This application seeks support for the continuation of a successful grant designed to study the effects of phosphorylation on opioid receptor activation of G-protein coupled potassium channels (Kit3) In the previously funded period, the effects of both serine/threonine phosphorylation and tyrosine phosphorylation on opioid receptor desensitization and Kir3 channel activation were described. We propose to extend these studies by focusing our effort on the specific effects of tyrosine phosphorylation (Y-PO4) on mu opioid receptor (MOR) activation of Kir3. Our previous site directed mutagenesis studies identified potential Y-PO4 Isites within the receptor and channel likely to control the response to opioid agonists. Initially, the mechanisms of this regulation will be studied using cDNA expression of MOR and Kir3.1 in AtT20 cells and primary hippocampal cultures. Studies would be extended by in vitro electrophysiological recording oi DAMGO activated Kir3 responses and confocal imaging of receptor and channel trafficking. We would use pharmacological inhibitors to identify the kinases and phosphatases responsible the Y-PO4 mediated effects in these malleable in vitro systems. Results of this aim would test the hypothesis that Y-PO4 of specific sites within MOR and Kir3.1 regulates the efficiency of opioid signaling. The phosphorylation state of specific sites within the MOR and Kir3.1 sequences would be assessed by 32p-incorporation and by probing with novel phosphospecific antibodies. Regulation of opioid receptor signaling by tyrosine phosphorylation has important implications for understanding opioid responses during physiological stress;thus, moving from simple in vitro analyses to more complex, in vivo systems would be a priority. After characterizing the specificity and utility of the phosphospecific antibodies (MOR-YP and Kir3.1-YP) in the in vitro systems, we will test the hypothesis that nerve trauma results in growth factor-induced changes in tyrosine phosphorylation of MOR and Kir3.1 detectable by changes in MOR-YP and Kir3.1-YP immunostaining within nociceptive circuits in spinal cord and brain. Results of the proposed studies are likely to provide additional understanding of the mechanisms mediating the plasticity of opioid signaling.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DA011672-15
Application #
8444726
Study Section
Special Emphasis Panel (NSS)
Program Officer
Wu, Da-Yu
Project Start
1999-05-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
15
Fiscal Year
2013
Total Cost
$241,302
Indirect Cost
$79,971
Name
University of Washington
Department
Pharmacology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Chavkin, Charles; Schattauer, Selena S; Levin, Jamie R (2014) Arrestin-mediated activation of p38 MAPK: molecular mechanisms and behavioral consequences. Handb Exp Pharmacol 219:281-92
Chavkin, Charles (2013) Dynorphin--still an extraordinarily potent opioid peptide. Mol Pharmacol 83:729-36
Chavkin, Charles (2011) The therapeutic potential of *-opioids for treatment of pain and addiction. Neuropsychopharmacology 36:369-70
Melief, Erica J; Miyatake, Mayumi; Carroll, F Ivy et al. (2011) Duration of action of a broad range of selective ?-opioid receptor antagonists is positively correlated with c-Jun N-terminal kinase-1 activation. Mol Pharmacol 80:920-9
Melief, Erica J; Miyatake, Mayumi; Bruchas, Michael R et al. (2010) Ligand-directed c-Jun N-terminal kinase activation disrupts opioid receptor signaling. Proc Natl Acad Sci U S A 107:11608-13
Aita, Megumi; Byers, Margaret R; Chavkin, Charles et al. (2010) Trigeminal injury causes kappa opioid-dependent allodynic, glial and immune cell responses in mice. Mol Pain 6:8
Clayton, Cecilea C; Bruchas, Michael R; Lee, Michael L et al. (2010) Phosphorylation of the mu-opioid receptor at tyrosine 166 (Tyr3.51) in the DRY motif reduces agonist efficacy. Mol Pharmacol 77:339-47
Beaudry, H; Proteau-Gagne, A; Li, Shuang et al. (2009) Differential noxious and motor tolerance of chronic delta opioid receptor agonists in rodents. Neuroscience 161:381-91
Clayton, Cecilea C; Xu, Mei; Chavkin, Charles (2009) Tyrosine phosphorylation of Kir3 following kappa-opioid receptor activation of p38 MAPK causes heterologous desensitization. J Biol Chem 284:31872-81
Bruchas, Michael R; Xu, Mei; Chavkin, Charles (2008) Repeated swim stress induces kappa opioid-mediated activation of extracellular signal-regulated kinase 1/2. Neuroreport 19:1417-22

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