Purification and recognition of recombinant mouse P2X1 receptors expressed in a baculovirus system

Chen, Liping; Hardwick, James P.; McPhie, Peter; Sitkovsky, Michail V.

doi:10.1002/1098-2299(20000901)51:1<7::aid-ddr2>3.0.co;2-w

Cited by 3 publications

(3 citation statements)

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“…), look at agonist binding (Chen et al . ), and in single particle transmission electron microscopy studies shown marked conformational changes in the receptor on agonist binding (Roberts et al . ).…”

Section: Discussionmentioning

confidence: 99%

Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

Roberts

Bottrill

Mistry

et al. 2012

Journal of Neurochemistry

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Recombinant FlagHis6 tagged Human P2X1 receptors expressed in HEK293 cells were purified, digested with trypsin and analysed by mass spectroscopy (96% coverage following de-glycosylation and reduction). The receptor was basally phosphorylated at residues S387, S388 and T389 in the carboxyl terminus, a triple alanine mutant of these residues had a modest ∼ 25% increase in current amplitude and recovery from desensitization. Chemical modification showed that intracellular lysine residues close to the transmembrane domains and the membrane stabilization motif are accessible to the aqueous environment. The membrane-impermeant cross-linking reagent 3,3′-Dithiobis (sulfosuccinimidylpropionate) (DTSSP) reduced agonist binding and P2X1 receptor currents by > 90%, and modified lysine residues were identified by mass spectroscopy. Mutation to remove reactive lysine residues around the ATP-binding pocket had no effect on inhibtion of agonist evoked currents following DTSSP. However, agonist evoked currents were ∼ 10-fold higher than for wild type following DTSSP treatment for mutants K199R, K221R and K199R-K221R. These mutations remove reactive residues distant from the agonist binding pocket that are close enough to cross-link adjacent subunits. These results suggest that conformational change in the P2X1 receptor is required for co-ordination of ATP action.

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Section: Discussionmentioning

confidence: 99%

Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

Roberts

Bottrill

Mistry

et al. 2012

Journal of Neurochemistry

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“…We have prepared several amine functionalized congeners, that may potentially be conjugated to fluorescent probes for receptor characterization, or immobilized on a solid support for isolation of the receptor by affinity chromatography. Among P2X receptors, only the P2X 1 subtype has so far been isolated in this manner (35). Effects of tyrosyl derivatives on P2X 7 Table 1 Antagonistic Effects of Tyrosine Derivatives on Function of Human P2X …”

Section: Discussionmentioning

confidence: 99%

“…10H), 7.21 (d, J ) 8.5 Hz, 2H), 7.00 (d, J ) 7.4 Hz, 2H), 6.60-6.46 (m, 1H), 6.44-6.34 d, J ) 15.7 Hz, 1H), 5.40-5.06 (b, 1H), 4.00-2.80 (m, 10H). FAB-MS: m/z (relative intensity) 669.2 (M + 1, 20), 434.2(35), 131.1 (100). Compound 10 (R f ) 0.56, CH 3 OH/CHCl 3 ) 5: 95) was shown to be pure by TLC.…”

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Functionalized Congeners of Tyrosine-Based P2X₇ Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization

et al. 2002

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Chemically funtionalized analogues of antagonists of the P2X 7 receptor, an ATP-gated cation channel, were synthesized as tools for biophysical studies of the receptor. These functionalized congeners were intended for use in chemical conjugation with retention of biological potency. The antagonists were L-tyrosine derivatives, related to [N-benzyloxycarbonyl-O-(4-arylsulfonyl)-Ltyrosyl]benzoylpiperazine (such as MRS2409, 2). The analogues were demonstrated to be antagonists in an assay of human P2X 7 receptor function, consisting of inhibition of ATP-induced K + efflux in HEK293 cells expressing the recombinant receptor. The analogues were of the general structure R 1 -Tyr(OR 2 )-piperazinyl-R 3 , in which three positions (R 1 -R 3 ) were systematically varied in structure through introduction of chemically reactive groups. Each of the three positions was designed to incorporate a 3-or 4-nitrophenyl group. The nitro groups were reduced using NaBH 4 -copper(II) acetylacetonate to amines, which were either converted to the isothiocyanate groups, as potential affinity labels for the receptor, or acylated, as models for conjugation. An alternate route to N α -3-aminobenzyloxycarbonyl functionalization was devised.The various positions of functionalization were compared for effects on biological potency, and the R 2 and R 3 positions were found to be most amenable to derivatization with retention of high potency. Four dimeric permutations of the antagonists were synthesized by coupling each of the isothiocyanate derivatives to either the precursor amine or to other amine congeners. Only dimers linked at the R 2 -position were potent antagonists. In concentration-response studies, two derivatives, a 3-nitrobenzyloxycarbonyl derivative 18 and a 4-nitrotoluenesulfonate 26b, displayed IC 50 values of roughly 100 nM as antagonists of P2X 7 receptor-mediated K + flux.

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Pharmacochemistry of the platelet purinergic receptors

Jacobson

Deflorian

Mishra

et al. 2011

Purinergic Signalling

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Platelets contain at least five purinergic G protein-coupled receptors, e.g., the pro-aggregatory P2Y 1 and P2Y 12 receptors, a P2Y 14 receptor (GPR105) of unknown function, and anti-aggregatory A 2A and A 2B adenosine receptor (ARs), in addition to the ligand-gated P2X1 ion channel. Probing the structure-activity relationships (SARs) of the P2X and P2Y receptors for extracellular nucleotides has resulted in numerous new agonist and antagonist ligands. Selective agents derived from known ligands and novel chemotypes can be used to help define the subtypes pharmacologically. Some of these agents have entered into clinical trials in spite of the challenges of drug development for these classes of receptors. The functional architecture of P2 receptors was extensively explored using mutagenesis and molecular modeling, which are useful tools in drug discovery. In general, novel drug delivery methods, prodrug approaches, allosteric modulation, and biased agonism would be desirable to overcome side effects that tend to occur even with receptor subtype-selective ligands. Detailed SAR analyses have been constructed for nucleotide and nonnucleotide ligands at the P2Y 1 , P2Y 12 , and P2Y 14 receptors. The thienopyridine antithrombotic drugs Clopidogrel and Prasugrel require enzymatic pre-activation in vivo and react irreversibly with the P2Y 12 receptor. There is much pharmaceutical development activity aimed at identifying reversible P2Y 12 receptor antagonists. The screening of chemically diverse compound libraries has identified novel chemotypes that act as competitive, non-nucleotide antagonists of the P2Y 1 receptor or the P2Y 12 receptor, and antithrombotic properties of the structurally optimized analogues were demonstrated. In silico screening at the A 2A AR has identified antagonist molecules having novel chemotypes. Fluorescent and other reporter groups incorporated into ligands can enable new technology for receptor assays and imaging. The A 2A agonist CGS21680 and the P2Y 1 receptor antagonist MRS2500 were derivatized for covalent attachment to polyamidoamine dendrimeric carriers of MW 20,000, and the resulting multivalent conjugates inhibited ADP-promoted platelet aggregation. In conclusion, a wide range of new pharmacological tools is available to control platelet function by interacting with cell surface purine receptors.

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Purification and recognition of recombinant mouse P2X1 receptors expressed in a baculovirus system

Cited by 3 publications

References 45 publications

Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

Functionalized Congeners of Tyrosine-Based P2X₇ Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization

Pharmacochemistry of the platelet purinergic receptors

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Purification and recognition of recombinant mouse P2X1 receptors expressed in a baculovirus system

Cited by 3 publications

References 45 publications

Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

Functionalized Congeners of Tyrosine-Based P2X7 Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization

Pharmacochemistry of the platelet purinergic receptors

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Product

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Functionalized Congeners of Tyrosine-Based P2X₇ Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization