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PubMed1992

Effects of repeated injections of cocaine on catecholamine receptor binding sites, dopamine transporter binding sites and behavior in rhesus monkey.

In order to determine if repeated injections of cocaine produced long-lasting alterations in catecholaminergic binding sites, rhesus monkeys were treated with saline (1.0 ml/15 kg) or cocaine (3.0-4.0 mg/kg) four times daily for 14 consecutive days and sacrificed two weeks after the last injection. The densities of dopamine D1 receptor binding sites, dopamine transporter binding sites and beta adrenergic receptor binding sites were significantly decreased in caudate nucleus to 51%, 17% and 61% of control, respectively, two weeks after repeated cocaine injections. There were no differences in D2 receptor binding site densities in the caudate, nor were there differences in binding sites between groups in the other brain regions examined: prefrontal cortex (D1, D2, dopamine transporter, beta), nucleus accumbens (D1, D2, dopamine transporter) and substantia nigra (D2). Behavioral observation showed that the cocaine-treated monkeys became sensitized to the repeated injections. Early in the regimen, these animals displayed stereotypic grooming, buccal movements and visual checking after each injection that differed significantly from the saline-treated animals. As the regimen progressed, the frequency of grooming decreased while the frequencies of visual tracking and splayed legs increased in a manner consistent with the development of behavioral sensitization. Together, these findings suggest that the caudate nucleus may be more sensitive than other dopamine-containing brain regions to long-lasting pre- and post-synaptic effects of repeated cocaine administration, and that the changes seen in dopaminergic neurons may be related to behavioral sensitization.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1995

The membrane-proximal scavenger receptor cysteine-rich domain of CD6 contains the activated leukocyte cell adhesion molecule binding site.

Binding studies with a CD6 immunoglobulin fusion protein (CD6 Rg) resulted in the identification and cloning of a CD6 ligand. This ligand was found to be a member of the immunoglobulin supergene family and was named ALCAM (activated leukocyte cell adhesion molecule). Cell adhesion assays showed that CD6-ALCAM interactions mediate thymocyte-thymic epithelium cell binding. ALCAM is also expressed by activated leukocytes and neurons and may be involved in interactions between T cells and activated leukocytes and between cells of the immune and nervous systems, respectively. Herein we describe the preparation of domain-specific murine CD6 Rg fusion proteins and show that the membrane-proximal SRCR (scavenger receptor cysteine-rich) domain of CD6 contains the ALCAM binding site. We also show that mAbs which bind to this domain preferentially block CD6-ALCAM binding. These results demonstrate that the membrane-proximal SRCR domain of CD6 is necessary for CD6 binding to ALCAM and provide the first direct evidence for the interaction of an SRCR domain with a ligand.

Activated-Leukocyte Cell Adhesion Molecule↗

PubMed2004

The predicted 3D structure of the human D2 dopamine receptor and the binding site and binding affinities for agonists and antagonists.

Dopamine neurotransmitter and its receptors play a critical role in the cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's disease and schizophrenia would be significantly enhanced with the availability of the 3D structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. We report here the 3D structure of the long isoform of the human D2 dopamine receptor, predicted from primary sequence using first-principles theoretical and computational techniques (i.e., we did not use bioinformatic or experimental 3D structural information in predicting structures). The predicted 3D structure is validated by comparison of the predicted binding site and the relative binding affinities of dopamine, three known dopamine agonists (antiparkinsonian), and seven known antagonists (antipsychotic) in the D2 receptor to experimentally determined values. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies, and give relative binding affinities that correlate well with experiments. The predicted binding site for dopamine and agonists is located between transmembrane (TM) helices 3, 4, 5, and 6, whereas the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM helix 5. We identify characteristic differences between the binding sites of agonists and antagonists.

Binding Sites↗

PubMed1994

Deformed protein binding sites and cofactor binding sites are required for the function of a small segment-specific regulatory element in Drosophila embryos.

How each of the homeotic selector proteins can regulate distinct sets of DNA target elements in embryos is not understood. Here we describe a detailed functional dissection of a small element that is specifically regulated by the Deformed homeotic protein. This 120 bp element (module E) is part of a larger 2.7 kb autoregulatory enhancer that maintains Deformed (Dfd) transcription in the epidermis of the maxillary and mandibular segments of Drosophila embryos. In vitro binding assays show that module E contains only one Dfd protein binding site. Mutations in the Dfd binding site that increase or decrease its in vitro affinity for Dfd protein generate parallel changes in the regulatory activity of module E in transgenic embryos, strong evidence that the in vitro-defined binding site is a direct target of Dfd protein in embryos. However, a monomer or multimer of the Dfd binding region alone is not sufficient to supply Dfd-dependent, segment-specific reporter gene expression. An analysis of a systematic series of clustered point mutations in module E revealed that an additional region containing an imperfect inverted repeat sequence is also required for the function of this homeotic protein response element. The Dfd binding site and the putative cofactor binding site(s) in the region of the inverted repeat are both necessary and in combination sufficient for the function of module E.

Animals↗

PubMed1983

Binding of insulin to solubilized insulin receptor from human placenta. Evidence for a single class of noninteracting binding sites.

Binding of 125I-insulin to insulin receptor was studied at equilibrium using plasma membrane protein, from human placenta, solubilized in Triton X-100. Measured at 1 nM 125I-insulin, the amount of radioligand bound specifically was dependent upon pH, the optimal association at 4 degrees C occurring in the pH range 8.2-8.7. The Adair equation was employed for analysis of binding isotherms generated at several pH values for concentrations of radioligand varying from 0.01-8.0 nM. Association constants derived from these analyses showed the same pH dependence described above and were found to be independent of receptor concentration. Hill plots derived from these titrations consistently yielded Hill coefficients of 1 and Scatchard plots gave virtually straight lines. Close correspondence was found between theoretical analyses and observations under all experimental conditions and graphical methods employed. These results are consistent with a single class of noninteracting 125I-insulin binding sites on the solubilized insulin receptor.

Binding Sites↗

PubMed1975

Protein interactions with small molecules. Relationships between stoichiometric binding constants, site binding constants, and empirical binding parameters.

The multiple equilibria for the binding of a ligand A by a macromolecule P with n binding sites may be formulated in terms of a stoichiometric analysis or on the basis of a site-oriented scrutiny. The dependence of binding on ligand concentration can always be correlated in terms of n stoichiometric binding constants,Ki, even if there are interactions between sites that accentuate or attenuate binding affinities. A corresponding correlation in terms of site binding constants, kj, under the most general circumstances depends on the definition of n2n-1 different constants of which 2n-1 are independent. If experimental data are correlated in terms of n parameters kalpha, kbeta ... klambda in an equation of the site-binding form, (see article for formular) then there is no guarantee that the values of ka, kb, etc., have any unique relationships to site binging constants. Examples are given to illustrate this point. Equation are derived for relating stoichiometric binding constants to site binding constants, for the general case and for various special circumstances. These equations make it possible to define and analyze binding insystems with interactions and conformational accommodations. Accordingly, a graphical procedure is described (in which iKi is plotted against i, the stoichiometric binding step) that provides an affinity profile for concise representation of magnitudes of binding constants and for detecting interactions that accentuate or attenuate site binding affinities.

Binding Sites↗

PubMed2003

Consensus sequence of transcription factor SF-1 binding site and putative binding site in the 5' flanking regions of genes encoding mouse steroidogenic enzymes 3betaHSDI and Cyp17.

Using 42 nucleotide sequences extracted from the Transcription Regulatory Regions Database (TRRD) containing SF-1 transcription factor binding site, we have determined the decanucleotide (GTCAAGGTCA) consensus sequence for SF-1 binding. In the frequency matrix of this sequence nucleotides between the 3rd and the 7th position had the highest frequency and guanine nucleotides at the 6th and the 7th positions were recognized in all nucleotide sequences. The latter suggests a crucial role of these guanines for the interaction of DNA with SF-1 protein. The determined consensus and frequency matrix were used for search of putative SF-1 binding sites in regulatory regions of two genes, encoding mouse Cyp17 (17alpha-hydroxylase/17-20-lyase) and 3betaHSDI (3beta-hydroxysteroid dehydrogenase/4delta-5delta-isomerase I), the microsomal enzymes involved in steroidogenesis. 5;-Flanking regions of genes encoding Cyp17 and 3betaHSDI were shown to contain six and five such binding sites, respectively. The presence of the putative SF-1 binding sites in the regulatory regions of mouse Cyp17 and 3betaHSDI suggests that gene SF-1 could represent one of the putative genes which (as we predicted earlier) determine coordinated inheritable variability of hormonal activity in mouse Leydig cells.

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Clathrin assembly protein AP180: primary structure, domain organization and identification of a clathrin binding site.

Binding of AP180 to clathrin triskelia induces their assembly into 60-70 nm coats. The largest rat brain cDNA clone isolated predicts a molecular weight of 91,430 for AP180. Two cDNA clones have an additional small 57 bp insert. The deduced molecular weight agrees with gel filtration results provided the more chaotropic denaturant 6 M guanidinium thiocyanate is substituted for the weaker guanidinium chloride. The sequence and the proteolytic cleavage pattern suggest a three domain structure. The N-terminal 300 residues (pI 8.7) harbour a clathrin binding site. An acidic middle domain (pI 3.6, 450 residues), interrupted by an uncharged alanine rich segment of 59 residues, appears to be responsible for the anomalous physical properties of AP180. The C-terminal domain (166 residues) has a pI of 10.4. AP180 mRNA is restricted to neuronal sources. AP180 shows no significant homology to known clathrin binding proteins, but is nearly identical to a mouse phosphoprotein (F1-20). This protein, localized to synaptic termini, has so far been of unknown function.

Adaptor Proteins, Vesicular Transport↗

PubMedDate not supplied

High and low affinity opioid binding sites: relationship to mu and delta sites.

Binding and pharmacological studies suggest a common opiate and enkephalin binding site in addition to their previously reported selective sites. This common high affinity site has tentatively been named mu1, distinguishing it from the morphine-selective site (mu2) and enkephalin-selective site (delta). The existence of this additional common high affinity site and its association with opiate and opioid peptide analgesia may help explain some pharmacological observations, such as the cross tolerance between morphine and enkephalin analgesia and the lack of cross tolerance between them in the guinea pig ileum and mouse vas deferens bioassays.

Animals↗

PubMed1996

Multiple high-affinity binding sites for

Binding of [3H]serotonin (5-HT) to membranes prepared from Arctic charr brain homogenates was most consistent with a one-site model for [3H]5-HT binding, with KD and Bmax values of 5.7±0.3 nmol l-1 and 60.7±7.3 fmol mg-1 protein, respectively. Similarly, 5-HT displacement of [3H]5-HT was best explained by a monophasic model with an apparent Ki of 4.3±0.7 nmol l-1. The ability of a number of synthetic 5-HT receptor ligands to displace [3H]5-HT was studied. 8OH-DPAT was found to interact with three [3H]5-HT binding sites, whereas buspirone, TFMPP, spiperone and mianserin all distinguish two sites. In the presence of 300 nmol l-1 buspirone, 8OH-DPAT and mianserin distinguished two [3H]5-HT binding sites, whereas spiperone interacted with only one. Moreover, 8OH-DPAT differentiated three [3H]5-HT binding sites even in the presence of 0.5 mmol l-1 GTP, making it unlikely that these sites represent different affinity states of G-protein-coupled receptors. GTP had no effect on apparent Ki values for 8OH-DPAT, but reduced the Bmax value of the high-affinity site by 60 %. GTP had a similar effect on the saturation binding curve for [3H]5-HT, reducing Bmax by approximately 50 %, whereas KD was unaffected. The results provide evidence for at least three different high-affinity [3H]5-HT binding sites, one of them showing a pharmacological profile strikingly similar to that of the mammalian 5-HT1A receptor.

Journal Article↗

PubMed1988

Action of polychlorocycloalkane insecticides on binding of [35S]t-butylbicyclophosphorothionate to Torpedo electric organ membranes and stereospecificity of the binding site.

Binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to Torpedo electric organ membranes was characterized. A dose- and pH-dependent binding (100.8 pmol/mg protein) was detected with a single affinity (Kd of 0.9 microM) in the presence of 150 mM KCl at pH 6.8. Other anions such as Br- and I- also increased binding affinity, but to a lower degree than Cl-, which increased the affinity by two- to threefold. In presence of 150 mM KCl, [35S]TBPS binding was inhibited noncompetitively by Zn2+ and by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) (IC50 of 9 microM). The gamma-isomer of hexachlorocyclohexane (BHC) was much more potent in inhibiting this [35S]TBPS binding and the inhibition was competitive (Ki = 40 nM). Like binding of [35S]TBPS to the gamma-aminobutyric acid (GABA) receptor, its binding to Torpedo membranes was inhibited by pentobarbital, mephobarbital, and hexobarbital (IC50 of 85, 225, and 300 microM), respectively), but not by phenobarbital. Binding was not inhibited by diazepam, GABA, bicuculline, or avermectin B1a, ligands that bind to the GABAA receptor. [35S]TBPS binding was inhibited by BHC isomers with the following decreasing order of potency alpha = gamma greater than sigma greater than beta, and by cyclodiene insecticides. Endrin was more potent than dieldrin, but endosulfan I and II had similar effects. The data suggest that the binding site for polychlorocycloalkane insecticides on this protein is much less stereoselective than that of the Cl- channel of the GABAA receptor. Also, even though this Torpedo protein has higher affinity for insecticides, such as gamma-BHC, than does the GABAA receptor, it is the latter whose specificity correlates best with polychlorocycloalkane toxicity. Nevertheless, because of its high affinity for gamma-BHC such a protein in muscles or brain may be an important target for the action of this insecticide.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

New inhibitors of thrombin and other trypsin-like proteases: hydrogen bonding of an aromatic cyano group with a backbone amide of the P1 binding site replaces binding of a basic side chain.

Highly effective thrombin inhibitors have been obtained by preparing boronic acid analogues of m-cyano-substituted phenylalanine and its incorporation into peptides. The cyano group enhances binding by several orders of magnitude. For example, Ac-(D)Phe-Pro-boroPheOH binds to thrombin with a Ki of 320 nM and the Ki of Ac-(D)Phe-Pro-boroPhe(m-CN)-OH is 0.79 nM. Protein crystal structure determination of trypsin complexed to H-(D)Phe-Pro-boroPhe(m-CN)-OH indicates that the aromatic side chain is bound in the P1 binding site and that the cyano group can act as a H-bond acceptor for the amide proton of Gly219. Enhanced binding for inhibitors containing the m-cyano group was observed for coagulation factor Xa and for the factor VIIa.tissue factor complex [Ki values of Ac-(D)Phe-Pro-boroPhe(mCN)-OH are 760 and 3.3 nM, respectively]. This result is consistent with the sequence homology of these two enzymes in the P1 binding site. Two enzymes lacking the strict homology in the P1 binding site, pancreatic kallikrein and chymotrypsin, did not exhibit significantly enhanced binding.

Amides↗

PubMed1986

Heterogeneity of [3H]ethyl beta-carboline-3-carboxylate binding sites and [3H]gamma-aminobutyric acid binding sites.

Binding properties of [3H]flunitrazepam ([3H]FNZ), [3H]ethyl beta-carboline-3-carboxylate ([3H]beta CCE), [3H]muscimol ([3H]MUSC) and [3H]gamma-aminobutyric acid ([3H]GABA) to bovine cortical membranes and to their Triton extracts were studied. GABA, 1 X 10(-5) M, stimulated [3H]FNZ binding of frozen, thawed and washed membranes by an increase in affinity without alteration of the maximal number of binding sites, and this GABA stimulated [3H]FNZ binding can be inhibited by bicuculline methobromide. Freeze, thaw and wash with Triton X-100 removed the low affinity [3H]MUSC binding sites. The ratios of [3H]FNZ binding sites to [3H]beta CCE binding sites and [3H]MUSC binding sites to [3H]GABA binding sites were always found to be about 1:2 in both membranes and soluble extracts. The fact that [3H]beta CCE, after displaced by clonazepam, can be further displaced by unlabelled beta CCE from its binding sites and that a portion of [3H]beta CCE binding sites can be survived from FNZ-photolysis implied that there are at least two subclasses of beta CCE binding sites, one is sensitive to beta CCE only and the other is sensitive to both beta CCE and benzodiazepines (BZs). [3H]GABA, after displaced by MUSC, can be further displaced by unlabelled GABA from its binding sites. The results also support that there are two subclasses of BZ-related GABA binding sites, one is sensitive to GABA only and the other is sensitive to both GABA and MUSC. Furthermore, the decay rates of [3H]beta CCE and [3H]GABA binding activities exposed to various degree of electron bombardment are identical, which is evident that these two binding sites are believed to be functional associated in a macromolecular complex with molecular mass about 220,000 Mr.

Animals↗

PubMed1990

Possible binding site of thyrotropin binding inhibitor immunoglobulin (TBII) on the thyrotropin (TSH) receptor, which is different from TSH binding site.

A synthetic decapeptide, P-194, which has the sequence No. 103 to 111 of hTSH receptor structure with an additional N-terminal tyrosine, did not bind TSH nor affected its receptor binding and thyroid stimulating activity. Preincubation of P-194 with sera from thyroid patients caused a significant decrease in TBII activity in almost all 12 TBII positive sera and an increase of thyroid stimulating activity in 3 of 7 Graves' IgG studied. In addition, [125I] P-194 bound to serum IgG fraction from thyroid patients with a positive correlation with TBII (N = 35, r = 0.509, p less than 0.01). The P-194 portion may be, at least a part of, TBII binding site distinct from the TSH binding site on the TSH receptor.

Animals↗

PubMed1987

Recognition of neuropeptides FMRFamide and LPLRFamide by chicken cerebellum avian pancreatic polypeptide binding sites.

Binding isotherms were constructed for the binding of synthetic tetrapeptide and pentapeptide fragments to membranes prepared from chicken cerebellar tissue. Both the tetrapeptide (FMRFamide), which was originally isolated from ganglia of mollusks, and the pentapeptide (LPLRFamide) previously isolated from chicken brain are known to increase blood pressure and modulate brain neurons in rats. The C-terminal dipeptide sequences of the two peptides are identical and both show similarity to the dipeptide sequence established for the pancreatic polypeptide (PP) family. Specific high-affinity binding sites exist for the latter peptide, sites which are competed for (though with less affinity) by neuropeptide Y (NPY). Affinity for cerebellar membranes was virtually equivalent for the synthetic peptide LPLRFamide and FRMFamide; the binding affinities (IC50) of all fragments tested (C-terminal pentapeptides of avian PP and NPY, and FMRFamide and LPLRFamide) fell in the same approximate range. Since the N-terminal residues of FMRFamide and LPLRFamide are not homologous with equivalent residues of APP or NPY, our results indicate that only Arg-Tyr-NH2 or Arg-Phe-NH2 sequences are necessary for binding of the carboxy terminus peptides of the PP family. In this respect, these sequences are functionally equivalent.

Amino Acid Sequence↗

PubMed1983

Alpha 2-macroglobulin binding to cultured fibroblasts: identification by affinity chromatography of high-affinity binding sites.

Binding sites having the properties of high-affinity receptors for activated alpha 2-macroglobulin (alpha 2M) have been purified over 100-fold from membranes of spontaneously transformed NIH-3T3 cells (J. A. Hanover, S.-y. Cheng, M. C. Willingham, and I. H. Pastan [1983] J. Biol. Chem. 258, 370-377). To identify the molecular species involved in high-affinity binding, the solubilized receptor has been purified 500-fold by conventional procedures and further purified by affinity chromatography. After radioiodination of the 500-fold-purified preparation, the detergent-solubilized extract was applied to alpha 2M-Sepharose and an 85,000 +/- 5000 Mr species was selectively retained by the column. Binding of the 85,000 +/- 5000 Mr species to the affinity resin was inhibited by EDTA and by excess alpha 2M. Elution from the affinity column could be accomplished with bacitracin, a competitive inhibitor of alpha 2M binding, or with EDTA. Consistent with the previously reported characteristics of the high-affinity alpha 2M receptor, the 85,000 Mr species bound much more efficiently to methylamine-activated alpha 2M-Affigel than to alpha 2M-Affigel which had not been amine-activated. The present data suggest that a protein with a subunit Mr of 85,000 +/- 5000 may represent a component of the high-affinity alpha 2M receptor present on cultured fibroblasts.

Animals↗

PubMed1984

Insulin bound to the insulin receptor of IM-9 lymphocytes is more accessible to antiinsulin antibodies after treatment with dithiothreitol: bound insulin is deeply buried in the receptor-binding site.

Binding of insulin to its receptor followed by covalent cross-linking with disuccinimidyl suberate (DSS) dramatically impairs the ability of antiinsulin antibodies (both polyclonal and monoclonal) to bind to the insulin moiety. We have used dithiotheitol, which has major effects on the oligomeric structure of the insulin receptor, to determine if this decreased antibody recognition is due to alteration in the conformation of insulin itself or to steric factors. Treatment of the covalently cross-linked insulin-receptor complex with dithiothreitol (DTT) increased the ability of the polyclonal and two monoclonal antiinsulin antibodies to immunoprecipitate the insulin-receptor complex. This treatment decreased immunoprecipitation by antireceptor antibodies. The effect of DTT may have been due to a reversal of either a binding-induced conformational change in the insulin moiety or an alteration in the conformation of the insulin-receptor complex, thereby decreasing steric hindrance. In an effort to choose between these two possible explanations, we prepared a biotinylated derivative of insulin which was cross-linked to the receptor. Since the biotin moiety is relatively rigid, it seemed improbable that binding to the receptor would alter the conformation of the epitope recognized by antibiotin antibodies and that the change would be reversed by DTT. Treatment of the cross-linked biotin-insulin-receptor complex with DTT did increase the ability of both antiinsulin and antibiotin antibodies to immunoprecipitate the cross-linked receptor complex. Identification of the cross-linked receptor on reduced sodium dodecyl sulfate-polyacrylamide gels confirms that the DTT treatment alters the distribution of the oligomeric forms of the receptor. These studies favor the hypothesis that when bound to its receptor, most of the insulin molecule is sequestered within the receptor-binding site such that there is steric hindrance to the approach of antiinsulin antibodies. Moreover, DTT alters the conformation of the cross-linked insulin-receptor complex so as to decrease this steric hindrance.

Cell Line↗

PubMed1990

Isolation-induced changes in radioligand binding to benzodiazepine binding sites.

Binding of [3H]flunitrazepam was studied in brain tissues of isolated Wistar rats and compared to group-reared animals. Modifications were observed in hippocampus and cortex (Kd increased) and in cerebellum (Bmax decreased) and when brain sections of control rats were incubated in the bath fluid that had served to incubate sections from isolated rats, a flattening of the saturation curve was observed. Results are discussed in terms of possible modulators of benzodiazepine binding sites, mainly tissue GABA concentrations.

Animals↗