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PubMed2000

Altered ligand binding and transcriptional regulation by mutations in the PML/RARalpha ligand-binding domain arising in retinoic acid-resistant patients with acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by a specific translocation, t(15;17), that fuses the promyelocytic leukemia (PML) gene with the RA receptor RARalpha. Pharmacologic doses of retinoic acid (RA) induce differentiation in human APL cells and complete clinical remissions. Unfortunately, APL cells develop resistance to RA in vitro and in vivo. Recently, mutations in PML/RARalpha have been described in APL cells from patients clinically resistant to RA therapy. The mutations cluster in 2 regions that are involved in forming the binding pocket for RA. These mutant PML/RARalpha proteins have been expressed in vitro, which shows that they cause a diversity of alterations in binding to ligand and to nuclear coregulators of transcription, leading to varying degrees of inhibition of retinoid-induced transcription. This contrasts with the nearly complete dominant negative activity of mutations in PML/RARalpha previously characterized in cell lines developing RA resistance in vitro. Current data from this study provide additional insight into the molecular mechanisms of resistance to RA and suggest that alterations in the ability of mutants to interact with coregulators can be determinant in the molecular mechanism of resistance to RA. In particular, ligand-induced binding to the coactivator ACTR correlated better with transcriptional activation of RA response elements than the ligand-induced release of the corepressor SMRT. The diversity of effects that are seen in patient-derived mutations may help explain the partial success to date of attempts to overcome this mechanism of resistance in patients by the clinical use of histone deacetylase inhibitors.

Amino Acid Substitution↗
PubMed1996

Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains.

The urokinase plasminogen activator receptor (uPAR) is a membrane protein comprised of three extracellular domains. In order to study the importance of this domain organization in the ligand-binding process of the receptor we subjected a recombinant, soluble uPAR (suPAR) to specific proteolytic cleavages leading to liberation of single domains. Treatment of the receptor with pepsin resulted in cleavage between residues 183 and 184, thus separating the third domain (D3) from the rest of the molecule, which was left as an intact fragment (D(1 + 2)). D(1 + 2) proved capable of ligand binding as shown by chemical cross-linking, but quantitative binding/competition studies showed that the apparent ligand affinity was 100- to 1000-fold lower than that of the intact suPAR. This loss of affinity was comparable with the loss found after cleavage between the first domain (D1) and D(2 + 3), using chymotrypsin. This result shows that in addition to D1, which has an established function in ligand binding (Behrendt, N., Ploug, M., Patthy, L., Houen, G., Blasi, F., and Dano, K. (1991) J. Biol. Chem. 266, 7842-7847), D3 has an important role in governing a high affinity in the intact receptor. Real-time biomolecular interaction analysis revealed that the decrease in affinity was caused mostly by an increased dissociation rate of the ligand complex of D(1 + 2). Zero length cross-linking, using carbodiimide-induced, direct condensation, was used to identify regions within suPAR engaged in molecular ligand contact. The purified suPAR was cross-linked to the radiolabeled amino-terminal fragment (ATF) of urokinase, followed by cleavage with chymotrypsin. In accordance with the cleavage pattern found for the uncomplexed receptor, this treatment led to cleavage between D1 and D(2 + 3). Analysis of the radiolabeled fragments revealed the expected ligand labeling of D1 but a clear labeling of D(2 + 3) was also found, indicating that this part of the molecule is also situated in close contact with ATF in the receptor-ligand complex. The latter contact site may contribute to the role of molecular regions outside D1 in high affinity binding.

Amino Acid Sequence↗
PubMed1993

A simple fluorimetric method for the estimation of ligand binding parameters in ligand-enzyme complexes.

The theory and practice of a simple fluorimetric method for the estimation of ligand binding parameters in ligand-enzyme complexes is described. In this method, the concentration of a ligand-binding enzyme and the dissociation constant of a ligand-enzyme complex were estimated solely from the total concentration of a ligand and the total fluorescence of the ligand in the absence and in the presence of enzyme.

Alcohol Dehydrogenase↗
PubMed1997

Identification of common ligand binding determinants of the insulin and insulin-like growth factor 1 receptors. Insights into mechanisms of ligand binding.

Insulin and insulin-like growth factor 1 (IGF-1) are peptides that share nearly 50% sequence homology. However, although their cognate receptors also exhibit significant overall sequence homology, the affinity of each peptide for the non-cognate receptor is 2-3 orders of magnitude lower than for the cognate receptor. The molecular basis for this discrimination is unclear, as are the molecular mechanisms underlying ligand binding. We have recently identified a major ligand binding site of the insulin receptor by alanine scannning mutagenesis. These studies revealed that a number of amino acids critical for insulin binding are conserved in the IGF-1 receptor, suggesting that they may play a role in ligand binding. We therefore performed alanine mutagenesis of these amino acids to determine whether this is the case. cDNAs encoding alanine-substituted secreted recombinant IGF-1 receptors were expressed in 293 EBNA cells, and the ligand binding properties of the expressed proteins were evaluated. Mutation of Phe701 resulted in a receptor with undetectable IGF-1 binding; alanine substitution of the corresponding amino acid of the insulin receptor, Phe714, produces a 140-fold reduction in affinity for insulin. Mutation of Asp8, Asn11, Phe58, Phe692, Glu693, His697, and Asn698 produces a 3.5-6-fold reduction in affinity for IGF-1. In contrast, alanine mutation of the corresponding amino acids of the insulin receptor with the exception of Asp12 produces reductions in affinity that are 50-fold or greater. The affinity of insulin for these mutants relative to wild type receptor was similar to that of their relative affinity for IGF-1 with two exceptions; the IC50 values for insulin binding to the mutants of Arg10, which has normal affinity for IGF-1, and His697, which has a 6-fold reduction in affinity for IGF-1, were both at least 2 orders of magnitude greater than for wild type receptor. The Kd values for insulin of the corresponding alanine mutants of the insulin receptor, Arg14 and His710, are 2-3 orders of magnitude greater than for wild type receptor. However, in contrast, the relative affinity of des(25-30)[PheB25 alpha-carboxamide]insulin for these IGF-1 receptor mutants is reduced only 4- and 50-fold, respectively.

Binding Sites↗
PubMed2002

Homology modelling of the ligand binding domain of mineralocorticoid receptor: close structural kinship with glucocorticoid receptor ligand binding domain and their similar binding mode with DOC (de-oxy corticosterone).

Mineralocorticoids play a major role in regulating sodium and potassium homeostasis and also contribute to the control of blood pressure and in some physiological disorders. The physiological effects of this class of corticosteroids are mediated by ligand-induced nuclear transcription factor, the mineralocorticoid receptor(MR) / glucocorticoid receptor(GR), a member of the steroid / nuclear receptor superfamily. Although the MR interacts with both glucocorticoids and mineralocorticoids, the GR interacts specifically with glucocorticoids. The three dimensional structure of progesterone complexed to its receptor revealed in X-ray diffraction method is utilised to develop a homology model of human mineralocorticoid receptor ligand binding domain (hMR LBD) in a similar fashion as mouse GR LBD was developed previously. The secondary structure of hMR LBD contains eleven helices, eight turns and four sheets. This receptor contains a long helix, H9, with thirty four residues. The 12-residue C-terminal extension (residues 973-984) of hMR LBD, which is essential for hormone binding, is tightly fixed in position by an antiparallel b-sheet interaction. The three dimensional model reveals two polar sites located at the extremities of the elongated hydrophobic ligand-binding pocket (LBP). De-oxy corticosterone (DOC) is docked to the LBs of both hMR LBD and mGR LBD. The difference accessible surface area (DASA) study revealed the interaction zones of both the receptors in complex with DOC. Observations relating to the native and complex proteins revealed a close structural kinship between hMR LBD and mGR LBD.

Amino Acid Sequence↗
PubMed1976

[Measurement of thyroxine binding globulin by competitive ligand binding assay (CLBA) (author's transl)].

The competitive ligand binding assay (CLBA) first described by Chopra et al. ((1972) J. Clin. Endocrinol. Metab. 35, 565-573) is a convenient routine method for the accurate measurement of thyroxine binding globulin in large numbers of serum samples. The assay is based on the partition of a constant quantity of radiolabelled T3 between a fixed quantity of rabbit T3 antibodies and the thyroxine binding globulin of the serum, after prior removal of T3 and T4 from the serum with an anion exchange resin (Amberlite IRA 400). In euthyroid subjects serum thyroxine binding globulin was 25.5 +/- 5.0 mg/1, in hyperthyroid patients thyroxine binding globulin was significantly decreased to 13.0 +/- 4.0 mg/1 and was significantly increased in hypothyroid patients to 36.8 +/- 6.2 mg/1 as well as in pregnant women to 41.3 +/- 6.2 mg/1. No difference was found between normal subjects and young women taking contraceptive pills with low oestrogen content. There were significant negative correlations between the thyroxine binding globulin in serum on the one hand side and the free T4-and free T3-fraction on the other. The low thyroxine binding globulin estimates in hyperthyroid patients increased gradually to normal during treatment with thyroid blocking drugs, the elevated thyroxine binding globulin in hypothyroid patients decreased to normal during treatment with thyroid hormones. The competitive ligand binding assay used here seems to be convenient as a routine method for the precise and reproducible measurement of thyroxine binding globulin in serum.

Animals↗
PubMed2003

Low resolution structures of the retinoid X receptor DNA-binding and ligand-binding domains revealed by synchrotron X-ray solution scattering.

Nuclear receptors are ligand-inducible transcription factors that share structurally related DNA-binding (DBD) and ligand-binding (LBD) domains. Biochemical and structural studies have revealed the modular nature of DBD and LBD. Nevertheless, the domains function in concert in vivo. While high-resolution crystal structures of nuclear receptor DBDs and LBDs are available, there are no x-ray structural studies of nuclear receptor proteins containing multiple domains. We report the solution structures of the human retinoid X receptor DBD-LBD (hRXRalphaDeltaAB) region. We obtained ab initio shapes of hRXRalphaDeltaAB dimer and tetramer to 3.3 and 1.7 nm resolutions, respectively, and established the position and orientation of the DBD and LBD by fitting atomic coordinates of hRXRalpha DBD and LBD. The dimer is U-shaped with DBDs spaced at approximately 2 nm in a head to head orientation forming an angle of about 10 degrees with respect to each other and with an extensive interface area provided by the LBD. The tetramer is a more elongated X-shaped molecule formed by two dimers in head to head arrangement in which the DBDs are extended from the structure and spaced at about 6 nm. The close proximity of DBDs in dimers may facilitate homodimer formation on DNA; however, for the homodimer to bind to a DNA element containing two directly repeated half-sites, one of the DBDs would need to rotate with respect to the other element. By contrast, the separation of DBDs in the tetramers may account for their decreased ability to recognize DNA.

Binding Sites↗
PubMed1991

A region of the insulin receptor important for ligand binding (residues 450-601) is recognized by patients' autoimmune antibodies and inhibitory monoclonal antibodies.

Chimeric receptors containing different portions of the homologous human insulin receptor, insulin-like growth factor I receptor, and insulin receptor-related receptor were utilized to identify the epitopes recognized by various anti-insulin receptor antibodies. The antibodies studied included 12 monoclonal antibodies to the extracellular domain of the human insulin receptor as well as 15 patients' sera with autoimmune anti-insulin receptor antibodies. All of the patients' sera and all 8 monoclonal antibodies that inhibit insulin binding were found to recognize an epitope contained within residues 450-601 of the alpha subunit of the receptor. In contrast, 2 monoclonal antibodies that do not inhibit insulin binding were found to recognize the cysteine-rich region of the alpha subunit. Chimeric insulin receptors that had residues 450-601 replaced with the homologous residues of the insulin-like growth factor I receptor exhibited a decreased ability to bind insulin. In contrast, insulin-like growth factor I receptors that have had the comparable region replaced with that of the insulin receptor showed no decrease in their ability to bind ligand. These results indicate that residues 450-601 of the insulin receptor are important for insulin binding and include the major site for recognition by inhibitory monoclonal antibodies and patients' autoimmune anti-receptor antibodies.

Antibodies, Monoclonal↗
PubMed2001

N-terminal carboxyl and tetrazole-containing amides as adjuvants to Grb2 SH2 domain ligand binding.

High affinity binding of peptides to Src homology 2 (SH2) domains, often requires the presence of phosphotyrosyl (pTyr) or pTyr-mimicking moieties in the N-terminal position of the binding ligand. Several reports have shown that N(alpha)-acylation of the critical pTyr residue can result in increased SH2 domain binding potency. For Grb2 SH2 domains which recognize pTyr-Xxx-Asn-NH(2) motifs, significant potency enhancement can be incurred by N(alpha)-(3-amino)Z derivatization of tripeptides such as pTyr-Ile-Asn-NH(2). Using ligands based on the high affinity pY-Ac(6)c-Asn-(naphthylpropylamide) motif, (where Ac(6)c=1-aminocyclohexanecarboxylic acid), additional reports have shown moderate potentiating effects of N(alpha)-oxalyl derivatization. The current study examined variations of the N(alpha)-oxalyl theme in the context of a Xxx-Ac(6)c-Asn-(naphthylpropylamide) platform, where Xxx=the hydrolytically stable pTyr mimetics phosphonomethyl phenylalanine (Pmp) or carboxymethyl phenylalanine (Cmf). The effects of N(alpha)-(3-amino)Z derivatization were also investigated for this platform, to ascertain whether the large binding enhancement reported for tripeptides such as pTyr-Ile-Asn-NH(2) could be observed. In ELISA-based extracellular Grb2 SH2 domain binding assays, it was found for the Pmp-based series, that extending the oxalyl carboxyl out by one methylene unit or replacing carboxyl functionality with a tetrazole isostere, resulted in binding potency greater than the parent N(alpha)-acetyl-containing compound, with enhancement approximating that observed for the N(alpha)-oxalyl derivative. When Cmf was used as the pTyr mimetic, only modest differences in IC(50) values were observed for the series. Examination of the N(alpha)-(3-amino)Z derivatized Pmp-Ac(6)c-Asn-(naphthylpropylamide), showed that binding affinity was reduced relative to the parent N(alpha)-acetyl analogue, in contrast to the reported significant enhancement of affinity observed with other peptide ligands. Treatment of MDA-453 tumor cells, which are mitogenically driven through erbB-2 tyrosine kinase-dependent pathways, with Pmp-containing inhibitors resulted in growth inhibition, with the N(alpha)-oxalyl and N(alpha)-malonyl-containing compounds exhibiting IC(50) values (4.3 and 4.6 microM, respectively) approximately five-fold lower than the parent N(alpha)-acetyl-containing compound. Tetrazole and N(alpha)-(3-amino)Z-containing inhibitors were from two- to four-fold less potent than these latter analogues in the growth inhibition assays.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Control of estrogen receptor ligand binding by Hsp90.

The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.

Animals↗
PubMed2003

Homology modelling of the nuclear receptors: human oestrogen receptorbeta (hERbeta), the human pregnane-X-receptor (PXR), the Ah receptor (AhR) and the constitutive androstane receptor (CAR) ligand binding domains from the human oestrogen receptor alpha (hERalpha) crystal structure, and the human peroxisome proliferator activated receptor alpha (PPARalpha) ligand binding domain from the human PPARgamma crystal structure.

We have generated by homology the three-dimensional structures of the ligand binding domain (LBD) of several interrelated human steroid hormone receptors (SHRs). These are the oestrogen receptor beta (hERbeta), the pregnane-X-receptor (PXR), the Ah receptor (AhR) and the constitutive androstane receptor (CAR). They were produced by homology modelling from the human oestrogen receptor alpha (hERalpha) crystallographic coordinates [Nature 389 (1997) 753] as a template together with the amino acid sequences for hERbeta [FEBS Lett. 392 (1996) 49], PXR [J. Clin. Invest. 102 (1998) 1016], AhR [Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 815] and CAR [Nature 395 (1998) 612; Mol. Cell. Biol. 14 (1994) 1544], respectively. The selective endogenous ligand, in each case, was docked interactively within the putative ligand binding site using the position of oestradiol in hERalpha as a guide, and the total energy was calculated. In each receptor model a number of different ligands known to fit closely within the ligand binding site were interactively docked and binding interactions noted. Specific binding interactions included combinations of hydrogen bonding and hydrophobic contacts with key amino acid sidechains, which varied depending on the nature of the ligand and receptor concerned. We also produced the human peroxisome proliferator activated receptor alpha (PPARalpha) by homology modelling using the human PPARgamma (hPPARgamma) LBD crystallographic coordinates summarised in [Toxicol. In Vitro 12 (1998) 619] as a template together with the amino acid sequence for hPPARalpha [Toxicol. In Vitro 12 (1998) 619; Nature 395 (1998) 137]. The models will provide a useful tool in unravelling the complexity in the physiologic response to xenobiotics by examining the ligand binding interactions and differences between the steroid hormone receptors activation or inactivation by their ligands.

Amino Acid Sequence↗
PubMed2004

Separation of mismatched DNA by using the affinity column immobilizing mismatch-binding ligands.

Mismatch binding ligands (MBL), ND and NA, bind to DNA containing mismatch base pairs. These MBLs were immobilized to NHS-activated affinity column via amine linker. Affinity chromatographic analyses of mismatched DNAs by using the ND-immobilized column showed clear separation of G-G and G-A from other mismatched DNAs. The results of the NA-immobilized column also showed good separation of A-A, G-A, G-G, A-C from other mismatched DNAs. The order of mobility of mismatched duplexes on MBL-immobilized column was in good agreement with that of the binding affinity in a solution phase obtained by the melting temperature measurement.

Base Pair Mismatch↗
PubMed1993

Characterization of ligand binding to acyl-CoA-binding protein.

Ligand binding to recombinant bovine acyl-CoA-binding protein (rACBP) was examined using a Lipidex 1000 competition assay and an e.p.r. spectroscopy displacement assay. Of all putative ligands tested, rACBP exhibited a high binding affinity only for acyl-CoA esters. No alternative ligands could be found in rat liver fractions purified on an affinity of column on which ACBP was coupled to Sepharose 4B. E.p.r. data indicate that both the acyl chain and the CoA head group of acyl-CoA are involved in binding and that the 3'-phosphate group on the ribose moiety of acyl-CoA esters plays a crucial role in the binding of acyl-CoA to ACBP. E.p.r. competition binding studies show that the binding affinity of acyl-CoA esters for rACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-CoA esters with 14-22 carbon atoms in the acyl chain. No correlation between the number of double bonds in the acyl chain and the binding affinity was observed. The experimental results strongly indicate that ACBP specifically binds long-chain acyl-CoA esters with a very high affinity, results that indicate that ACBP is likely to be involved in the intracellular transport and pool formation of these compounds.

Acyl Coenzyme A↗
PubMed1996

Ligand-binding characteristics and related structural features of the expressed goldfish kainate receptors: identification of a conserved disulfide bond and three residues important for ligand binding.

Low-molecular-weight kainate receptors from nonmammalian vertebrate brain belong structurally to the ionotropic glutamate receptor superfamily. In this study, two previously cloned goldfish kainate receptor subunits (GFKAR alpha and GFKAR beta) were transiently expressed in human embryonic kidney 293 cells, and their ligand-binding properties and some associated structural features were characterized, resulting in the following findings. 1) Both subunits form homomeric receptors with high affinity for [3H]kainate (KD = 16 and 31 nM, respectively) and L-glutamate (KD = 2 and 40 microM, respectively). 2) A deletion mutant lacking the originally proposed second-transmembrane domain was efficiently expressed and retains the overall ligand-binding properties of wild-type GFKAR alpha, strongly indicating that this region is not a transmembrane domain. 3) Mutations of Q12, A53, and Y54 of GFKAR beta indicate that these three residues are important for ligand binding (particularly L-glutamate), which is consistent with the sequence homology to bacterial periplasmic binding proteins. 4) Mutation of the three extracellular cysteine residues of GFKAR beta indicated that the two conserved cysteine residues (C305 and C385), located between two transmembrane segments, form a solvent-accessible disulfide bond. Analysis of [3H]kainate binding to wild-type and cysteine mutations of GFKAR beta indicate that in the absence of the disulfide bond, the affinity for kainate is increased 3-fold. These data lend further evidence in support of a model of glutamate receptor topology with three transmembrane segments and reveal several general structural features of the extracellular ligand-binding domain of the kainate receptors. These results are consistent with the notion that the ligand-binding domain has close structural similarities to bacterial periplasmic binding proteins.

Amino Acid Sequence↗
PubMed1986

Rat liver iodothyronine monodeiodinase. Evaluation of the iodothyronine ligand-binding site.

Ligand binding characteristics of rat liver microsomal type I iodothyronine deiodinase were evaluated by measuring dose-response inhibition and apparent Michaelis-Menten or inhibitor constants of iodothyronine analogues to compete as substrates or inhibitors for the natural substrate L-thyroxine. These data show strong correlations with the binding requirements of hormone analogues to serum thyroxine-binding prealbumin since iodothyronine analogues with a negatively charged side chain, a negative charge or hydrogen bonding function in the 4'-position, tetraiodo ring substitution, and a skewed hormone conformation are structural features shared in common which markedly affect enzyme activity and protein binding affinity. 3,3',5'-Triiodo-L-thyronine is the most potent natural substrate (IC50 = 0.3 microM) and tetraiodothyroacetic acid is the most potent inhibitor (IC50 = 0.2 microM). Both thyroxine (T4)-5'- and T4-5-deiodination pathways are inhibited by these potent analogues, providing further evidence for a single enzyme catalyzing the rat liver microsomal deiodination reactions. These data also show that L-hormone analogues are preferentially deiodinated via the T4-5'-deiodination pathway, whereas D-analogues produce products via the T4-5-deiodination pathway. The thyroxine-binding prealbumin complex was used to model the interaction of thyroid hormones with the deiodinase active site. Computer graphic modeling of the prealbumin complex showed that only those analogues which are potent deiodinase inhibitors or substrates can be accommodated in the hormone binding site. This model suggests the design of functionally specific ligands which can modulate peripheral thyroid hormone metabolism and act as antithyroidal drugs.

Animals↗
PubMed1996

Dimerization of the extracellular domain of granuloycte-colony stimulating factor receptor by ligand binding: a monovalent ligand induces 2:2 complexes.

Granulocyte-colony stimulating factor (G-CSF) binds to a specific cell surface receptor and induces signals for growth and differentiation in cells of granulocyte hematopoietic lineage. In order to understand how G-CSF binding initiates signals into these cells, we have studied its interactions with the entire extracellular domain of the receptor (sG-CSFR). The sG-CSFR was purified from CHO cell conditioned media with a G-CSF affinity column, resting in a preparation fully competent for ligand binding. However, when sG-CSFR was purified by conventional means, i.e., without affinity chromatography, only about half was competent. Therefore, all studies were carried out using affinity-purified material. The sG-CSFR exhibited a weak self-association into a dimer with a dissociation constant of 200microM in the absence of G-CSF. Addition of G-CSF dimerizes the receptor, with a preferred stoichiometry of 2 G-CSF molecules plus 2 receptors. Unexpectedly, receptor-receptor interactions rather than through two receptors binding to the same G-CSF molecule; i.e., G-CSF is a monovalent ligand. G-CSF binding to the receptor monomer occurs with high affinity. The binding of G-CSF also enhances the receptor-receptor dimerization; when G-CSF is bound to both receptors, dimerization is enhanced 2000-fold, while the interaction of a 1:1 receptor-ligand complex with a second ligand-free receptor is enhanced 80-fold. Thus, the mechanism of receptor dimerization is fundamentally different than that of related cytokine receptors such as growth hormone and erythropoietin receptors. Circular dichroic spectra showed a small but significant conformational change of receptor upon binding G-CSF. This is consistent with the idea that G-CSF binding alters the conformation of the receptor, resulting in an increase in receptor-receptor interactions.

Animals↗
PubMed1997

Transcriptional repression of the alpha-subunit gene by androgen receptor occurs independently of DNA binding but requires the DNA-binding and ligand-binding domains of the receptor.

The pituitary glycoprotein hormones LH and FSH regulate the reproductive cycle and are sensitive to feedback by gonadal steroids. The common alpha-subunit shared by these hormones is transcriptionally repressed by androgen receptor (AR) in the presence of its ligand dihydrotestosterone. This identifies at least one mechanism that contributes to AR-dependent suppression of gonadotropin synthesis. Repression of alpha-subunit transcription by AR requires only the sequences within the first 480 bp of the promoter. While this region contains a high-affinity binding site for AR, this element does not mediate the suppressive effects of androgens. Instead, two other elements within the promoter-regulatory region (alpha-basal element and cAMP-regulatory element), which are important for expression of the alpha-subunit gene in gonadotropes, mediate the effects of AR. This suggests that AR inhibits activity of the alpha-subunit promoter by interfering with the transcriptional properties of the proteins that bind to alpha-basal element and the cAMP-regulatory elements. Furthermore, transfection analysis of various mutant ARs identified both the DNA-binding and ligand-binding domains of the receptor as critical for repression. Comparisons with the MMTV promoter revealed distinct structural requirements that underlie the transactivation and transrepression properties of AR.

Animals↗
PubMed1986

Carbodiimide-mediated conjugation of hippuric acid to concanavalin A: retention of ligand binding and hemagglutinating activities.

Conditions for conjugating small molecules with reactive carboxyl groups to concanavalin A (ConA), with retention of biological activity of the lectin, are described. Hippuric acid was conjugated to reactive amino groups on ConA with N-ethyl-N'-(dimethylaminopropyl) carbodiimide under conditions in which neither inter- nor intra-molecular cross-linking was detectable. These same conditions provided for the loading of variable amounts of hippurate as a function of reaction time; conjugates were synthesized with 9.6 mol hippurate.mol ConA-1. Conjugation in the presence of 0.1 M phosphate provided a condition for limiting the extent of coupling; only amide linkages were formed. These conjugates retained both the ability to bind ligands and the hemagglutination titer of the native lectin.

Carbodiimides↗