooperativity. Through this effect occupancy may play a significant role in regulating ligand-induced cell responses.