The mu-opioid receptor (MOR) mediates most of the actions of morphine and other clinically relevantanalgesics as well as drugs of abuse such as heroin. The proteomic and functional studies proposed in thisapplication are designed to elucidate the protein components of MOR signaling complexes. Identification ofMOR interacting proteins will allow us to test our hypothesis that protein-protein interactions play animportant role in regulating the MOR signal transduction pathway.
In Aim I, we will identify and characterizeMOR interacting proteins. A split-ubiquitin screen we have performed identified several novel MORIPsincluding GPR177, the mammalian ortholog of Drosophila Wntless. To identify additional MORIPs, each ofthe intracellular domains of the human MOR will be used as bait to separately screen a human brain cDNAlibrary. We also propose to utilize highly selective anti-MOR antibodies combined with proteomics and massspectrometry to identify a spectrum of MORIPs from immunoprecipitated mouse brain lysates. The majorgoal in Aim 2 will be to validate the MOR-protein interactions identified in Aim I. Cellular colocalizationstudies will confirm whether or not the MOR and candidate interactors are expressed within the same cellsand intracellular compartments. Pull-down and coimmunoprecipitation will substantiate the MOR-proteininteraction, while deletion mapping will permit identification of sites within the proteins that are necessary forthe interactions to occur. Preliminary studies we have so far performed indicate that GPR177 meets allinclusion criteria and appears to represent a bona-fide MORIP. The goal in Aim 3 is to understand thefunctional significance of MOR/GPR177 interaction. We will examine the requirement for MOR/GPR177interaction in MOR trafficking, desensitization, and signal transduction. We will also examine the role of theMOR/GPR177 interaction in regulating Wnt2 secretion from cells. To accomplish this goal, we will determinewhether disrupting the MOR/GPR177 interaction, by expressing dominant negative forms of GPR177 or byknocking down GPR177 expression, affects the functional properties of the MOR. Identification of MORinteracting proteins will provide new insights into the etiology of drug abuse and dependence.
This project seeks to identify and evaluate the function of proteins that interact with and regulate the muopioidreceptor (MOR), the cell associated receptor that mediates most of the analgesic actions of morphineas well as drugs of abuse. Understanding the function of GPR177, a novel interacting protein we identified,is likely to provide new insight into the etiology of drug abuse and dependence. MOR-interacting proteinsmay also represent novel therapeutic targets for the treatment of these important public health problems.
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