Drug addiction is a major medical problem in the United States. Opioid addiction is associated with a variety of behaviors that are detrimental to both the individual and society. While much has been learned about opioid pharmacology and signal transduction over the last three decades, the molecular mechanisms that are responsible for opioid tolerance and dependence due to chronic opioid drug use are complex and much remains to be learned. Acute administration of opioids triggers intracellular signal transduction, initiated by receptor-mediated heterotrimeric G protein activation. Effectors include adenylyl cyclase, potassium and calcium ion channels, and MAP kinase, all of which contribute to the pharmacological effects of opioids. Agonist efficacy diminishes rapidly when multiple doses are given over a short period of time. This homologous desensitization is due to uncoupling of the receptor from the G protein, due to receptor phosphorylation by G protein-coupled receptor kinases (GRKs). Arrestin binds preferentially to GRK phosphorylated receptors and precludes further activation of G proteins. Chronic administration of opioid agonists results in receptor down regulation, involving proteolysis of the receptor protein and a concomitant decrease in the number of functional receptors. It is highly probable that agonist-induced down regulation of opioid receptors contributes to opioid tolerance. The PI has provided compelling evidence that the ubiquitin/proteasome system is involved in basal turnover and agonist-induced down regulation of opioid receptors. Pulse-chase analysis revealed that agonist treatment accelerates proteolysis of the receptor. Preincubation with proteasome inhibitors, but not other protease inhibitors, blocked agonist-induced receptor down regulation. Immunoprecipitation of opioid receptors revealed that opioid receptors are polyubiquitinated prior to degradation. This research proposal will focus on the purification of opioid receptors and analysis of post-translational modification using mass spectrometry. Sites of phosphorylation and ubiquitination of opioid receptors will be mapped by mas spectrometric analysis and site-directed mutagenesis. The hypothesis that inhibition of agonist-induced down regulation with proteasome inhibitors will attenuate the developments of tolerance will be tested.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
2R01DA009113-04A1
Application #
6631096
Study Section
Molecular, Cellular and Developmental Neurosciences 2 (MDCN)
Program Officer
Koustova, Elena
Project Start
1997-07-15
Project End
2008-01-31
Budget Start
2003-04-01
Budget End
2004-01-31
Support Year
4
Fiscal Year
2003
Total Cost
$194,375
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Biochemistry
Type
Schools of Medicine
DUNS #
623946217
City
Newark
State
NJ
Country
United States
Zip Code
07107
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Chaturvedi, K; Bandari, P; Chinen, N et al. (2001) Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors. J Biol Chem 276:12345-55
Chaturvedi, K; Jiang, X; Christoffers, K H et al. (2000) Pharmacological profiles of selective non-peptidic delta opioid receptor ligands. Brain Res Mol Brain Res 80:166-76

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