Bridging the Gap Between Structural Bioinformatics and Receptor Research: The Membrane-embedded, Ligand-gated, P2X Glycoprotein Receptor

Mager, Peter P.; Weber, Anje; Illéš, Peter

doi:10.2174/1568026043387197

Cited by 19 publications

(56 citation statements)

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“…In view of the presence of consensus phosphorylation sites in the extracellular loop of the P2X 3 receptor (Mager et al, 2004), we hypothesized about the functional significance of these sites as potential targets of ecto-PKC-induced phosphorylation. Initially, two selective PKC activators were superfused with a protocol also used for the application of nucleotides.…”

Section: Resultsmentioning

confidence: 99%

“…To identify the functionally relevant PKC phosphorylation site(s) at the hP2X 3 receptor, Ser and Thr residues situated within five of the seven consensus sites (see Fig. 7A) were consecutively mutated to the neutral amino acid Ala. An intracellular PKC site highly conserved in all P2X subunits is located N terminally (PKC1, 12-14), whereas additional sites are extracellularly localized (PKC2,(134)(135)(136)PKC3,(178)(179)(180)PKC4,(196)(197)(198); and PKC6, 269 -271) (Mager et al, 2004). Furthermore, two Ser residues (S110, S267) that are present in the human but not in the rat P2X 3 receptor were also mutated to Ala. None of the mutations abolished the membrane expression of the hP2X 3 receptor in HEK293 cells or its sensitivity to extracellular nucleotides, because the application of ␣,␤-meATP (3 M) caused reproducible current amplitudes in all cases (for the second current response in the usual series, see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Naturally occurring attachment sites were predicted by the ScanPROSITE regular expression search via the interface of PROSITE. Additional details of the molecular modeling techniques have been described previously (Mager et al, 2004).…”

Section: Methodsmentioning

confidence: 99%

“…It has been hypothesized that the completely conserved PKC phosphorylation site of the P2X 3 signaling peptide within the subsequence 1-44 is essential to pass the endoplasmatic reticulum or the plasma membrane (Mager et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

“…The aim of the present study was to find out whether ectoPKCs are able to modulate human (h) recombinant P2X 3 receptor channels by phosphorylating consensus PKC sites at the extracellular loop of this receptor (Mager et al, 2004). hP2X 3 receptors transfected into human embryonic kidney HEK293 cells were superfused with relatively low concentrations of UTP, which fail to activate the receptor but supply terminal phosphate residues for phosphorylation reactions.…”

Section: Introductionmentioning

confidence: 99%

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Regulation of Human Recombinant P2X₃Receptors by Ecto-Protein Kinase C

et al. 2005

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The whole-cell patch-clamp technique was used to record current responses to nucleotides and nucleosides in human embryonic kidney HEK293 cells transfected with the human purinergic P2X 3 receptor. When guanosine 5Ј-O-(3-thiodiphosphate) was included into the pipette solution, UTP at concentrations that did not alter the holding current facilitated the ␣,␤-methylene ATP (␣,␤-meATP)-induced current. ATP and GTP, but not UDP or uridine, had an effect similar to that of UTP. Compounds known to activate protein kinase C (PKC) acted like the nucleoside triphosphates investigated, whereas various PKC inhibitors invariably reduced the effects of both PKC activators and UTP. The substitution by Ala of Ser/Thr residues situated within PKC consensus sites of the P2X 3 receptor ectodomain either abolished (PKC2 and PKC3; T134A, S178A) or did not alter (PKC4 and PKC6; T196A, S269A) the UTP-induced potentiation of the ␣,␤-meATP current. Both the blockade of ecto-protein kinase C activity and the substitution of Thr-134 or Ser-178 by Ala depressed the maximum of the concentration-response curve for ␣,␤-meATP without altering the EC 50 values. Molecular simulation of the P2X 3 receptor structure indicated no overlap between assumed nucleotide binding domains and the relevant phosphorylation sites PKC2 and PKC3. ␣,␤-meATP-induced currents through native homomeric P2X 3 receptors of rat dorsal root ganglia were also facilitated by UTP. In conclusion, it is suggested that low concentrations of endogenous nucleotides in the extracellular space may prime the sensitivity of P2X 3 receptors toward the effect of subsequently applied (released) higher agonistic concentrations. The priming effect of nucleotides might be attributable to a phosphorylation of PKC sites at the ectodomain of P2X 3 receptors.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Regulation of Human Recombinant P2X₃Receptors by Ecto-Protein Kinase C

et al. 2005

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Interaction of P2 purinergic receptors with cellular macromolecules

Köles

Gerevich

Oliveira

et al. 2007

Naunyn-Schmied Arch Pharmacol

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Ionotropic P2X and metabotropic P2Y receptors interact with a number of macromolecules in the cell membrane which may contribute to their functional plasticity. P2X receptors are homomeric or heteromeric assemblies of three subunits. P2Y receptors may form oligomeric complexes either with the same or with other P2Y receptor types. Although the signalling mechanism of P2X receptor channels is fast (within milliseconds) and relatively simple, by originating from the opening of an ion channel permeable to mono- and divalent cations, various macromolecules may modify the trafficking of these receptors to and from the cell membrane, as well as their activation and desensitization kinetics, and the possible opening of membrane pores induced by long-lasting exposure to agonists. P2X and Cys-loop receptors may physically interact with each other, resulting in mutual current occlusion. Heteromeric P2Y receptors may, via G(s), G(q/11) or G(i/o) protein-coupling and activation of the respective transduction mechanisms, mediate responses in the range of a few seconds. However, P2Y receptors may also interact with the signalling cascade of, e.g. receptor tyrosine kinases, and thereby mediate responses on a much slower time scale (within hours to days). In addition, P2Y receptors may interact with small, homomeric G proteins, integrins, and PDZ proteins. Eventually, P2Y receptors may cross-talk via Galpha-dependent signalling with other G protein-coupled receptors and via Gbetagamma (or indirectly Galpha)-dependent signalling with various ion channels. Thus, the activation of P2X and P2Y receptors by extracellular adenosine triphosphate/adenosine diphosphate or uridine triphosphate/uridine diphosphate may trigger specific chains of events which interact at the level of the individual elements both with each other and with the transduction mechanisms of other receptors, creating a huge diversity of the possible effects.

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The Cdk5 Kinase Downregulates ATP-Gated Ionotropic P2X3 Receptor Function Via Serine Phosphorylation

et al. 2009

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Cdk5 is an endogenous kinase activated by the neuronal-specific protein p35 and implicated in multiple neuronal functions, including modulation of certain pain responses. We investigated whether Cdk5 could regulate ATP-gated P2X(3) receptors that are members of the family of membrane proteins expressed by sensory neurons to transduce nociception in baseline and chronic pain. To study the potential P2X(3) receptor modulation by Cdk5, we co-transfected rat P2X(3) receptors and Cdk5 into HEK cells and observed increased P2X(3) receptor serine phosphorylation together with downregulation of receptor currents only when these genes were transfected together with the gene of the Cdk5 activator p35. The changes in receptor responses were limited to depressed current amplitude as desensitization and recovery were not altered. Transfection of p35 with P2X(3) similarly downregulated receptor responses, suggesting that this phenomenon could be observed even with constitutive Cdk5. The present data indicate a novel target to express the action of Cdk5 on membrane proteins involved in pain perception.

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Bridging the Gap Between Structural Bioinformatics and Receptor Research: The Membrane-embedded, Ligand-gated, P2X Glycoprotein Receptor

Abstract: The geometry optimized P2X3 receptor subunit is freely available for academic researchers on e-mail request (PDB format).

Cited by 19 publications

References 0 publications

Regulation of Human Recombinant P2X₃Receptors by Ecto-Protein Kinase C

Regulation of Human Recombinant P2X₃Receptors by Ecto-Protein Kinase C

Interaction of P2 purinergic receptors with cellular macromolecules

The Cdk5 Kinase Downregulates ATP-Gated Ionotropic P2X3 Receptor Function Via Serine Phosphorylation

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Bridging the Gap Between Structural Bioinformatics and Receptor Research: The Membrane-embedded, Ligand-gated, P2X Glycoprotein Receptor

Abstract: The geometry optimized P2X3 receptor subunit is freely available for academic researchers on e-mail request (PDB format).

Cited by 19 publications

References 0 publications

Regulation of Human Recombinant P2X3Receptors by Ecto-Protein Kinase C

Regulation of Human Recombinant P2X3Receptors by Ecto-Protein Kinase C

Interaction of P2 purinergic receptors with cellular macromolecules

The Cdk5 Kinase Downregulates ATP-Gated Ionotropic P2X3 Receptor Function Via Serine Phosphorylation

Contact Info

Product

Resources

About

Regulation of Human Recombinant P2X₃Receptors by Ecto-Protein Kinase C

Regulation of Human Recombinant P2X₃Receptors by Ecto-Protein Kinase C