Interaction of the D-2short dopamine receptor with G proteins: analysis of receptor G protein selectivity

The human D-2short (D-2S) dopamine receptor has been expressed together with the G proteins Gi2 and Go in insect cells using the baculovirus system. Levels of receptor were determined using [H-3]spiperone binding. Levels of G protein heterotrimer were determined using quantitative Western blot and using [S-35]GTPgammaS saturation binding experiments. Levels of the receptor and G protein and the receptor/G protein ratio were similar in the two preparations. Stimulation of [S-35]GTPgammaS binding by a range of agonists occurred with higher relative efficacy and in some cases higher potency in the preparation expressing Go, indicating that interaction of the D-2S receptor is more efficient with this G protein. The effects of various G protein-selective agents on 10,11-dihydroxy-N-n-propylnorapomorphine ([H-3]NPA) binding were used to examine the receptor/G protein complex in the two preparations. Suramin inhibited [H-3]NPA binding with slightly higher potency in the Gi2 preparation, whereas GppNHp inhibited [H-3]NPA binding with greater potency (similar to6-fold) in the Go preparation. This may imply that the G protein is more readily activated in the D-2S/Go preparation. [H-3]Spiperone binding occurred with an increased B-max in the presence of suramin in the Go preparation but not in the Gi2 preparation, suggesting a higher affinity interaction between the free receptor and this G protein. It is concluded that the higher efficiency activation of Go by the D-2S receptor may be a function of higher affinity receptor/G protein interaction as well as a greater ability to activate the G protein. (C) 2003 Elsevier Science Inc. All rights reserved.

Inverse agonism of the antipsychotic drugs at the D-2 dopamine receptor

The antipsychotic drugs had been assumed to act as antagonists at D-2 dopamine receptors but recently these drugs have been shown to possess inverse agonist properties at this receptor. Inverse agonism may be demonstrated from the ability of these drugs to potentiate forskolin-stimulated cAMP accumulation or to suppress agonist-independent [S-35]GTPgammaS binding. The antipsychotic drugs tested generally appear as full inverse agonists in these assays regardless of chemical or therapeutic class. The mechanism of inverse agonism of the antipsychotic drugs is still unclear but may involve stabilisation of the ground state of the D-2 receptor. (C) 2003 Elsevier Science B.V All rights reserved.

A genomewide survey of developmentally relevant genes in Ciona intestinalis - III. Genes for Fox, ETS, nuclear receptors and NF kappa B

A survey against the draft genome sequence and the cDNA/EST database of Ciona intestinalis identified a number of genes encoding transcription factors regulating a variety of processes including development. In the present study, we describe almost complete sets of genes for Fox, ETS-domain transcription factors, nuclear receptors, and NFkappaB as well as other factors regulating NFkappaB activity, with their phylogenetic nature. Vertebrate Fox transcription factors are currently delineated into 17 subfamilies: FoxA to FoxQ. The present survey yielded 29 genes of this family in the Ciona genome, 24 of which were Ciona orthologues of known Fox genes. In addition, we found 15 ETS aenes, 17 nuclear receptor genes, and several NFkappaB signaling pathway genes in the Ciona genome. The number of Ciona genes in each family is much smaller than that of vertebrates, which represents a simplified feature of the ascidian genome. For example, humans have two NFkappaB genes, three Rel genes, and five NFAT genes, while Ciona has one gene for each family. The Ciona genome also contains smaller numbers of genes for the NFkappaB regulatory system, i.e. after the split of ascidians/vertebrates, vertebrates evolved a more complex NFkappaB system. The present results therefore provide molecular information for the investigation of complex developmental processes, and an insight into chordate evolution.

Supramolecular aggregates between carboxylate anions and an octaaza macrocyclic receptor

The 28-membered octaazamacrocycle Me-2[28]py(2)N(6) was used as a receptor for the molecular recognition of aromatic and aliphatic carboxylate substrates. The receptor-substrate binding behaviour of (H6Me2[28]py(2)N(6))(6+) with an aliphatic (-O2C(CH2)(n)CO2-, n=0 to 4) and an aromatic (phthalate, isophthalate, terephthalate, 4,4'-dibenzoate, benzoate, 3- and 4-nitrobenzoate) series of carboxylate anions was evaluated by H-1 NMR spectroscopy (carried out in DMSO-d(6) at 300 K). Two association constants were found for most of the studied cases, except for 3- and 4-nitrobenzoate for which only K-1 was determined. For oxalate, malonate, benzoate and dibenzoate anions only the beta(2) constants could be obtained. The values of the first association constant cover a range from 2.86 to 3.69 (log units), and the second stepwise constant from 2.15 to 2.89 (also in log units). No special selectivity was found but the highest values were determined for adipate and the lowest for the monoprotic 3- and 4-nitrobenzoates. Single crystal X-ray structures of H6Me2[28]py(2)N(6)(6+) with terephthalate, 1, and 4,4'-dibenzoate (2) were determined showing supramolecular entities with general formula (H6Me2[28]py(2)N(6)).(substrate)(2)(PF6)(2).4H(2)O. These anions are the building blocks of an extensive 3-D network of hydrogen bonds.

Evaluation of the binding ability of a novel dioxatetraazamacrocyclic receptor that contains two phenanthroline units: selective uptake of carboxylate anions

The novel dioxatetraaza macrocycle [26]phen(2)N(4)O(2), which incorporates two phenanthroline units, has been synthesized, and its acid-base behavior has been evaluated by potentiometric and H-1 NMR methods. Six protonation constants were determined, and the protonation sequence was established by NMR. The location of the fifth proton on the phen nitrogen was confirmed by X-ray determinations of the crystal structures of the receptor as bromide and chloride salts. The two compounds have the general molecular formula {(H-5[26]phen(2)N(4)O(2))X-n(H2O)(5-n)}X(n-1)(.)mH(2)O, where X = Cl, n = 3, and m = 6 or X = Br, n = 4, and m = 5.5. In the solid state, the (H-5[26]phen(2)N(4)O(2))(5+) cation adopts a "horseshoe" topology with sufficient room to encapsulate three or four halogen anions through the several N-(HX)-X-... hydrogen-bonding interactions. Two supermolecules {(H-5[26]phen(2)N(4)O(2))X-n(H2O)(5-n)}((5-n)+) form an interpenetrating dimeric species, which was also found by ESI mass spectrum. Binding studies of the protonated macrocycle with aliphatic (ox(2-), mal(2-), suc(2-), cit(3-), cta(3-)) and aromatic (bzc(-), naphc(-), anthc(-), pyrc(-), ph(2-), iph(2-), tph(2-), btc(3-)) anions were determined in water by potentiometric methods. These studies were complemented by H-1 NMR titrations in D2O of the receptor with selected anions. The H-i[26]phen(2)N(4)O(2)(i+) receptor can selectively uptake highly charged or extended aromatic carboxylate anions, such as btc(3-) and pyrc(-), in the pH ranges of 4.0-8.5 and < 4.0, respectively, from aqueous solution that contain the remaining anions as pollutants or contaminants. To obtain further insight into these structural and experimental findings, molecular dynamics (MD) simulations were carried out in water solution.

Carboxylate anions binding and sensing by a novel tetraazamacrocycle containing ferrocene as receptor

A tetraazamacrocycle containing ferrocene moieties has been synthesized and characterized. The tetraprotonated form of this compound was evaluated as a receptor (R) for anion recognition of several substrates (S), Cl-, PF6-, HSO4-, H2PO4- and carboxylates, such as p-nitrobenzoate (p-nbz(-)), phthalate (ph(2-)), isophthalate (iph(2-)) and dipicolinate (dipic(2-)). H-1 NMR titrations in CD3OD indicated that this receptor is not suitable for recognizing HSO4- and H2PO4-, but weakly binds p-nbz(-), and strongly interacts with ph(2-), dipic(2-), and iph(2-) anions forming 1 : 2 assembled species. The largest beta(2) binding constant was determined for ph(2-), followed by dipic(2-) and finally iph(2-). The effect of the anionic substrates on the electron-transfer process of the ferrocene units of R was evaluated using cyclic voltammetry (CV) and square wave voltammetry (SWV) in methanol solution and 0.1 mol dm(-3) (CH3)(4)NCl as the supporting electrolyte. Titrations of the receptor were undertaken by addition of anion solutions in their tetrabutylammonium or tetramethylammonium forms. The protonated ligand exhibits a reversible voltammogram, which shifts cathodically in the presence of the substrates. The data revealed kinetic constraints in the formation of the receptor/substrate entity for dipic(2-), ph(2-) and iph(2-) anions, but not for p-nbz(-). In spite of the slow kinetics of assembled species formation with the ph(2-) substrate, this anion provides the largest redox-response when the supramolecular entity is formed, followed by dipic(2-), iph(2-) and finally p-nbz(-) anions. This trend is in agreement with the H-1 NMR results and the values of the binding constants. Single crystal X-ray structures of the receptor with PF6-, ph(2-), iph(2-) and p-nbz(-) were carried out and showed that supermolecules with a RS2 stoichiometry are formed with the first three anions, but RS4 with p-nbz(-). In all cases the binding occurs outside the macrocyclic cavity via N-H center dot center dot center dot O=C hydrogen bonds for carboxylate anions and N - H center dot center dot center dot F hydrogen bonds for the PF6- anion, which is in agreement with the solution results. The macrocyclic framework adopts different conformations in order to interact with each substrate having Fe center dot center dot center dot Fe intramolecular distances ranging from 10.125(14) to 12.783(15) angstrom.

Synthesis and evaluation of a solid supported molecular tweezer type receptor for cholesterol

A new macroporous stationary phase bearing 'tweezer' receptors that exhibit specificity for cholesterol has been constructed from rigid multifunctional vinylic monomers derived from 3,5-dibromobenzoic acid, propargyl alcohol and cholesterol. The synthesis of the novel tweezer monomer that contains two cholesterol receptor arms using palladium mediated Sonogashira methodologies and carbonate couplings is reported. The subsequent co-polymerisation of this tweezer monomer with a range of cross-linking agents via a 'pseudo' molecular imprinting approach afforded a diverse set of macroporous materials. The selectivity and efficacy of these materials for cholesterol binding was assessed using a chromatographic screening process. The optimum macroporous stationary phase material composition was subsequently used to construct monolithic solid phase extraction columns for use in the selective extraction of cholesterol from multi-component mixtures of structurally related steroids.

Facile bisurethane supramolecular polymers containing flexible alicyclic receptor units

This paper details the synthesis, characterisation and physical analyses of a series of hydrogen bonded urethane supramolecular polymer systems that are created by a facile one-step synthesis from inexpensive and commercially available starting materials. We report the synthesis and characterisation of a series of low molecular weight bisurethanes (<650 a.m.u.) that exhibit physical properties in the bulk that are characteristic of polyurethane materials possessing far higher molecular weight. The physical characteristics of these low molecular weight bisurethanes were investigated by using temperature-dependent rheological analysis and viscometry and the nature in which these compounds assembled was assessed using IR and NMR spectroscopies. These studies reveal that these simple bisurethanes self-assemble via hydrogen bonding interactions.

Genistein affects the expression of genes involved in blood pressure regulation and angiogenesis in primary human endothelial cells

Background: Several lines of evidence suggest that the dietary isoflavone genistein (Gen) has beneficial effects with regard to cardiovascular disease and in particular on aspects related to blood pressure and angiogenesis. The biological action of Gen may be, at Least in part, attributed to its ability to affect cell signalling and response. However, so far, most of the molecular mechanisms underlying the activity of Gen in the endothelium are unknown. Methods and results: To examine the transcriptional response to 2.5 mu M Gen on primary human endothelial cells (HUVEC), we applied cDNA array technology both under baseline condition and after treatment with the pro-atherogenic stimulus, copper-oxidized LDL. The alteration of the expression patterns of individual transcripts was substantiated using either RT-PCR or Northern blotting. Gen significantly affected the expression of genes encoding for proteins centrally involved in the vascular tone such as endothelin-converting enzyme-1, endothetin-2, estrogen related receptor a and atria[ natriuretic peptide receptor A precursor. Furthermore, Gen countered the effect of oxLDL on mRNA levels encoding for vascular endothelial growth factor receptor 165, types 1 and 2. Conclusions: Our data indicate that physiologically achievable levels of Gen change the expression of mRNA encoding for proteins involved in the control of blood pressure under baseline conditions and reduce the angiogenic response to oxLDL in the endothelium. (c) 2005 Elsevier B.V. All rights reserved.

Investigation of cooperativity in the binding of ligands to the D-2 dopamine receptor

The D 2 dopamine receptor exists as dimers or as higher-order oligomers, as determined from data from physical experiments. In this study, we sought evidence that this oligomerization leads to cooperativity by examining the binding of three radioligands ([H-3] nemonapride, [H-3] raclopride, and [H-3] spiperone) to D 2 dopamine receptors expressed in membranes of Sf9 cells. In saturation binding experiments, the three radioligands exhibited different B-max values, and the B-max values could be altered by the addition of sodium ions to assays. Despite labeling different numbers of sites, the different ligands were able to achieve full inhibition in competition experiments. Some ligand pairs also exhibited complex inhibition curves in these experiments. In radioligand dissociation experiments, the rate of dissociation of [H-3] nemonapride or [H-3] spiperone depended on the sodium ion concentration but was independent of the competing ligand. Although some of the data in this study are consistent with the behavior of a cooperative oligomeric receptor, not all of the data are in agreement with this model. It may, therefore, be necessary to consider more complex models for the behavior of this receptor.

Large-scale functional expression of WT and truncated human adenosine A(2A) receptor in Pichia pastoris bioreactor cultures

Background: The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 similar to 15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A(2A)R) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures. Results: Bioreactor cultures yielded an approximately five times increase in cell density (OD600 similar to 75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A(2A)R, and therefore more suitable for further functional and structural studies. Conclusion: Large-scale expression of the A(2A)R in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.

Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor beta (TGF beta) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24-25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n = 8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnP,H-R), LH beta subunit, FSH beta subunit and GAPDH. Levels of mRNA for inhibin/activin beta A and beta B subunits, inhibin alpha subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (< 2 amol/reaction). Significant changes in expression (P < 0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r=0.77; P < 0.001) and betaglycan (r=0.45; P < 0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P < 0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r=0.33; P=0.06) and ActRIIA (r=0.34; P=0.06) mRNA levels. Expression of mRNAs encoding LH beta and FSH beta subunit were both lowest (P < 0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.