Chronic pain causes debilitation and suffering in millions of Americans. Treatments to provide relief from this chronic pain are often ineffective. A critical and rate-limiting step for developing new treatments is that the underlying biological cause for the transition from acute to chronic pain is poorly understood. This research application is targeted directly at filling this void. We recently discovered that G protein-coupled receptor kinase 2 (GRK2) is key to preventing transition to chronic hyperalgesia. Chronic inflammation causes a decrease in GRK2 in pain transmitting neurons (nociceptors) that we mimicked in a mouse model using Cre-Lox technology. Using this model, we have now demonstrated that low nociceptor GRK2 markedly prolongs thermal hyperalgesia induced by the prototypic inflammatory mediator prostaglandin E2 (PGE2) without affecting baseline sensitivity. This project aims at determining the molecular pathways that underlie the transition to chronic pain that occurs when nociceptor GRK2 is low and understanding how chronic inflammation reduces GRK2 in nociceptors. Our hypothesis is that an inflammation-induced reduction in nociceptor GRK2 switches signaling in response to cAMP-inducing mediators like PGE2 from protein kinase A towards the cAMP target known as "exchange protein directly activated by cAMP" (Epac) and its downstream targets leadin to transition to chronic pain. To test our hypothesis we will answer four specific questions: 1. What is the contribution of nociceptor GRK2 to mechanical hyperalgesia? 2. How do GRK2 and Epac1 interact to regulate Epac signaling to its downstream effectors? 3. When and how does inflammation reduce GRK2 and increase Epac1 in nociceptors? and 4. Can we generate proof of principle that targeting GRK2/Epac prevents transition to chronic pain? In aim 1, we will use an in vivo approach with SNS-GRK2 mice that have low nociceptor GRK2.
In aim 2, in vitro approaches using GRK2 deletion mutants, a kinase dead GRK2 mutant, kinase assays and co-immuno-precipitations will be used.
In aim 3, we will use immuno-fluorescence and qPCR analysis of nociceptor GRK2/Epac in response to inflammation.
In aim 4, we will use a classic genetic approach to develop and use novel mouse models with nociceptor specific deletion of Epac1 and overexpression of GRK2. The proposed research is innovative for three major reasons: (a) This is the first time GRK2 has been found to function as an endogenous inhibitor of Epac activation;(b) We will directly test our hypothesis that the newly discovered GRK2/Epac interface functions as an intracellular molecular switch regulating hyperalgesic signaling and thereby the transition to chronic pain;(c) Identifying the downstream GRK2/Epac interface to combat chronic pain represents a completely novel approach that avoids the disadvantages of targeting upstream receptors or mediators. This contribution is significant because it is the first step in a continuum of research that is very likely to lead to development of novel pharmacologic strategies that specifically target the newly identified GRK2/Epac interface to prevent chronic pain.
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