Roberts JA et al. (2012), Mass spectrometry analysis of human P2X1 recept...

XB-ART-46547

J Neurochem 2012 Dec 01;1235:725-35. doi: 10.1111/jnc.12012.

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

Roberts JA , Bottrill AR , Mistry S , Evans RJ .

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Recombinant FlagHis(6) 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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Species referenced: Xenopus laevis
Genes referenced: dsp p2rx1 prss1 sst.1 tbx2

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	Fig. 1. Purification and mass spectrometry analysis of the Human P2X1 receptor. (a) Anti-P2X1 receptor antibody western blot analysis of 3X FLAG peptide eluted fractions from anti-FLAG agarose beads. FT, flow through; W1–2, washes; E1–E10, FLAG peptide eluted fractions; E11–13, 0.1 M Glycine pH 3.5 eluted fractions. P2X1 protein of the correct mass is observed in fractions E2–E8. (b) InstantBlue (Expedeon) stained gel of eluted fractions from anti-FLAG agarose beads (lanes labelled as Fig. 1a). (c) Combined and concentrated fractions were run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with InstantBlue (Expedeon). Clean purified FLAG tagged P2X1 protein can be observed along with PNGase F deglycosylated P2X1 receptor protein and the PNGase F protein (star indicates confirmed by mass spectrometry) (d) Identification of the P2X1 receptor with 26.8 ± 6% percentage coverage on single mass spectrometry runs was achieved with trypsin digest though other enzymes were tested (e.g. chymotrypsin, 11.2 ± 5% coverage, data not shown). Coverage of the P2X1 receptor protein was increased (26.8 ± 6% vs. 37.8 ± 3%) by deglycosylating the receptors with PNGase F. Use of Orbitrap versus Qtrap mass spectrometer increased mass accuracy and peptide identification increasing coverage even further on average to 59.2 ± 2%. (e) Human P2X1 receptor protein sequence showing the total coverage of all observed peptides (26 runs). Transmembrane regions 1 and 2 are highlighted with a bold line. Amino acid residues shown in bold lowercase were not observed on mass spectrometry of the P2X1 receptor protein most probably because their masses were below the limit of detection (∼500 Da) (Table S1). Other areas of predicted low mass were only observed as a result of partial digestion and therefore identified as part of a larger peptide mass. Vertical lines indicate sites for trypsin digestion at arginine and lysine residues.
	Fig. 2. Phosphorylation status of the Human P2X1 receptor. (a) P2X1 receptor protein sequence of the N- and C-termini. Intracellular modifications made by membrane permeable Dithiobis(sulfosuccinimidylpropionate) (DSP) were identified by mass spectrometry and are indicated on the protein sequence. Membrane impermeable 3,3′-Dithiobis (sulfosuccinimidylpropionate) (DTSSP) modifications were only observed on extracellular P2X1 protein residues. DSP modification highlights the lipid/transmembrane boundaries with K27 and K28 modified showing accessibility. In silico analysis of the N-terminal protein sequence reveals residues Y16 and T18 as potential phosphorylation sites (bold type). Mass spectrometry analysis did not show modification at these residues even after enriching for phosphorylated peptides. In silico analysis of the C-terminal protein sequence reveals tyrosine residues Y362, 363 and 370 and serine and threonine residues 386–389 and 398–399 as potential phosphorylation sites (bold type). Phosphorylation was initially observed at residues S387 and S388 and residues S388, T389 and S399 were identified on mass spectrometry runs enriched for phosphorylated peptides (P ). These residues were the only phosphorylated residues observed on mass spectrometry of the P2X1 receptor protein. (b) ATP evoked currents (period indicated by bar) from P2X1 wildtype and mutant receptors (S387A and S388A – SS-AA, S387A, S388A and T389A – SST-AAA). Traces show reproducible response evoked at a 5-minute interval and level of recovery from desensitization at 60 s between ATP applications. (c, d) Time to peak (10–90%) and decay time (100–50%) of currents evoked by 100 μM ATP for wildtype and mutant P2X1 receptors. (e) Recovery from desensitization at 60 s for wildtype and mutant P2X1 receptors. p < 0.05, p < 0.01, *p < 0.001, (n = 4–6).
	Fig. 3. Localization of 3,3′-Dithiobis (sulfosuccinimidylpropionate) (DTSSP) modification of the human P2X1 receptor. (a) P2X1 homology model (based on zP2X4 crystal structure model – Kawate et al. 2009) depicted as a trimeric cartoon. Lysine residues modified DTSSP and identified by mass spectrometry are indicated in red. Lysine residue K70 that was not observed in any mass spectrometry runs is shown in dark grey. Residues observed and not modified by DTSSP in some runs are shown in grey. Residue K68 that was not observed to be modified by DTSSP (11/11) is shown in black. The serine residues 130, 286 (blue spheres) and tyrosine 274 (green spheres) were also modified by DTSSP. (b) Human P2X1 protein sequence with DTSSP modification of the P2X1 receptor marked as observed from mass spectrometry data. Transmembrane regions 1 and 2 are highlighted with a bold line. Residues are coloured as indicated in panel (a). Arrows indicate residues modified by DTSSP, an arrow with a cross show a residue that was not modified by DTSSP, and arrows with a question mark where it remains unknown either because of non-coverage of the protein sequence or modification not detected. Note that all the DTSSP modifications occur in the extracellular region.
	Fig. 4. Effect of 3,3′-Dithiobis (sulfosuccinimidylpropionate) (DTSSP) modification on Human P2X1 receptor function. (a) Application of 100 μM ATP to Xenopus oocytes expressing P2X1 wildtype receptors evoked a large inward current recorded by two electrode voltage clamp. Pre-incubation with 100 μM DTSSP for 30 min almost abolished responses. Inset traces are representative of normalized responses to 100 μM ATP in the presence or absence of 100 μM DTSSP indicating no significant change in the current time course. (b) Pooled electrophysiology data depicting the decrease in channel function post-DTSSP treatment. (c) P2X1 receptor ATP-binding site analysis utilizing uv cross-linked 32P 2Azido ATP (2AzATP) shows a marked reduction in radioactivity of the P2X1 receptor protein band following pre-treatment with 100 μM DTSSP. (d) Pooled densitometry data collected from autoradiography of the DTSSP treated and non-treated 2AzATP radioactive P2X1 receptor bands (n = 4) ***p < 0.001.
	Fig. 5. Site directed mutagenesis of human P2X1 receptor to discover the molecular basis for 3,3′-Dithiobis (sulfosuccinimidylpropionate) (DTSSP) inhibition. (a) Cartoon representation of P2X1 receptor structure highlighting the residues K199 and K221 (blue spheres) positioned on different subunits approximately 12 angstroms apart. The diameter of the circle shown is approximately 24 angstroms. Residues in yellow correspond to positions 196 and 320 in adjacent subunits where introduced cysteine residues form a disulphide bond and inhibit channel activation. Red spheres correspond to lysine residues that when mutated had no effect on DTSSP inhibition of ATP evoked responses. (b) Combining mass spectrometry and crystal structure to map possible cross-linked pairs, multiple mutations were designed to discover the residues responsible for DTSSP inhibition and cross-linking. Residues 70 : 140; 70 : 309; 70 : 286; 140 : 215; 190 : 283; 190 : 286; 199 : 221 and 322 : 322 are all within 12 angstroms and on separate P2X1 subunits and therefore possible candidates for causing functional inhibition and dimer/trimer formation with DTSSP. Only double mutant K199R:K221R showed a significant reduction of DTSSP inhibition also reflected in the single mutants K199R and K221R (n = 3–25). No mutations were observed to disrupt the DTSSP formation of dimers/trimers on western blot (data not shown). Inset example trace data for human P2X1 wildtype and P2X1 double mutant K199R: K221R in the presence and absence of 100 μM DTSSP p < 0.01, *p < 0.001.

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