# Presentation: 107130

## 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

## arrestin

******arrestin**

- agonist

- G Protein-Coupled Receptor Signaling and Desensitization

## 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

## What Receptor?  What Agonist?

- 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.

- 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)

## 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

## Summary of Behavioral Responses to Morphine, Cocaine, Amphetamine and Apomorphine in genetically modified mice.

|  | GRK2
HT | GRK3
KO | GRK4
KO | GRK5
KO | arr1
KO |
| --- | --- | --- | --- | --- | --- |
| Antinociception |   |   |   |   |   |
| Morphine | = | = | = | = | = |
| Locomotor Activity |  |  |  |  |  |
| Cocaine | =()* | () | = | = | =() |
| Amphetamine | = | N.D. | N.D. | N.D. | N.D. |
| Apomorphine | = | =() | N.D. | = | =() |

**=** 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*

## 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

## Βarr2-KO mice in response to morphine:

- Morphine, 10 mg/kg s.c.

## Effects of arrestin-2 on G protein coupling

- Effects of arrestin-2 on G protein coupling

## Morphine Antinociceptive Tolerance 

- Hot plate (56C)

*Bohn et al.,  (2000)  Nature 408.*

## Slide 11

## 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 **β**arr2-KO mice?*

## Morphine’s rewarding properties via dopaminergic and non-dopaminergic systems

- Dopamine system is most evident- stimulate release of DA

- Removal of Barr2 could have effects on Dopamine Receptor signaling as well
- Compare cocaine and morphine

- μOR

- DAR, μOR

## Increased DA release = Increased Locomotor Activity

- Openfield Locomotor Activity- acute drug treatment

## 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?

## Locomotor Sensitization after Morphine and Cocaine

- Both WT and βarr2-KO mice become sensitized to morphine and cocaine

## Drug Reinforcement and Dopamine

- Blocks reuptake of DA, leads to elevated extracellular DA levels in striatum.

- Cocaine:

- 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

## Extracellular Dopamine levels following Morphine or Cocaine

- Morphine, 10 mg/kg s.c.

- Microdialysis in Striata of freely moving conscious mice

- Drug Reinforcement?

## Conditioned Place Preference with Morphine and Cocaine

## The reinforcing properties of morphine are enhanced

|  | Morphine | Cocaine |
| --- | --- | --- |
| Locomotor Activity | Not enhanced, Decreased | Slightly Decreased |
| Sensitization |  |  |
| Microdialysis | Elevated DA | Same DA |
| Conditioned Place Preference | More CPP | Same CPP |

- Specific for morphine, not cocaine (dopamine)

- Summary of Dopaminergic effects in βarr2-KO mice

## GRK6-KO mice and cocaine:

- Gainetdinov, et al.  Dopaminergic Supersensitivity in G Protein-Coupled Receptor Kinase 6 - Deficient Mice. (2003) Neuron 38:291-303.

## Cocaine Sensitization in GRK6-KO mice

**Sensitization**

**Day 1**

**Day 7**

## 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.

## Morphine effects in GRK6-KO mice:

- Locomotor Activity

- acute

- sensitization

- GRK6-KO mice are supersensitive to morphine in the
- locomotor activity test.

## What about morphine effects on antinociception?

- Morphine (10 mg/kg, i.p.)

- In the hot plate analgesia test the genotypes look the same...

- Morphine Tolerance (10 mg/kg, i.p., daily)

## In the absence of arr2:

- Enhanced and prolonged morphine analgesia

- Lack of morphine tolerance in Hot Plate test- μOR – G protein coupling maintained

**In the absence of ********arr2:**

- Decreased Locomotor Activity with Morphine

- Enhanced CPP with Morphine

- Increased Dopamine levels 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

## 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.

- GPCR desensitization by GRK and βarrestin mechanisms plays an important role in the actions of drugs of abuse.

- Specifically:

## Aknowledgements:

- _Marc G. Caron_
- Raul Gainetdinov
- Tatyana Sotnikova
- Ivan Medvedev

- _Robert J. Lefkowitz _
- Richard Premont

- _Linda A. Dykstra_
- Psychology/Pharmacology
- UNC-Chapel Hill

- _National Institute _
- _on Drug Abuse_
- F32 DA06023
- K01 DA 14600
- & Narcotic Supply

- _Duke University Medical Center_

- _University of North Carolina- Chapel Hill_

## Join US!

- http://medicine.osu.edu/pharmacology

- POST-DOCTORAL POSITIONS-
- New Laboratories in the Department of Pharmacology-
- Forming Focus Group on Addiction within the Pharmacogenomics Program

- Howard Gu    Wolfgang Sadee     Kirk Mykytyn      Laura Bohn

*Join US!*

- The Ohio State University College of Medicine and Public Health

## Slide 30

## Receptor Signaling/ Agonist Efficacy Depends upon a Balance Between Activation and Desensitization

- arr2

- G

## arr2-GFP

- ___arr2-GFP_

- ___arr1-GFP_

- Morphine

- Etorphine

- 0

- 0

- 5 min

- 5 min

- arr2 translocation more pronounced in the absence of endogenous arrestins in arr1/arr2 KO Mouse Embryonic Fibroblasts

**Part IV:   Specificity: Agonists/ GRKs & ****arrestins**