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 #
5R37DA011672-14
Application #
8248316
Study Section
Special Emphasis Panel (NSS)
Program Officer
Wu, Da-Yu
Project Start
1999-05-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
14
Fiscal Year
2012
Total Cost
$251,356
Indirect Cost
$83,303
Name
University of Washington
Department
Pharmacology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Kuhar, Jamie Rose; Bedini, Andrea; Melief, Erica J et al. (2015) Mu opioid receptor stimulation activates c-Jun N-terminal kinase 2 by distinct arrestin-dependent and independent mechanisms. Cell Signal 27:1799-806
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
Lemos, Julia C; Roth, Clarisse A; Chavkin, Charles (2011) Signaling events initiated by kappa opioid receptor activation: quantification and immunocolocalization using phospho-selective KOR, p38 MAPK, and K(IR) 3.1 antibodies. Methods Mol Biol 717:197-219
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
Chavkin, Charles (2011) The therapeutic potential of ?-opioids for treatment of pain and addiction. Neuropsychopharmacology 36:369-70
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
Lyssand, John S; Whiting, Jennifer L; Lee, Kyung-Soon et al. (2010) Alpha-dystrobrevin-1 recruits alpha-catulin to the alpha1D-adrenergic receptor/dystrophin-associated protein complex signalosome. Proc Natl Acad Sci U S A 107:21854-9
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
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

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