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

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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
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G Protein-Coupled Receptor Signaling and
Desensitization
agonist
?arrestin
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What are the contributions of the desensitization
mechanisms to determining the receptors
potential to signal?
No specific inhibitors of GRKs (GRK 1-7, 17 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
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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) 276R1214-21. 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) 27225425-8.
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, 241029-1036)
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) 28997478-83.
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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 ßarr1 KO mice
• ßarr2-KO
• GRK6-KO mice

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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
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ßarrestin2 and GRK6 knockout mice presented
different responses compared to their WT
littermates
1. ßarr2-KO mice Morphine antinociception
Dopaminergic function Morphine Cocaine 2.
GRK6-KO mice Dopaminergic function Cocaine
Morphine Morphine antinociception
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?arr2-KO mice in response to morphine
Morphine, 10 mg/kg s.c.
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Effects of ?arrestin-2 on G protein coupling
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Morphine Antinociceptive Tolerance Hot plate
(56?C)
Bohn et al., (2000) Nature 408.
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Summary of ßarr2-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 ßarr2-KO mice?
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Morphines 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
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Increased DA release Increased Locomotor
Activity Openfield Locomotor Activity- acute drug
treatment
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Morphine decreased locomotor activity Decrease
d Reinforcement? Cocaine No difference Same
Reinforcement?
Perhaps a better indicator of the potential for
drug rewarding properites is sensitization. Sensi
tization Increased responsiveness to the drug
over extended treatment (reversed tolerance) Do
both WT and ßarr2-KO mice become sensitized to
either drug?
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Locomotor Sensitization after Morphine and Cocaine
Both WT and ßarr2-KO mice become sensitized to
morphine and cocaine
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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 -gt associated with drug rewarding
properties
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Extracellular Dopamine levels following Morphine
or Cocaine
Microdialysis in Striata of freely moving
conscious mice
Morphine, 10 mg/kg s.c.
Drug Reinforcement?
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Conditioned Place Preference with Morphine and
Cocaine
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Summary of Dopaminergic effects in ßarr2-KO mice
The reinforcing properties of morphine are
enhanced Specific for morphine, not cocaine
(dopamine)
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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
38291-303.
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Cocaine Sensitization in GRK6-KO mice
Sensitization
Day 7
Day 1
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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.
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Morphine effects in GRK6-KO mice
Locomotor Activity
acute
sensitization
GRK6-KO mice are supersensitive to morphine in
the locomotor activity test.
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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.)
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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

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GPCR desensitization by GRK and ßarrestin
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 ßarr2 appears to
be selective for enhancing opioid
receptor-mediated behaviors.
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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-Chape
l Hill
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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
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Receptor Signaling/ Agonist Efficacy Depends upon
a Balance Between Activation and Desensitization
G?
?arr2
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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
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5 min
5 min
Etorphine
Morphine