Receptor dynamics of closely related ligands: "fast'' and "slow'' interferons.

Two related human alpha interferons with 83% homology in their primary sequences show a similar specific activity on nonhuman cells, but a striking difference on human cells, on which alpha-1 shows 1-5% of the specific molar activity displayed by alpha-2. Both interferons were labelled with 125I, and their binding kinetics followed on growing cultures of the human Burkitt line Daudi. Binding of alpha-1 showed slower rates of association and faster rates of dissociation implying that differences in apparent binding affinity were responsible for the differences in specific molar activity. However, binding was shown to reach steady-state rather than an equilibrium, so differences in the dynamics of the ligand-receptor complexes may represent amplification of differences in the initial binding constant. alpha-2, but not alpha-1, induces a marked loss of binding sites leading to a high affinity steady-state binding. Inhibition of cell multiplication by both interferons depends on a continued stimulation by free ligands at steady-state. It is proposed that the differences in specific molar activity are, in the main, kinetic and cause alpha-1 and alpha-2 to behave respectively as "slow' and "fast' interferons.     

Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects

To study the molecular basis of differences in the insecticidal spectrum of Bacillus thuringienesis delta-endotoxins, we have performed binding studies with three delta-endotoxins on membrane preparations from larval insect mid-gut. Conditions for a standard binding assay were established through a detailed study of the binding of 125I-labeled Bt2 toxin, a recombinant B. thuringiensis delta-endotoxin, to brush border membrane vesicles of Manduca sexta. The toxins tested (Bt2, Bt3 and Bt73 toxins) are about equally toxic to M. sexta but differ in their toxicity against Heliothis virescens. Equilibrium binding studies revealed saturable, high-affinity binding sites on brush border membrane vesicles of M. sexta and H. virescens. While the affinity of the three toxins was not significantly different on H. virescens vesicles, marked differences in binding site concentration were measured which reflected the differences in in vivo toxicity. Competition experiments revealed heterogeneity in binding sites. For H. virescens, a three-site model was proposed. In M. sexta, one population of binding sites is shared by all three toxins, while another is only recognized by Bt3 toxin. Several other toxins, non-toxic or much less toxic to M. sexta than Bt2 toxin, did not or only marginally displace binding of 125I-labeled Bt2 toxin in this insect. No saturable binding of this toxin was observed to membrane preparations from tissues of several non-susceptible organisms. Together, these data provide new evidence that binding to a specific receptor on the membrane of gut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of B. thuringiensis insecticidal crystal proteins.     

Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens

Human galectins have functionally divergent roles, although most of the members of the galectin family bind weakly to the simple disaccharide lactose (Galbeta1-4Glc). To assess the specificity of galectin-glycan interactions in more detail, we explored the binding of several important galectins (Gal-1, Gal-2, and Gal-3) using a dose-response approach toward a glycan microarray containing hundreds of structurally diverse glycans, and we compared these results to binding determinants on cells. All three galectins exhibited differences in glycan binding characteristics. On both the microarray and on cells, Gal-2 and Gal-3 exhibited higher binding than Gal-1 to fucose-containing A and B blood group antigens. Gal-2 exhibited significantly reduced binding to all sialylated glycans, whereas Gal-1 bound alpha2-3- but not alpha2-6-sialylated glycans, and Gal-3 bound to some glycans terminating in either alpha2-3- or alpha2-6-sialic acid. The effects of sialylation on Gal-1, Gal-2, and Gal-3 binding to cells also reflected differences in cellular sensitivity to Gal-1-, Gal-2-, and Gal-3-induced phosphatidylserine exposure. Each galectin exhibited higher binding for glycans with poly-N-acetyllactosamine (poly(LacNAc)) sequences (Galbeta1-4GlcNAc)(n) when compared with N-acetyllactosamine (LacNAc) glycans (Galbeta1-4GlcNAc). However, only Gal-3 bound internal LacNAc within poly(LacNAc). These results demonstrate that each of these galectins mechanistically differ in their binding to glycans on the microarrays and that these differences are reflected in the determinants required for cell binding and signaling. The specific glycan recognition by each galectin underscores the basis for differences in their biological activities.     

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.     

Thermodynamic studies on anion binding to apotransferrin and to recombinant transferrin N-lobe half molecules

Equilibrium constants for the binding of anions to apotransferrin, to the recombinant N-lobe half transferrin molecule (Tf/2N), and to a series of mutants of Tf/2N have been determined by difference UV titrations of samples in 0.1 M Hepes buffer at pH 7.4 and 25 degrees C. The anions included in this study are phosphate, sulfate, bicarbonate, pyrophosphate, methylenediphosphonic acid, and ethylenediphosphonic acid. There are no significant differences between anion binding to Tf/2N and anion binding to the N-lobe of apotransferrin. The binding of simple anions like phosphate appears to be essentially equivalent for the two apotransferrin binding sites. The binding of pyrophosphate and the diphosphonates is inequivalent, and the studies on the recombinant Tf/2N show that the stronger binding is associated with the N-terminal site. Anion binding constants for phosphate, pyrophosphate, and the diphosphonates with the N-lobe mutants K206A, K296A, and R124A have been determined. Anion binding tends to be weakest for the K296A mutant, but the variation in log K values among the three mutants is surprisingly small. It appears that the side chains of K206, K296, and R124 all make comparable contributions to anion binding. There are significant variations in the intensities of the peaks in the difference UV spectra that are generated by the titrations of the mutant apoproteins with these anions. These differences appear to be related more to variations in the molar extinction coefficients of the anion-protein complexes rather than to differences in binding constants.     

Measurement of the relative binding affinity of zearalenone, alpha-zearalenol and beta-zearalenol for uterine and oviduct estrogen receptors in swine, rats and chickens: an indicator of estrogenic potencies

1. The relative binding affinity of zearalenone, alpha-zearalenol, and beta-zearalenol for estrogen receptors was determined in the pig, rat and chicken. 2. Similar relative binding patterns were observed, with alpha-zearalenol exhibiting greater affinity than zearalenone and beta-zearalenol the least binding affinity in all species. 3. The relative binding affinity of alpha-zearalenol was greater in pig, than in rat and significantly greater than in chicken. 4. Interspecies differences in zearalenone sensitivity may be due to the binding affinity of alpha-zearalenol for estrogen receptors and differences in zearalenone metabolites formed.     

Different cation binding to the I domains of alpha1 and alpha2 integrins: implication of the binding site structure.

In the present work, we studied the interactions of recombinant alpha1 and alpha2 integrin I domains with cations Tb(3+), Mn(2+), Mg(2+) and Ca(2+). We observed that alpha1 and alpha2 I domains bind these cations with significantly different characteristics. The binding of Mg(2+) by the alpha1 I domain was accompanied by significant changes of tryptophan fluorescence which could be interpreted as a conformational change. Comparison of the alpha1 integrin I domain structure obtained by comparative modeling with a known structure of the alpha2 integrin I domain shows distinct differences in the metal ion binding sites which could explain the differences in cation binding.     

Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

Receptor binding studies on sarbecoviruses would benefit from an available toolkit of recombinant spike proteins, or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric Receptor Binding Domain (RBD) proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI-/- mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric, complex glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that complex glycosylated trimeric RBD proteins are attractive to analyze sarbecovirus receptor binding and explore ACE2 expression profiles in tissues.     

Measurement of the relative binding affinity of zearalenone, α-zearalenol and β-zearalenol for uterine and oviduct estrogen receptors in swine,rats and chickens: An indicator of estrogenic potencies

1. The relative binding affinity of zearalenone, α-zearalenol and β-zearalenol for estrogen receptors was determined in the pig, rat and chicken.2. Similar relative binding patterns were observed, with a-zearalenol exhibiting greater affinity than zearalenone and β-zearalenol the least binding affinity in all species.3. The relative binding affinity of α-zearalenol was greater in pig, than in rat and significantly greater than in chicken.4. Interspecies differences in zearalenone sensitivity may be due to the binding affinity of α-zearalenol for estrogen receptors and differences in zearalenone metabolites formed.     

Differential binding properties of human pregnancy zone protein- and alpha2-macroglobulin-proteinase complexes to low-density lipoprotein receptor-related protein.

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Both alpha-macroglobulins (alpha-Ms) covalently bind proteinases, which is accompanied by the exposure of carboxy terminal receptor recognition domains important for the rapid clearance from the circulation and tissues. It is accepted that the molecule responsible for the clearance of alpha2-M- and PZP-proteinase complexes is the low-density lipoprotein receptor-related protein (LRP). Although both alpha-M-proteinase complexes bind to the same receptor, differences in the binding properties have been reported. In addition, although it is known that the binding of alpha2-M-proteinase complexes to LRP can be blocked by Ni2+, the effect on PZP-proteinase has never been examined. In order to investigate differences in the binding properties of both alpha-Ms to the receptor, we purified LRP from human placenta by affinity chromatography and then analyzed the specificity and affinity of binding of alpha2-M- and PZP-proteinase complexes to the receptor by enzyme immunoassay. Our results clearly established that although both alpha-M-proteinase complexes specifically bind to LRP, PZP-chymotrypsin complexes bind to the receptor with lesser apparent affinity (Kd approximately equal 320 nM) than alpha2-M-chymotrypsin complexes (Kd approximately equal 40 nM). We also demonstrated that Ni2+ blocks the binding of alpha2-M-chymotrypsin complexes, but not PZP-chymotrypsin complexes, to LRP. These data suggest that the binding to LRP involves conformational differences between both alpha-Ms in a region immediately upstream of the carboxy terminal receptor recognition domain. The possibility that PZP-proteinase complexes interact with other receptors not available to alpha2-M-proteinase complexes could be considered.     

Agonist Binding to α2-Adrenoceptors Is Elevated in the Locus Coeruleus from Victims of Suicide

Abstract: The binding of an agonist, p-[¹²⁵I]iodoclonidine, and an antagonist, [³H]yohimbine, to α₂-adrenoceptors was measured autoradiographically in the locus coeruleus from 10 pairs of antidepressant-free victims of suicide and age-matched controls. Agonist binding to α₂-adrenoceptors was significantly greater in the locus coeruleus from victims of suicide compared with control subjects. In contrast, antagonist binding to α₂-adrenoceptors in the locus coeruleus did not differ significantly between control and suicide subjects. HPLC analysis of norepinephrine in tissue sections of the locus coeruleus did not reveal any differences between control subjects and suicide victims, suggesting that differences in agonist binding are not a result of differences in retention of the endogenous agonist norepinephrine in tissue sections. The increase in agonist binding to α₂-adrenoceptors in the locus coeruleus of victims of suicide links an altered expression of the high-affinity state of autoinhibitory α₂-adrenoceptors with suicide.     

Impact of IgG2 high molecular weight species on neonatal Fc receptor binding assays

A cell-based assay and a solution neonatal Fc receptor (FcRn) binding assay were implemented for the characterization of an IgG₂ antibody after observation that different product lots exhibited unexpected differences in FcRn binding in the cell-based format with membrane-bound FcRn. The experiments described here suggest that the apparent differences observed in the FcRn binding across different product lots in the cell-based format can be attributed to the different levels of the higher order high molecular weight species (HMWs) in them. A strong correlation between FcRn binding in the cell-based format and the percentage (%) higher order HMWs suggests that small amounts (∼0.1%) of the latter could cause the enhanced apparent FcRn binding (% relative binding ranging from 50 to 100%) in the format. However, when the binding was assessed with recombinant FcRn in soluble form, avidity effects were minimal and the assay format exhibited less sensitivity toward the differences in higher order HMWs levels across product lots. In conclusion, a solution-based assay may be a more appropriate assay to assess FcRn binding of the dominant species of an Fc-fusion protein or monoclonal antibody if minor differences in product variants such as higher order HMWs are shown to affect the binding significantly.     

Molecular comparison of monocot and dicot U1 and U2 snRNAs

To elucidate differences between the pre-mRNA splicing components in monocots and dicots, we have cloned and characterized several U1 and U2 snRNA sequence variants expressed in wheat seedling nuclei. Primer extension sequencing on wheat and pea snRNA populations has demonstrated that two 5'-terminal nucleotides found in most other U1 snRNAs are missing/modified in many plant U1 snRNAs. Comparison of the wheat U1 and U2 snRNA variants with their counterparts expressed in pea nuclei has defined regions of structural divergence between monocot and dicot U1 and U2 snRNAs. The U1 and U2 snRNA sequences involved in RNA:RNA interaction with pre-mRNAs are absolutely conserved. Significant differences occur between wheat and pea U1 snRNAs in stem I and II structures implicated in the binding of U1-specific proteins suggesting that the monocot and dicot U1-specific snRNP proteins differ in their binding specificities. Stem III structures, which are required in mammalian systems for splicing complex formation but not for U1-specific protein binding, differ more extensively than stems I, II, or IV. In U2 snRNAs, the sequence differences between these two species are primarily localized in stem III and in stem IV which has been implicated in snRNP protein binding. These differences suggest that monocot and dicot U1 and U2 snRNPs represent distinct entities that may have monocot- and dicot-specific snRNP protein variants associated with each snRNA.     

3-(4-Aminobutyn-1-yl)pyridines: binding at alpha 4 beta 2 nicotinic cholinergic receptors

The binding of a series pyridylbutynylamines 6 was examined at alpha4beta2 nACh receptors. Structural modifications, comparing 6 with pyridyl ethers 2, did not consistently result in parallel effects on receptor affinity, suggesting possible differences in their modes of binding. Furthermore, the binding of amine 6a seemed to be accounted for by the newer vector pharmacophore models.     

Ligand association with the rabbit kidney and brain Y1, Y2 and Y5-like neuropeptide Y (NPY) receptors shows large subtype-related differences in sensitivity to chaotropic and alkylating agents

The binding to rabbit kidney or hypothalamic particulates of the subtype-selective neuropeptide Y (NPY) receptor ligands [125I](Leu31,Pro34)hPYY (as Y1 site label at 2 nM human pancreatic polypeptide (hPP)), [125I]-hPYY(3-36) (Y2 label), and [125I]-hPP (Y5 label) displayed great differences in sensitivity to alkylators and chaotropic agents. Sensitivity to a nonionic chaotrope, urea, was much higher for the Y1 binding than for the Y5-like binding or the Y2 binding. The non-selective alkylator N-ethylmaleimide (NEM) and several alkylators selective for aminergic receptors were much more efficacious against the Y1 relative to the Y2 binding. Similar differences could be confirmed with the attachment of Y1 and Y2-selective tracers to CHO cells expressing the cloned guinea-pig Y1 or Y2 receptors. The Y5-like binding was quite insensitive to NEM, but sensitive to chloroethylclonidine (CEC) and prazobind, which were less potent at the Y1, and especially at the Y2 site. The unrestricted-access alkylator 2-aminoethyl methanethiosulfonate inhibited the binding to all subtypes, while the restricted-access agent 2-(trimethylammonium)ethylmethanethiosulfonate poorly inhibited the Y5-like binding, or the guanine nucleotide-insensitive Y2 binding. These results are compatible with an active conformation of the Y5-like site dependent on maintenance of a shared hydrophobic cavity. The Y2 sites resistant to guanosine polyphosphates and restricted-access alkylators were detected mainly in particulates slowly solubilized by cholate at 0-5 degrees C; these sites could be clustered.     

Site-directed mutagenesis of a single residue changes the binding properties of the serotonin 5-HT2 receptor from a human to a rat pharmacology.

Mesulergine displays approximately 50-fold higher affinity for the rat 5-HT2 receptor than for the human receptor. Comparison of the deduced amino acid sequences of cDNA clones encoding the human and rat 5-HT2 receptors reveals only 3 amino acid differences in their transmembrane domains. Only one of these differences (Ser----Ala at position 242 of TM5) is near to regions implicated in ligand binding by G protein-coupled receptors. We investigated the effect of mutating Ser242 of the human 5-HT2 receptor to an Ala residue as is found in the rat clone. Both [3H]mesulergine binding and mesulergine competition of [3H]ketanserin binding showed high affinity for rat membranes and the mutant human clone but low affinity for the native human clone, in agreement with previous studies of human postmortem tissue. These studies suggest that a single naturally occurring amino acid change between the human and the rat 5-HT2 receptors makes a major contribution to their pharmacological differences.     

Lysine binding to activated human platelets and its similarity to fibrinogen binding

Platelet surface glycoproteins IIb-IIIa are considered to function as the binding site for fibrinogen. Fibrinogen binding is essential for platelet aggregation and several amines have been shown to inhibit this binding. The present study compares the binding properties of 125I-fibrinogen and [3H]lysine with platelets activated by the Ca2+ ionophore A23187. Many lines of similarities in the binding properties are apparent; however, several differences were also found. The similarities are listed below and the differences are pointed out in parentheses. Marked enhancement by platelet activation; deficiency of binding by thrombasthenic platelets lacking the glycoproteins IIb-IIIa; saturability (fibrinogen binding approaches saturation at more than 12 microM, within 10 min; lysine binding at more than 100 mM within 1 min); Ca2+-dependence (at 1 mM Ca2+ lysine binding is minute and fibrinogen binding is half-saturated); reversibility; the binding achieved within 10 min is exchangeable; dissociation depends upon time and external ligand concentration; inhibition by the oligoamines His-Lys and Lys4; inhibition by serum from a thrombasthenic patient who developed anti-glycoproteins IIb-IIIa antibodies; specificity; alanine neither binds to activated platelets nor inhibits fibrinogen binding; it thus appears that the lysine which associates with activated platelets is mostly bound onto the surface of the cells rather than being incorporated. Moreover, the major site of lysine binding seems to be the complexed glycoproteins IIb-IIIa.     

Binding of spermidine and putrescine to energized liver mitochondria.

The binding of spermidine and putrescine to mitochondrial membranes was studied by applying a thermodynamic model of ligand-receptor interactions developed both for equilibrium and far-from-equilibrium binding processes (V. Di Noto, L. Dalla Via, A. Toninello, and M. Vidali Macromol. Theory Simul. 5, 165-181, 1996). Results demonstrate the presence of two monocoordinated binding sites (S1 and S2) for spermidine and one monocoordinated binding site (S2) for putrescine, all exhibiting high capacity and low affinity. It is proposed that differences in the polyamines' flexibility and hydrophilicity perhaps contributes to the observed variations in their interactions with the two sites. A comparison of the binding parameters of these polyamines with those of spermine reveals differences in the specific function of the S1 and S2 sites, identified in studies of spermine binding (L. Dalla Via, V. Di Noto, D. Siliprandi, and A. Toninello Biochim. Biophys. Acta 1284, 247-252, 1996).     

Evidence for Differential Binding of Growth Hormones to Membrane and Cytosolic GH Binding Proteins of Rabbit Liver

Abstract

The existence of three GH binding proteins in rabbit liver membranes has been adduced from binding studies with a panel of monoclonal antibodies (1)˙ Immunologically cross-reactive analogues of ‘type 2’ binding proteins were shown to exist in rabbit liver cytosol and in affinity purified receptor from liver microsomes. We now report differences in the binding of human and ovine GH with respect to two antigenic determinants on the ‘type 1″ GH binding protein. The discovery of these differences has enabled the detection of cross-reactive analogues of both binding protein types ‘1″ and ‘2’ in liver cytosol and in affinity purified preparations from liver membranes. These findings show a) a close structural relationship between the pool of cytosolic GH binding proteins and those present in the membranes; and b) differential ligand binding to, as well as absolute ligand selection by GH binding proteins, which could reflect the ability of GH to trigger a range of biological responses either through different receptors or differential interaction with particular receptor subtypes.     

Classification of the insulin-like growth factor binding proteins into three distinct categories according to their binding specificities

Competitive binding experiments with insulin-like growth factor (IGF)-1, IGF-2 and des-(1-3)-IGF-1 have confirmed the interpretation based on limited amino-terminal sequence analysis that at least three types of IGF binding protein occur. In addition to the acid stable subunit of the large serum binding protein which exhibits des-(1-3)-IGF-1 binding only slightly less than IGF-1, the small IGF binding proteins can be separated into two classes based on differences in des-(1-3)-IGF-1 and IGF-2 binding potencies.