The ? opioid receptor (KOPR) is one of the three major types (?, ? and ?) of opioid receptors that mediate effects of opioids in vivo. Activation of KOPR produces analgesia, dysphoria, water diuresis, hypothermia and modulation of immune responses. KOPR antagonists may be potentially useful for curbing cocaine craving and as anti-depressants and anti-anxiety agents. KOPR, a member of the 7TMR family, is coupled through pertussis toxin-sensitive G proteins to a variety of effectors. For 7TMRs. the capacity of agonists to modulate downstream signaling molecules depends on the availability of the receptors on cell surface. The number of cell surface 7TMRs reflects a balance between biosynthesis and endocytosis pathways. The post-activation endocytic events have been well-documented;however, regulation along the biosynthesis pathway is much less understood. The focus of this grant application is to characterize the regulation of the KOPR trafficking along the biosynthesis pathway, in particular by the proteins GEC1, sortilin and 14-3-3 proteins, which we found to interact with the KOPR and to be involved in KOPR export trafficking. The central hypothesis is that the export trafficking is regulated by molecules interacting with the KOPR.
The specific aims are as follows. (1) To delineate mechanisms underlying GEC1-promoted expression and trafficking of the KOPR. The hypothesis that GEC1 enhances KOPR expression by enhancing ATPase activity of NSF, but not by membrane association by lipid conjugation will be tested. (2) To investigate the role of 14-3-3 in the trafficking and cellular pharmacology of the KOPR. The hypothesis is that 14-3-3 proteins bind to KOPR C-terminal domain and/or i3 loop, which masks COPI binding, allowing the KOPR to sort to plasma membranes. (3) To examine the interaction of the KOPR with sortilin and its functional consequences. The hypothesis to be tested is that sortilin binding to the KOPR sorts the receptor to endosomes and lysosomes and this represents a novel post-ER quality control mechanism. We will also examine if the interactions of the KOPR with these proteins affect signaling and regulation. The proposed studies will provide better understanding of cell biology of the KOPR and provide mechanistic insights into regulation of export of the KOPR. In addition, such understanding will have important implications for other membrane bound receptors since these regulatory mechanisms of export are likely to be applicable to some other proteins.

Public Health Relevance

Program narrative The proposed research will provide better understanding on how cell surface receptor numbers are regulated, which play important roles in response to external stimuli, including drugs and neurotransmitters.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA017302-09
Application #
8261953
Study Section
Molecular Neuropharmacology and Signaling Study Section (MNPS)
Program Officer
Thomas, David A
Project Start
2004-05-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
9
Fiscal Year
2012
Total Cost
$288,090
Indirect Cost
$96,030
Name
Temple University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
057123192
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman et al. (2016) Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization. Biochem J 473:497-508
Huang, Peng; Yakovleva, Tatyana; Aldrich, Jane V et al. (2016) Two short-acting kappa opioid receptor antagonists (zyklophin and LY2444296) exhibited different behavioral effects from the long-acting antagonist norbinaltorphimine in mouse anxiety tests. Neurosci Lett 615:15-20
Huang, Peng; Chen, Chongguang; Liu-Chen, Lee-Yuan (2015) Detection of mu opioid receptor (MOPR) and its glycosylation in rat and mouse brains by western blot with anti-μC, an affinity-purified polyclonal anti-MOPR antibody. Methods Mol Biol 1230:141-54
Chen, Chongguang; Huang, Peng; Liu-Chen, Lee-Yuan (2015) Identification and verification of proteins interacting with the kappa opioid receptor (KOPR). Methods Mol Biol 1230:129-40
DiMattio, Kelly M; Ehlert, Frederick J; Liu-Chen, Lee-Yuan (2015) Intrinsic relative activities of κ opioid agonists in activating Gα proteins and internalizing receptor: Differences between human and mouse receptors. Eur J Pharmacol 761:235-44
DiMattio, Kelly M; Chen, Chongguang; Shi, Lei et al. (2015) K303⁶·⁵⁸ in the μ opioid (MOP) receptor is important in conferring selectivity for covalent binding of β-funaltrexamine (β-FNA). Eur J Pharmacol 748:93-100
Wang, Yu-Jun; Huang, Peng; Blendy, Julie A et al. (2014) Brain region- and sex-specific alterations in DAMGO-stimulated [(35) S]GTPγS binding in mice with Oprm1 A112G. Addict Biol 19:354-61
Dimattio, K M; Yakovleva, T V; Aldrich, J V et al. (2014) Zyklophin, a short-acting kappa opioid antagonist, induces scratching in mice. Neurosci Lett 563:155-9
Xu, Wei; Wang, Yujun; Ma, Zhongze et al. (2013) L-isocorypalmine reduces behavioral sensitization and rewarding effects of cocaine in mice by acting on dopamine receptors. Drug Alcohol Depend 133:693-703
Huang, Peng; Chiu, Yi-Ting; Chen, Chongguang et al. (2013) A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor-tdTomato fusion protein (KOPR-tdT) in neuronal cells. J Pharmacol Toxicol Methods 68:340-5

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