GPCR Regulatory Mechanisms: Effects on Tolerance, Sensitization, and Reinforcement

1. GPCR Regulatory Mechanisms: Effects on Tolerance, Sensitization, and Reinforcement Laura M. Bohn, Ph.D. The Ohio State University College of Medicine and Public Health Departments of Pharmacology & Psychiatry, Program in Pharmacogenomics, Columbus, Ohio

2. agonist G -P Desensitization Decreased Response Activation Biological Response GRK G Protein-Coupled Receptor Signaling and Desensitization agonist arrestin

3. What are the contributions of the desensitization mechanisms to determining the receptor’s potential to signal? No specific inhibitors of GRKs (GRK 1-7, 1&7 are visual) No specific inhibitors of arrestins (arr1 & arr2) Use a genetic knockout mouse approach: GRK2-HT, GRK3-KO, GRK4-KO, GRK5-KO, GRK6-KO arr1-KO, arr2-KO

4. What Receptor? What Agonist? GRK2-KO: Embryonic Lethal GRK3-KO: Airway and Cardiac Responses Walker et al., Altered airway and cardiac responses in mice lacking G protein-coupled receptor kinase 3. Am J Physiol. (1999) 276:R1214-21. Mild olfactory supersensitivity Peppel et al., G protein-coupled receptor kinase 3 (GRK3) gene disruption leads to loss of odorant receptor desensitization. J Biol Chem. (1997) 272:25425-8. GRK5-KO: Supersensitive to Muscarinic agonists (M2 receptors) Gainetdinov, et al.,Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice. (1999) Neuron, 24:1029-1036) GRK6-KO & Barr2-KO: Deficient in chemotaxis. Fong et al., Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6- deficient mice. Proc Natl Acad Sci U S A. (2002) 28;99:7478-83.

5. Screening for differential sensitivities to drugs of abuse Two major drugs of abuse chosen: Opiate Narcotics (Morphine) Psychostimulants (Cocaine) General Screen for differences in sensitivity: Morphine: Antinociception- Hot Plate test Cocaine: Locomotor activity- open field activity monitor • GRK2 HT, GRK3, GRK4, GRK5 and βarr1 KO mice • βarr2-KO • GRK6-KO mice

6. Summary of Behavioral Responses to Morphine, Cocaine, Amphetamine and Apomorphine in genetically modified mice. = Equivalent to WT response; () Slight decrease; =()* no difference at 10, 15, 25, 30 mg/kg., increased at 20 mg/kg, i.p.; N.D. Not Determined From NeuroMolecular Medicine Review, in press

7. βarrestin2 and GRK6 knockout mice presented different responses compared to their WT littermates 1. βarr2-KO mice Morphine antinociception Dopaminergic function: Morphine & Cocaine 2. GRK6-KO mice Dopaminergic function: Cocaine & Morphine Morphine antinociception

8. Hot Plate (56 C) Bohn et al., (1999) Science 284 Βarr2-KO mice in response to morphine: Morphine, 10 mg/kg s.c.

9. M M M G G GRK -P arr2 PAG Brainstem Spinal Cord Effects of arrestin-2 on G protein coupling

10. Pellet implantation Daily injection Daily Tolerance Mice are injected daily with 10 mg/kg Morphine. 30 min after injection, test. Pellet Implantation 75 mg time-release Morphine pellet implanted s.c. Morphine Antinociceptive Tolerance Hot plate (56C) Bohn et al., (2000) Nature 408.

11. G protein coupling following chronic morphine M M M G G -P GRK arr2 Log [DAMGO], M Brainstem Membranes Bohn et al., (2000) Nature 408.

12. Summary of βarr2-KO responses to morphine previously described: Enhanced and prolonged morphine antinociception (Hot plate and Tail flick) Loss of morphine antinociceptive tolerance in hot plate (tail flick: significanlty attenuated) No difference in physical dependence (WT & KO mice exhibit same withdrawal symptoms) What are the effects of morphine on the reinforcing and dopaminergic properties in the βarr2-KO mice?

13. Morphine’s rewarding properties via dopaminergic and non-dopaminergic systems Dopamine system is most evident- stimulate release of DA μOR DAR, μOR Removal of Barr2 could have effects on Dopamine Receptor signaling as well Compare cocaine and morphine

14. Dose Response Morphine: Cocaine: Increased DA release = Increased Locomotor Activity Openfield Locomotor Activity- acute drug treatment

15. Morphine: decreased locomotor activity: Decreased Reinforcement? Cocaine: No difference: Same Reinforcement? Perhaps a better indicator of the potential for drug rewarding properites is sensitization. Sensitization: Increased responsiveness to the drug over extended treatment (reversed tolerance) Do both WT and βarr2-KO mice become sensitized to either drug?

16. Locomotor Sensitization: Increased locomotor activity over continued administration of the drug (“Reverse Tolerance”) Morphine: Daily injections days 1-7, rest day 8, inject and test day 9 Cocaine: Daily injections days 1-5, rest day 6, inject and test day 7 Locomotor Sensitization after Morphine and Cocaine Both WT and βarr2-KO mice become sensitized to morphine and cocaine

17. Drug Reinforcement and Dopamine Cocaine: Blocks reuptake of DA, leads to elevated extracellular DA levels in striatum. Morphine: DA release induced in striatum and nucleus accumbens by opioid receptor-mediated inhbition of GABAergic neurons in substantia nigra and VTA Increase DA release = Increased DA receptor activation -> associated with drug “rewarding” properties

18. Dopamine Dopamine metabolites Cocaine, 20 mg/kg i.p. Morphine= more DA release in arr2-KO mice Cocaine= genotypes same Extracellular Dopamine levels following Morphine or Cocaine Microdialysis in Striata of freely moving conscious mice Morphine, 10 mg/kg s.c. Drug Reinforcement?

19. B A corncob bedding tea bag Saline Saline Saline Drug Drug Drug Pre-conditioning Post-conditioning Day 1 2 3 4 5 6 7 8 record time spent in each compartment record time spent in each compartment A B A B A B Postconditioning time on drug side – Preconditioning time on drug side = Preference for the side paired with Drug Cocaine 10 mg/kg i.p. Conditioned Place Preference with Morphine and Cocaine

20. Summary of Dopaminergic effects in βarr2-KO mice The reinforcing properties of morphine are enhanced Specific for morphine, not cocaine (dopamine)

21. GRK6-KO mice and cocaine: GRK6 are supersenstivie to cocaine in the locomotor activity test: Gainetdinov, et al. Dopaminergic Supersensitivity in G Protein-Coupled Receptor Kinase 6 - Deficient Mice. (2003) Neuron 38:291-303.

22. Cocaine Sensitization in GRK6-KO mice Sensitization Day 7 Day 1

23. D D D G G arr1/2 GRK -P Striatum HEK-293 Cells Effect of GRK6 on Dopamine Receptor Coupling What about Morphine? If the DA receptor is more sensitive then morphine should produce more locomotor activity too.

24. Morphine effects in GRK6-KO mice: Locomotor Activity acute sensitization GRK6-KO mice are supersensitive to morphine in the locomotor activity test.

25. What about morphine effects on antinociception? In the hot plate analgesia test the genotypes look the same... Morphine Tolerance (10 mg/kg, i.p., daily) Morphine (10 mg/kg, i.p.)

26. In the absence of arr2: • Greater degree of OR and G protein coupling • Enhanced and prolonged morphine analgesia • Lack of morphine tolerance in Hot Plate test- μOR – G protein coupling maintained • Decreased Locomotor Activity with Morphine • Increased Dopamine levels with Morphine • Enhanced CPP with Morphine No differences observed with Cocaine In the absense of GRK6: • Enhanced locomotor activity: Cocaine & Morphine • Greater degree of DA Receptor-G protein coupling • No difference in morphine antinociception or tolerance

27. GPCR desensitization by GRK and βarrestin mechanisms plays an important role in the actions of drugs of abuse. Specifically: The deletion of GRK6 appears to selectively affect dopamine, rather than opioid receptor signaling While the deletion of βarr2 appears to be selective for enhancing opioid receptor-mediated behaviors.

28. Aknowledgements: Duke University Medical Center Marc G. Caron Raul Gainetdinov Tatyana Sotnikova Ivan Medvedev National Institute on Drug Abuse F32 DA06023 K01 DA 14600 & Narcotic Supply Robert J. Lefkowitz Richard Premont University of North Carolina- Chapel Hill Linda A. Dykstra Psychology/Pharmacology UNC-Chapel Hill

29. The Ohio State University College of Medicine and Public Health Join US! Howard Gu Wolfgang Sadee Kirk Mykytyn Laura Bohn POST-DOCTORAL POSITIONS- New Laboratories in the Department of Pharmacology- Forming Focus Group on Addiction within the Pharmacogenomics Program http://medicine.osu.edu/pharmacology http://pharmacogenomics.osu.edu/section1.html

31. M M M G GRK -P [agonist] G coupling GRK arrestin RF RB R* RP Rarr (activity, recruitment, specificity, levels?) resensitization Receptor Signaling/ Agonist Efficacy Depends upon a Balance Between Activation and Desensitization G arr2

32. Part IV: Specificity: Agonists/ GRKs & arrestins arr2 translocation more pronounced in the absence of endogenous arrestins in arr1/arr2 KO Mouse Embryonic Fibroblasts arr2-GFP arr1-GFP 0 5 min 0 5 min Etorphine Morphine