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Molecular mechanisms of inhibition of P2X7 receptor by novel antagonists and activation by ADP-ribosylating ectoenzymes

ReferenceBB/C517292/2
Principal Investigator / Supervisor Professor Annmarie Surprenant
Co-Investigators /
Co-Supervisors
Institution The University of Manchester
DepartmentLife Sciences
Funding typeResearch
Value (£) 87,967
StatusCompleted
TypeResearch Grant
Start date 01/04/2007
End date 28/02/2009
Duration23 months

Abstract

The P2X7 receptor ion channel is an integral membrane protein which has little or no precedent in terms of the molecular and cellular basis of its action. These ion channels are gated by extracellular ATP and are primarily expressed on antigen-presenting immune cells where they are rapidly up-regulated and activated in the initial response to inflammatory stimuli. They couple to rapid release of the pro-inflammatory cytokine, interleukin-1beta in activated macrophage and P2X7-deficient mice show reduced inflammatory responses. These findings have excited much interest in immune cell biology and drug development research as a promising target to treat inflammatory diseases, particularly arthritis. Equally exciting at the level of fundamental biological processes, P2X7 receptors are unique ion channels in that they couple directly not only to cation/calcium flux but also to rapid alterations in cell and mitochondrial morphology (membrane blebbing, phosphatidylserine exposure, microvesicle shedding, mitochondrial swelling with depolarisation). Such phenomena previously have been associated with active cell death (apoptosis) but brief activation of the P2X7R induces these events without subsequent cell death. Much work in the past 5 years has begun to delineate signal transduction pathways and potential physiological roles for these cellular events but a glaring lack of information persists in regard to mechanisms underlying agonist binding and activation of this receptor. Such information is of fundamental biological interest because the pharmacology of this receptor is as unusual as is its cell biology. Two recent advances provide new opportunities to delineate molecular mechanisms of these ectodomain actions at the P2X7 receptor: the development of high affinity, selective antagonists and the discovery that this receptor is activated not only by extracellular ATP but also by ADP ribosylation. ADP ribosylation of the P2X7 receptor results from activatin of specific ADP-ribosylating ectoenzymes (ARTs) by extracellular NAD+. Specific methods are to study wild-type and mutant P2X7 receptors expressed alone or co-expressed with mammalian ARTs in HEK293 cells. We will (1) make point mutations and/or chimeric constructs in the ectodomain of P2X7, (2) use radio-labelled antagonists in ligand-binding studies, (3) measure membrane currents, membrane blebbing, cytoplasmic and mitochondrial calcium, annexin-V binding and YPORO-1 fluorescence, in quantitative pharmacological and biophysical experiments, (4) perform immunoprecipitation, blue native and 2D gel electrophoresis, and GST fusion protein pull-down assays, and (5) use time-lapse confocal microscopy of GPF and MRFP- tagged P2X7Rs and ARTs. These combined approaches will allow us to achieve our main objective which is to determine the molecular mechanisms underlying the actions on P2X7 receptors by these new selective receptor antagonists and the novel agonist, NAD-ART. These studies are also likely to identify residues forming the ligand binding motif of this protein and to provide tools for use in protein purification. Joint with BB/C517317/1

Summary

unavailable
Committee Closed Committee - Biochemistry & Cell Biology (BCB)
Research TopicsImmunology
Research PriorityX – Research Priority information not available
Research Initiative X - not in an Initiative
Funding SchemeX – not Funded via a specific Funding Scheme
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