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Signalling pathways and scaffolding proteins modulating the activity of the heat and capsaicin receptor TRPV1

Reference	BB/C003217/1
Principal Investigator / Supervisor	Professor Peter Anthony McNaughton
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Pharmacology
Funding type	Research
Value (£)	258,159
Status	Completed
Type	Research Grant
Start date	16/01/2005
End date	15/01/2008
Duration	36 months

Abstract

The membrane ion channel TRPV1 is activated by heat, low pH and capsaicin (the active extract of chilli peppers), and is important in the detection of pain caused by heat or by acid produced in ischaemia or inflammation. Activation of TRPV1 is potentiated by a number of pro-inflammatory factors, amongst them bradykinin and nerve growth factor (NGF), thus causing sensitisation and enhancing pain. One major objective of the current proposal is to elucidate the molecular mechanism by which NGF causes sensitisation. Experiments in the applicant lab have identified PI3 kinase as an early participant in the pathway activated by TrkA, the NGF receptor, and have shown that TRPV1 is phosphorylated by TrkA activation. We will extend these studies as follows to identify the signalling pathway and phosphorylation sites on TRPV1. (1) We will identify other participants in the pathway by using a combined pharmacological and dominant negative approach to neutralise possible members of the TrkA signalling pathway (such as Akt or src kinase). We will measure the impact of these alterations on TRPV1 in two ways: (a) by quantifying the effect on phosphorylation of TRPV1, and (b) by using calcium imaging of intact cells to measure the effect on the enhanced activation of TRPV1 following activation of TrkA. We will perform experiments in expression systems and in intact sensory neurons transfected using a high-efficiency transfection system. (2) We will identify critical phosphorylation sites on TRPV1 by observing the effect on both phosphorylation and function, as in (1), of mutating potential serine/threonine or tyrosine residues. (3) We will then investigate the effect of removing critical phosphorylation sites on sensitisation by NGF in real sensory neurons by transfecting TRPV1 from which the critical sites have been removed into sensory neurons expressing no endogenous TRPV1, either isolated from TRPV1-/- animals or in which TRPV1 has been downregulated by the use of siRNA. A second major objective is to investigate the role of proteins which bind to TRPV1 to form a signalling complex. In preliminary experiments we have found that a number of proteins are bound to TRPV1, including TrkA, PKC beta and the scaffolding protein AKAP79. These studies suggest that other proteins will be associated with TRPV1 to form a large signalling complex in which several different signalling pathways can converge to activate TRPV1. (4) We will investigate which other proteins bind to TRPV1. We will focus on likely partners in signalling cascades, including serine/threonine kinases such as PKC, PKA and CAM kinase, tyrosine kinases such as src and TrkA, and phosphatases such as calcineurin and SHP-1. We will also investigate the possible involvement of scaffolding proteins such as members of the AKAP and RACK families. (5) We will use GST fragments of TRPV1 to identify regions of TRPV1 which bind to associated proteins. In expression systems we will explore the effect of deletion of identified binding regions from TRPV1, which should remove the signalling effect of this associated protein. (6) We will then explore the effect of removing the binding of signalling partners on sensitisation in sensory neurons. We will transfect mutated TRPV1, with identified binding regions or phosphorylation sites deleted, into sensory neurons from TRPV1-/- animals in order to identify the role of particular binding partners in the actions of agents which are known to cause sensitisation, such as ATP, bradykinin, and agonists at protease activated receptors (PARs) and SNSRs.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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