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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 HealthDepartments of Pharmacology & Psychiatry,
Program in Pharmacogenomics,Columbus, Ohio
arrestin
GRK
-P
DesensitizationDecreased Response
agonist
ActivationBiological Response
G
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 LethalGRK3-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
1. GRK2 HT, GRK3, GRK4, GRK5 and βarr1 KO mice2. βarr2-KO 3. GRK6-KO mice
Summary of Behavioral Responses to Morphine, Cocaine, Amphetamine and Apomorphine in genetically modified
mice.
GRK2HT
GRK3KO
GRK4KO
GRK5KO
arr1KO
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.
0 15 30 45 60 75 90 120 150 0
20
40
60
80
100
WT
arr2-KO
arr2 +/-
Time (minutes)
% M
PE
Bohn et al., (1999) Science 284
Hot Plate (56 C)
None
0
10
20
30
40
50
60
70
80
WT
arr2-KO
-8 -7 -6 -5
Log [DAMGO], M
% S
tim
ula
ted
35S
-GT
P S
bin
din
g
-7 -6 -50
10
20
30
40
50
60WT
arr2-KO
Log [DAMGO], M
% S
tim
ula
tio
n
[35S
]GT
P S
Bin
din
g
-7 -6 -5
0
20
40
60
80
100 WTarr2-KO
Log ([DAMGO], M)
%3
5S
-GT
P S
Bin
din
g(%
Sti
mu
lati
on
)
PAG Brainstem Spinal Cord
Effects of arrestin-2 on G protein coupling
G
M
GRK arr2
M
-PG
M
Morphine Antinociceptive Tolerance Hot plate (56C)
Bohn et al., (2000) Nature 408.
0 1 2 3 4 5 7 9 0
20
40
60
80
100
WT
arr2-KO
DAYS
% M
PE
Daily injection
0 1 2 3
0
20
40
60
80
100
WT placeboarr2-KO placebo
WT Morphinearr2- KO Morphine
DAYS
% M
PE
Pellet implantation
Daily ToleranceMice are injected daily with 10 mg/kg Morphine. 30 min after injection, test.
Pellet Implantation75 mg time-release Morphinepellet implanted s.c.
None
0
10
20
30
40
50
60
70
80
WT
arr2-KO
-8 -7 -6 -5
WT+Morphine
arr2-KO+Morphine
Log [DAMGO], M
% S
tim
ula
ted
35S
-GT
P S
bin
din
g
Bohn et al., (2000) Nature 408.
BrainstemMembranes
Log [DAMGO], M
arr2G
MM
-P
G
M
GRK
G protein coupling following chronic morphine
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?
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
μORDAR, μOR
Increased DA release = Increased Locomotor ActivityOpenfield Locomotor Activity- acute drug treatment
Morphine:
0 30 60 90 120 150 1800
250
500
750
1000
1250 Morphine 10mg/kg s.c.
arr2-HTarr2-KO
WT
Time (minutes)
Lo
com
oto
r A
ctiv
ity
(co
un
ts/
5 m
in)
5 10 20 40Sal 0
10000
20000
30000
40000WTarr2-KO
[Morphine], mg/kg, s.c.
Lo
com
oto
r A
ctiv
ity
Co
un
ts i
n 1
20 m
in
Dose Response
0 30 60 90 120 1500
500
1000
1500Cocaine (20 mg/kg, i.p.)
arr2-KOWT
Time (minutes)
Lo
com
oto
r A
ctiv
ity
(co
un
ts/
5min
)
10 20 30Sal
0
10000
20000
30000WT
arr2-KO
[Cocaine], mg/kg, i.p.
Bea
m B
reak
s in
90
minCocaine:
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?
0 30 60 90 1200
200
400
600
800
1000
1200
1400
arr2-KO-SWT-S
arr2-KOWT
Morphine (10 mg/kg, s.c.)
Time (minutes)
Lo
com
oto
r A
ctiv
ity
(co
un
ts/
5 m
in)
Locomotor Sensitization after Morphine and Cocaine
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 9Cocaine: Daily injections days 1-5, rest day 6, inject and test day 7
0 30 60 900
500
1000
1500
2000
2500
WT
arr2-KO-S
WT-Sarr2-KO
Cocaine (20 mg/kg, i.p.)
Time (min)
Lo
com
oto
r A
ctiv
ity
(co
un
ts/5
min
)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
0 20 40 60 80 100 120 140 160 1800
100
200
300WT+MOR
arr2-KO+MOR
WT+SALarr2-KO+SAL
Time (minutes)
DA
, % o
f b
as
al l
ev
el
0 20 40 60 80 100 120 140 160 1800
50
100
150
200
Time (minutes)
DO
PA
C, %
of
ba
sa
l le
ve
l
0 20 40 60 80 100 120 140 160 1800
50
100
150
200
Time (minutes)
HV
A, %
of
ba
sa
l le
ve
l
Dopamine Dopamine metabolites
Morphine, 10 mg/kg s.c.
Microdialysis in Striata of freely moving conscious miceExtracellular Dopamine levels following Morphine or Cocaine
Morphine= more DA release in arr2-KO mice
Cocaine= genotypes same
0 20 40 60 80 100 1200
100
200
300WT-COCarr2-KO-COC
WT-SALarr2-KO-SAL
Time (min)
DA
, % o
f bas
al le
vel
Cocaine, 20 mg/kg i.p.
Drug Reinforcement?
Conditioned Place Preference with Morphine and Cocaine
1 3 6 -100
0
100
200
300
400
500WTarr2-KO
Morphine (mg/kg, s.c.)
T
ime
Sp
ent
on
Mo
rph
ine-
Pai
red
Sid
e(s
eco
nd
s)
corncobbedding
tea bag
A B
1 2 3 4 5 6 7 8 Day
Pre-conditioning
record time spent in each compartment
record time spent in each compartment
Post-conditioningDrug
Salin
e
Drug
Drug
Salin
e
Salin
e
A A AB B B
Postconditioning time on drug side – Preconditioning time on drug side =
Preference for the side paired with Drug
0
100
200
300
400 WTarr2-KO
T
ime
Sp
ent
on
Co
cain
e-P
aire
d S
ide
(sec
on
ds)
Cocaine10 mg/kg i.p.
Morphine Cocaine
Locomotor ActivityNot enhanced,
DecreasedSlightly
Decreased
Sensitization Microdialysis Elevated DA Same DA
Conditioned Place Preference
More CPP Same CPP
The reinforcing properties of morphine are enhancedSpecific for morphine, not cocaine (dopamine)
Summary of Dopaminergic effects in βarr2-KO mice
GRK6 are supersenstivie to cocaine in the locomotor activity test:
0 30 60 90 1200
500
1000
1500
Cocaine (20 mg/kg, i.p.)WTGRK6-HETGRK6-KO
Time (min)
Hor
izon
tal a
ctiv
ity(d
ista
nce
in c
m/5
min
)
0 10 20 300
5000
10000
15000
Dose (mg/kg, i.p.)
Tota
l dis
tanc
e(c
m in
60
min
)
Gainetdinov, et al. Dopaminergic Supersensitivity in G Protein-Coupled Receptor Kinase 6 - Deficient Mice. (2003) Neuron 38:291-303.
GRK6-KO mice and cocaine:
0 30 60 90 1200
500
1000
1500
2000
2500 Cocaine (20 mg/kg, i.p.)
KOWT
Time (min)
Hor
izon
tal a
ctiv
ity(d
ista
nce
in c
m)
0 30 60 90 1200
500
1000
1500
2000
2500 Cocaine (20 mg/kg, i.p.)
KOWT
Time (min)
COCAINE INJ. 1 2 3 4 5 6 7 days
ActivityMonitor
ActivityMonitor
Sensitization
Day 1 Day 7
Cocaine Sensitization in GRK6-KO mice
-10 -9 -8 -7 -6 -5 -4
0
10
20
30
40
50
60 WTGRK6-KO
Log[Quinpirole], M
35S
-GT
P S
Bin
din
g(%
Sti
mu
lati
on
)
-12 -11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100 D2RD2R/GRK6
log [DA], M
35S
-GT
P S
Bin
din
g(%
Sti
mu
lati
on
)
-12 -11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100 D3RD3R/GRK6
log [DA], M
35S
-GT
P S
Bin
din
g(%
Sti
mu
lati
on
)
Striatum HEK-293 Cells
Effect of GRK6 on Dopamine Receptor Coupling
G
D
GRK arr1/2
D
-PG
D
What about Morphine?If the DA receptor is more sensitive then morphine should produce more locomotor activity too.
Morphine effects in GRK6-KO mice:
Horizontal Activity10 mg/kg i.p. Morphine
0 30 60 90 120 150 1800
500
1000
1500WTGRK6-KO
Time (minutes)
Bea
m B
reak
s(b
eam
bre
aks/
5 m
in) Morphine
(10 mg/kg, i.p.)
Horizontal Activity10 mg/kg i.p. Morphine
0 30 60 90 120 1500
300
600
900
1200
1500
1800 Morphine(10 mg/kg, i.p.)
WTGRK6-KOWT SensGRK6-KO Sens
Time (minutes)H
ori
zon
tal
Act
ivit
y(b
eam
bre
aks/
5 m
in)
Locomotor Activity
acute sensitization
GRK6-KO mice are supersensitive to morphine in the locomotor activity test.
Hotplate Latencies 56 CMorphine 10mg/kg i.p.
0 30 60 90 120
0
10
20
30
40 WTGRK6-KO
Time (minutes)
% M
PE
What about morphine effects on antinociception?
Morphine (10 mg/kg, i.p.)
In the hot plate analgesia test the genotypes look the same...
HotPlate (56 C) Tolerance Morphine10 mg/kg i.p./ day, 30 minutues
0 1 2 3 4 50
10
20
30
40WT
GRK6-KO
Days
% M
PE
Morphine Tolerance (10 mg/kg, i.p., daily)
•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
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. CaronRaul GainetdinovTatyana SotnikovaIvan Medvedev
Robert J. Lefkowitz Richard Premont
Linda A. DykstraPsychology/PharmacologyUNC-Chapel Hill
National Institute on Drug AbuseF32 DA06023 K01 DA 14600& Narcotic Supply
Duke University Medical Center
University of North Carolina- Chapel Hill
http://pharmacogenomics.osu.edu/section1.html
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
Receptor Signaling/ Agonist Efficacy Depends upon a Balance Between Activation and
Desensitization
G
M
GRK arr2
M
-PG
M
RF RB R* RP Rarr
[agonist] G coupling GRK
(activity, recruitment,specificity, levels?)
arrestin
resensitization
arr2-GFP arr1-GFP
Morp
hin
eE
torp
hin
e
0 05 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