A new bell-shaped function for idiotypic interactions based on cross-linking

Most recent models of the immune network are based upon a phenomenological log bell-shaped interaction function. This function depends on a single parameter, the “field”, which is the sum of all ligand concentrations weighted by their respective affinities. The typical behavior of these models is dominated by percolation, a phenomenon in which a local stimulus spreads globally throughout the network. The usual reason for employing a log bell-shaped interaction function is that B cells are activated by cross-linking of their surface immunoglobulin receptors. Here we formally derive a new phenomenological log bell-shaped function from the chemistry of receptor cross-linking by bivalent ligand. Specifying how this new function depends on the ligand concentrations requires two fields: a binding field and a cross-linking field. When we compare the activation functions for ligand-receptor pairs with different affinities, the one-field and the two-field functions differ markedly. In the case of the one-field activation function, its graph is shifted to increasingly higher concentration as the affinity decreases but keeps its width and height. In the case of the two-field activation function, the graph of a low-affinity interaction is nested within the graphs of all higher-affinity interactions. We show that this difference in the relations among activation functions for different affinities radically changes the network behavior. In models that described B cell proliferation using the one-field activation function, network behavior was dominated by low-affinity interactions. Conversely, in our new model, the high-affinity interactions are the most significant. As a consequence, percolation is no longer the only typical network behavior.     

The chicken liver cation-independent mannose 6-phosphate receptor lacks the high affinity binding site for insulin-like growth factor II

The chicken liver cation-independent mannose 6-phosphate receptor has been purified to apparent homogeneity by affinity chromatography on pentamannose phosphate-Sepharose and tested for its ability to bind iodinated human IGF-I, human IGF-II, and chicken IGF-II. In contrast to the bovine, rat, and human cation-independent mannose 6-phosphate receptors, which bind human IGF-II and IGF-I with nanomolar and micromolar affinities, respectively, the chicken receptor failed to bind either radioligand at receptor concentrations as high as 1 microM. The bovine receptor binds chicken IGF-II with high affinity while the chicken receptor binds this ligand with only low affinity, which we estimate to be in the micromolar range. These data demonstrate that the chicken cation-independent mannose 6-phosphate receptor lacks the high affinity binding site for IGF-II. These results provide an explanation for the failure of previous investigators to identify the type II IGF receptor by IGF-II cross-linking to chicken cells and indicate that the mitogenic activity of IGF-II in chick embryo fibroblasts is most likely mediated via the type I IGF receptor.     

A biochemical and kinetic analysis of the interleukin-1 receptor. Evidence for differences in molecular properties of IL-1 receptors

This study describes the biochemical characterization and kinetic analysis of the interleukin-1 (IL-1) receptor in Raji human B-lymphoma and EL4 murine T-lymphoma cells. The internalization of 125I-IL-1 was studied in both cell types by an acid extraction technique which removes surface bound ligand. At 37 degrees C, binding to Raji IL-1 receptors was almost entirely cell surface (91%). EL4 cells, in contrast, internalized 59% of ligand at this temperature and this was almost totally inhibited by sodium azide. Receptor binding studies showed that the B-cells had a lower binding affinity but much higher receptor density per cell (KD = 2.1 nM, Ro = 7709) than the T-cells (KD = 0.4 nM, Ro = 241). The receptor binding affinity of two IL-1 analogs, Glu-4 and clone 18, was determined in competitive binding studies. In the B-cells the analogs had binding affinities of 25 and 90%, respectively, whereas in the T-cells the affinities were 0.2 and 200%, respectively. Chemical cross-linking studies showed that the IL-1 receptor in B-cells had a lower molecular weight than that in T-cells (68 kDa compared to 80 kDa). In summary these studies demonstrate that structural differences exist between IL-1 receptors in Raji and EL4 cells.     

Curves of ligand binding. The use of hyperbolic functions for expressing titration curves

1. The dependences of the concentrations of the non-ligated, uni-ligated and bi-ligated forms of a molecule that binds two molecules of ligand are expressed as functions of the logarithm of free ligand concentration by means of hyperbolic functions. Expressions are also given for the saturation both of an individual site and of the molecule as a whole. This form of expression allows derivation of the following points. 2. The sharpness of bell-shaped curves of concentration of the uni-ligated form is analysed in terms of the heights of their points of inflexion; these can rise to 1/ radical2 of the curve. 3. A single group can exhibit a doubly sigmoid saturation curve if this group and another have comparable affinities for a ligand, and if ligand binding at one of them diminishes the affinity at the other. If the molecular pK values pK(1) and pK(2) for the first and second molecules of ligand are called pK*+/-logm, so that K*(2)=K(1)K(2) and m(2)=K(1)/K(2), then the doubly sigmoid curve can be represented by the sum of two independent one-site saturation curves, in general of unequal height, of pK values pK*+/-log(1/2)[m+ radical(m(2)-4)]. The error in such representation is small either if the mutual interaction between the groups (i.e. m) is large, or if the groups have very similar affinities for the ligand. 4. The sum of two one-site saturation curves, again of pK values of pK*+/-log(1/2)[m+ radical(m(2)-4)] but of equal heights, gives a precise value for the total saturation, provided that the binding of one molecule does not promote the binding of a second, i.e. providing that m>/=2. Hence determinations of saturation cannot distinguish interacting and possibly identical sites from independent and different ones.     

Biochemical studies on muscarinic receptors

Muscarinic receptors have been characterized in smooth muscle and brain by the binding of reversible (e.g. atropine, quinuclidinylbenzylate) or irreversible (benzilylcholine or propylbenzilylcholine mustards) ligands. There is a close correlation between affinity constants derived from binding experiments and the affinities of muscarinic ligands for these sites obtained in pharmacological experiments on smooth muscle. Whereas atropine shows a single high affinity binding component (in subcellular preparations) several other ligands (QNB, ACh, oxotremorine) show multiple affinity binding. This indicated the existence of several types of binding sides which show selectivity toward certain cholinergic effectors. Most detergents inhibit the binding of ligands to the receptor site and therefore cannot be used to solubilize the receptor protein from the membrane. Treatment of brain subcellular membrane preparations with high salt concentrations (2M NaI) solubilize proteins which possess the muscarinic ligand binding properties observed in the membrane preparation. The affinities for muscarinic antagonists however are decreased, which suggests that a conformational change occurs in the protein upon solubilization.     

19F NMR studies of tryptophan/serum albumin binding

19F NMR provides direct measures of the Trp binding avidity of 'fatty acid free' bovine serum albumin when D- and L-6-fluorotryptophan are used as the probes. Both a high and low affinity binding site are present. The addition of octanoate either displaces the ligand from both sites or greatly decreases the affinity such that little binding occurs at 2 mM levels. In the case of L-6-fluorotryptophan separate signals are observed for the high and low affinity binding sites and titrations with competing ligands can be used to establish the relative affinities of ligands at the high affinity site. Binding at this site appears to be hydrophobic and shape specific with L-Phe being a very poor ligand (K(D)[L-Phe]/K(D)[L-Trp]=800) while both GHKalphaNal and GHKW displace L-6-fluorotryptophan from this site. In tripeptides of the general formula GHK[ epsilon NH(CH(2))(n)(CO)W], affinity increases with tether length and binding at the low affinity site is restored. This NMR assay appears well-suited for the discovery of selective binding agents in this and other biorecognition phenomena.     

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.     

Binding of GDNF and neurturin to human GDNF family receptor alpha 1 and 2. Influence of cRET and cooperative interactions

The members of the glial cell line-derived neurotrophic factor (GDNF) family signal via binding to the glycosyl phosphatidylinositol-anchored membrane proteins, the GDNF family receptors alpha (GFRalpha), and activation of cRET. We performed a detailed analysis of the binding of GDNF and neurturin to their receptors and investigated the influence of cRET on the binding affinities. We show that the rate of dissociation of (125)I-GDNF from GFRalpha1 is increased in the presence of 50 nm GDNF, an effect that can be explained by the occurrence of negative cooperativity. Scatchard plots of the ligand concentration binding isotherms reveal a pronounced downward curvature at low (125)I-GDNF concentrations suggesting the presence of positive cooperativity. This effect is observed in the range of GDNF concentrations responsible for biological activity (1-20 pm) and may have an important role in cRET-independent signaling. A high affinity site with a K(D) of 11 pm for (125)I-GDNF is detected only when GFRalpha1 is co-expressed with cRET at a DNA ratio of 1:3. These results suggest an interaction of GFRalpha1 and cRET in the absence of GDNF and demonstrate that the high affinity binding can be measured only when cRET is present.     

Receptor binding of asialoerythropoietin.

The interaction of 125I-asialoerythropoietin (asialoepo) with receptors has been characterized both by binding assay and affinity cross-linking. Purified spleen cells from mice infected with the anemia strain of Friend virus (FVA cells) have receptors for 125I-asialoepo with two classes of affinity constant: one with Kd = 0.02-0.03 nM and 300-400 per cell, the other with lower affinity (Kd = 0.9-1.2 nM) and 1,000-1,200 per cell. The Kd value for the high affinity site is one-third of that for the binding of native 125I-erythropoietin (125I-epo) to the same FVA cells (Kd = 0.08-0.1 nM). Using 125I-asialoepo or 125I-epo affinity cross-linking methods, we find two components with apparent molecular weights of 88 kDa and 105 kDa in FVA cells, and in the transformed mouse cell lines, 201, IW32, and NN10, in agreement with earlier studies using 125I-epo. These results indicate that 125I-asialoepo binds to the same receptors as 125I-epo, but with greater affinity for the high affinity site. Since 201 cells contain only a single class of lower affinity receptors for erythropoietin (epo), finding the same two components as found for FVA cells by cross-linking experiment indicates that the two components do not represent the two classes of receptor.     

Receptor–ligand molecular docking

Docking methodology aims to predict the experimental binding modes and affinities of small molecules within the binding site of particular receptor targets and is currently used as a standard computational tool in drug design for lead compound optimisation and in virtual screening studies to find novel biologically active molecules. The basic tools of a docking methodology include a search algorithm and an energy scoring function for generating and evaluating ligand poses. In this review, we present the search algorithms and scoring functions most commonly used in current molecular docking methods that focus on protein–ligand applications. We summarise the main topics and recent computational and methodological advances in protein–ligand docking. Protein flexibility, multiple ligand binding modes and the free-energy landscape profile for binding affinity prediction are important and interconnected challenges to be overcome by further methodological developments in the docking field.     

Relative ligand binding to small or large aggregates measured by scanning correlation spectroscopy.

Cell surface receptors transduce signals, required to produce cellular activity, that may be mediated by ligand-induced receptor aggregation. Several receptor systems exhibit both low and high ligand affinities and some models of receptor activation associate receptor clusters with high or low ligand binding affinity. In the present work succinyl concanavalin A, which binds with both high and low affinity to receptors, was studied on 3T3 Swiss mouse fibroblasts, where preaggregation of receptors has been postulated. Scanning fluorescence correlation spectroscopy measurements were used to determine the relationship between the degree of ligand binding and the state of receptor aggregation. Correlation analysis of fluorescence fluctuations across the cell surface reveal that the variance of the fluctuations (quantitated by g[0]) increased when the ligand concentration was varied from 0.33 to 67 mg/L. The g(0) values reached a plateau at concentrations greater than approximately 10 mg/L. These data are incompatible with homogeneous receptor distributions or equal affinity receptor binding but are compatible with a partly aggregated receptor system with high affinity binding to small aggregates, and low affinity binding to large aggregates. Computer simulated scanning fluorescence correlation spectroscopy experiments confirm that background fluorescence from the cell does not account for the experimentally observed effects.     

Site-site interactions in glycogen phosphorylase b probed by ligands specific for each site

Three ligand binding sites on glycogen phosphorylase b which were originally described by kinetic and physicochemical means, and more recently located and defined in molecular terms by X-ray crystallography, have been probed by ligands specific for each site. Kinetic analyses, supplemented by X-ray crystallographic binding studies, permit assignment of each ligand to a primary binding site, as well as determination of its dissociation constant and interaction with ligands binding to the other sites. 8-Anilino-1-naphthalenesulfonate binds most strongly to the activator site, in competition with adenosine 5'-phosphate, presumably because its sulfonate group interacts with several arginine residues, and binds only weakly to the hydrophobic inhibitor site, possibly because of charge repulsion. It is itself a weak activator and decreases binding affinities for compounds specific for the inhibitor site. Our results with 8-anilino-1-naphthalenesulfonate are not consistent with predictions of its expected behavior and suggest caution in the use of this reagent as an indicator of hydrophobicity. Our second major probe, caffeine, binds primarily to the inhibitor site, shows competitive inhibition with substrate binding to the catalytic site, and decreases the affinity for the activator at the activator site. The catalytic site was probed with two different types of ligand. Glucose, known to stabilize the inactive T conformation of the enzyme, competes with the substrate alpha-D-glucose 1-phosphate for the catalytic site and decreases the affinity of adenosine 5'-phosphate for the activator site. Glucose also improves the binding affinity of caffeine for the inhibitor site by 3-5-fold, both compounds synergistically stabilizing the inactive T conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding to opioid receptors of enkephalin derivatives taking alpha-helical conformation and its dimer.

The opioid receptor binding of [Leu]enkephalin derivatives with extended address segment to the C-terminal was studied. The extension peptide is designed to take an amphiphilic helical structure in order to evaluate effects of helical conformation and membrane affinity of enkephalin moiety of the derivatives on receptor binding. In the delta-receptor-selective binding assay, Tyr-Gly-Gly-Phe-Leu-Lys-Aib-Leu-Aib-OH (1) showed the same affinity as enkephalinamide, whereas in the mu-receptor-selective binding assay, a 7-fold reduction in affinity was observed. On the other hand, Tyr-Gly-Gly-Phe-Leu-(Lys-Aib-Leu-Aib)2-OH (2) showed 51- and 96-fold decreases in affinities for delta- and mu-receptors, respectively, compared with enkephalinamide. The low receptor affinity of derivative 2 is considered due to alpha-helical conformation, which might not be compatible with topological requirements of delta- and mu-receptors. A dimer, Tyr-Gly-Gly-Leu-Phe-(Lys-Aib-Leu-Aib)2-Lys(X)-Aib-OCH3 (X = Tyr-Gly-Gly-Phe-Leu-, (4], showed 2.5- and 3.0-fold increases in affinities respectively for delta- and mu-receptors compared with the monovalent derivative 2, possibly due to cross-linking of neighboring receptors. The Hill plot of the binding of the dimer to bovine brain membranes was composed of two phases, although such a heterogeneity of receptors was not observed in the presence of naloxone or in the binding to NG108-15 cell membranes. These findings indicate the presence of the bivalent-ligand-induced interactions between delta- and mu-receptors in bovine brain membranes.     

Identification of the ligand-binding site of the BMP type IA receptor for BMP-4

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. BMP induces its signal to regulate growth, differentiation, and apoptosis of various cells upon trimeric complex formation with two distinct type I and type II receptors on the cell surface: both are single-transmembrane serine/threonine kinase receptors. To identify the amino acid residues on BMP type I receptor responsible for its ligand binding, the structure-activity relationship of the extracellular ligand-binding domain of the BMP type IA receptor (sBMPR-IA) was investigated by alanine-scanning mutagenesis. The mutant receptors, as well as sBMPR-IA, were expressed as fusion proteins with thioredoxin in Escherichia coli, and purified using reverse phase high performance liquid chromatography (RP-HPLC) after digestion with enterokinase. Structural analysis of the parent protein and representative mutants in solution by CD showed no detectable differences in their folding structures. The binding affinity of the mutants to BMP-4 was determined by surface plasmon resonance biosensor. All the mutant receptors examined, with the exception of Y70A, displayed reduced affinities to BMP-4 with the rank order of decreases: I52A (17-fold) approximately F75A (15-fold) > T64A (4-fold) = T62A (4-fold) approximately E54A (3-fold). The decreases in binding affinity observed for the latter three mutants are mainly due to decreased association rate constants while alterations in rate constants both, for association and dissociation, result in the drastically reduced affinities for the former two mutants. These results allow us to conclude that sBMPR-IA recognizes the ligand using the concave face of the molecule. The major ligand-binding site of the BMP type IA receptor consists of Phe75 in loop 2 and Ile52, Glu54, Thr62 and Thr64 on the three-stranded beta-sheet. These findings should provide a general basis for the ligand/type I receptor recognition in the TGF-beta superfamily.     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

Characterization of nicotinic acetylcholine receptors from the insects Aphis craccivora, Myzus persicae, and Locusta migratoria by radioligand binding assays: relation to thiamethoxam action

Thiamethoxam, a new neonicotinoid insecticide acting at nicotinic acetylcholine receptors, was characterized in competition binding assays with [3-H]-imidacloprid, a specific nicotinic ligand, using membranes from the aphids Aphis craccivora and Myzus persicae, and from the locust Locusta migratoria. In all insects, Scatchard analysis suggested two binding sites for imidacloprid with Kd values in the range of 1 nM and 10 nM, respectively. The Hill values were significantly below 1 (range of 0.63 to 0.85). In contrast to imidacloprid and nicotine, the potency of thiamethoxam to displace [3-H]-imidacloprid varied considerably among these insects. Thiamethoxam was more active than nicotine on Aphis receptors but 100-fold less in Locusta, a nontarget insect. Comparable relations were found to nithiazine. In Myzus, the inhibition curve for thiamethoxam was shallow. This suggested a heterogeneous receptor population displaying a range of binding affinities to thiamethoxam in this aphid. In all three insects, the other neonicotinoid insecticides studied competed with [3-H]-imidacloprid in the same order: thiacloprid > imidacloprid > or = acetamiprid > nitenpyram. N-Methylation of imidacloprid strongly reduced the affinity to the imidacloprid site, whereas N-demethylation of thiamethoxam resulted in a comparable increase of affinity. Supplementary assays were performed with (-)-[3-H]-nicotine and [3-H]-alpha-bungarotoxin on locust membranes. Overall, the data suggested that the outstanding insecticidal properties of thiamethoxam may be due to either a different binding site on nicotinic receptors, or receptor isoforms, or specific pharmakokinetic behavior, rather than to exceptional affinity to one of the examined binding sites.     

Ligand selectivity and affinity of chemokine receptor CXCR1. Role of N-terminal domain

Glu-Leu-Arg ("ELR") CXC chemokines interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA) recruit neutrophils by binding and activating two receptors, CXCR1 and CXCR2. CXCR1 is specific, binding only IL-8 with nanomolar affinity, whereas CXCR2 is promiscuous, binding all ELRCXC chemokines with high affinity. Receptor signaling consists of two events: interactions between the ligand N-terminal loop (N-loop) and receptor N-terminal domain (N-domain) residues (site I), and between the ligand N-terminal ELR and the receptor juxtamembrane domain (J-domain) residues (site II). It is not known how these interactions mediate ligand affinity and selectivity, and whether binding at one site influences binding and function at the other. Sequence analysis and structure-function studies have suggested that the receptor N-domain plays an important role in ligand selectivity. Here, we report ligand-binding properties and structural characteristics of the CXCR1 N-domain in solution and in detergent micelles that mimic the native membrane environment. We find that IL-8 binds the N-domain with significantly higher affinity in micelles than in solution (approximately 1 microM versus approximately 20 microM) and that MGSA does not bind the N-domain in solution but does in micelles with appreciable affinity (approximately 3 microM). We find that the N-domain is structured in micelles and that the entire N-domain interacts with the micelle in an extended fashion. We conclude that the micellar environment constrains the N-domain, and this conformational restraint influences its ligand-binding properties. Most importantly, our data suggest that for both ligands, site I interaction provides similar affinity and that differential coupling between site I and II interactions is responsible for the observed differences in affinity.     

Role of insulin and IGF1 receptors in proliferation of cultured renal proximal tubule cells.

We have used a murine proximal tubule cell line (MCT cells) to determine the presence and binding characteristics of insulin and IGF1 receptors and to correlate these parameters with the concentration-response relationships for ligand-induced cellular proliferation. Separate insulin and IGF1 receptors were identified by equilibrium binding assays. Half-maximal displacement of either peptide occurred at 3-10 nM; crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Peptide effects on cellular proliferation were determined by measuring [3H]thymidine incorporation. Both insulin and IGF1 stimulate thymidine incorporation in a dose-dependent manner with similar increases above the basal level. The estimated half-maximal stimulation (EC50) occurred at 4 nM for IGF1 and 8 nM for insulin. A comparison of the receptor binding affinities with the dose-response relationships for [3H]thymidine incorporation reveals that each growth factor appears to be exerting its effect via binding to its own receptor. Therefore, in this cell line, physiologic concentrations of either insulin or IGF1 can modulate cellular growth. To our knowledge this is the first demonstration of a mitogenic effect which may be modulated by ligand binding to the insulin receptor in proximal tubule epithelia.