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