efficacy in cb 1 cannabinoid receptor signal transduction. nida november, 2003 allyn howlett, ph.d....
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Efficacy in CB1 Cannabinoid Receptor
Signal Transduction
.
NIDANovember, 2003
Allyn Howlett, Ph.D.Neuroscience/Drug Abuse Research ProgramJ. L. Chambers Biomedical/Biotechnology Research InstituteNorth Carolina Central University
Supported by Natl Institute on Drug Abuse
Frontiers in Addiction Research
Cannabinoid Receptor Subtypes
• CB1
– Found in neuronal cells and brain; other non-innervated tissue?
– Regulates neurotransmitter release
• CB1(A)
– Splice variant mRNA found in human brain, but not predicted in rodent gene (Sanofi Recherche)
– Similar pharmacology and signal transduction as CB1
• CB2
– Found in immune tissue (B cells, macrophages, T cells)– Activity not fully characterized
• CB?? or CB?– ? Antinociceptive effects of anandamide in CB1 (-/-) mice (Martin)– ? Vascular effects of anandamide not reproduced by other agonists
(Kunos)
Herkenham et al. (1991) J. Neurosci. 11: 563
CB1 Cannabinoid Receptor
NH2
V
P
A
N
Q
S
T
N
I
Q
F
A
T
S
V
T
V
V
V
V
Y
D
I
A
F
SF
F
D
H
F
S
R
H
V
L
K
F
A
D
N
T
LP
T
V
L
F
F
S
V
S
K
S
V
G
A
N
W
I
M
G
C
L
A
F
L
A
W
K
P
I
C
K
V
T
V
L
V
L
L
I
E
I
L
L
TW
L
MY
F
YA
IY
AA
V
RL
I
K W
A
QT
GK
V
R
T
QV
ST
HSI
I
K DE
H
K
G
V
V
I
T
S
R
P
R H
Q
K A IY
S
D
V
T
S
M
A
L
R
A
L
I
F
Y
R
I
P
H
L
I
A
YI
SF
S
PH
CR
RL
C
H
S
V
R
V
SI
PO4
Q
R
V
S
VL
V
I
C
L
P
DY
FG
P
S
D
S
I
F
T
T
D
M
R
I
MQ
COOH
IL
LD
A
H
PC
LV
AI
R
S
Y
R
IL
WI
VL
K
S
R
L
FD
ML
GI
V
PF
K
A
G
D
VV
A
A
K
S
F
A
T
TP
YR
M
M
ML
G
SN
I
E
N
A
L
S
C
CN
V
T
K
I
FC
L
L
L
IK
VF
L
LLLG
LELLL
L
GL
LG
L
CB1 Cannabinoid Receptor, A G-Protein Coupled Receptor
EC1
EC2EC3
IC1
IC2
IC33D structure recently determined (Biochemistry 2002, 41, 11344)
extracellular
intracellular
Homology Model of the CB1 Receptor
Biopolymers (Peptide Sciences), 2003, 71, 169-189
E2 loopas a part of binding site
extracellular
intracellular
Cannabinoid Receptor Agonists• Classical Cannabinoid
(ABC-tricyclic)
• Nonclassical Cannabinoid (AC-bicyclic; ACD-tricyclic)
– CP55940; CP55244 (Pfizer)
• Aminoalkylindole– WIN55212-2 (Sterling
Research Inst.)
• Eicosanoid– Arachidonylethanolamide
(anandamide)– 2-Arachidonoylglycerol
• Aryl Pyrazole analogs
– Organon analogs (Razdan and Martin)
O H
O H
H OC P 5 5 9 4 0
A
C 123
45
6
7
89
1 0
1 11 2
1 "2 "
3 "
1 '2 '
3 '
4 '
5 '
6 '
7 '8 ' 9 '
O H
O H
H OC P 5 5 2 4 4
A
C 123
45
6
7
89
1 0
1 2
1 51 31 4
1 '2 '
3 '
4 '
5 '
6 '
7 '8 ' 9 '1 7
D
O H
O
T H C
B
C
A
123
45
7
89
1 '2 '
3 '
4 '
5 '
1 0
1 1
W I N 5 5 2 1 2 - 2
NO
O
NO
13
5
8
1 0
A n a n d a m i d e
NH
OO H
2-Arachidonylglyceryl ether (noladin ether)
Some Other EndoCannabinoids
O-Arachidonoyl ethanol ester(Virodhamine)
2-Arachidonoylglycerol(2-AG)
OOH
OH
O2.5 >141
OOH
OH
Devane et al. (1992) Science 258: 1946Mechoulam et al. (1995) Biochem. Pharmacol. 50: 83 Hanus et al. (2001) PNAS 98: 3662Porter et al. (2002) J. Pharmacol. Exp. Ther. 301: 1020
CB1/CB2 Affiniy Ratio
O
OH O
???
CB1 Receptor Signal Via Gi/o Proteins
Signal Transduction Effector G protein Subunit
Ion Channels K+ currents Gi (1,2,3? Via cAMP?)Ca2+ currents Gi or Go beta-gamma?
Mitogen-Activated Protein Kinase Gi (1,2,3?) beta-gamma? or Go(1,2)?
Other?
PLA2 ? Ca2+ mobility? Focal Adhesion Kinase? PI3Kinase? NO synthesis? Sphingomyelin hydrolysis and ceramide?
250
105
50
35
75
N18TG2 Cells
C6 glioma Cells
CB1 R
CP52444
CP55940 9-THC
CBN
CBD
(+)isomers
Adenylyl Cyclase (types 5,6) Gi (1,2,3?)alpha (types 1,3,8 to inhibit? Or types 2,4,7 to stimulate?)
Cannabinoid receptor agonists inhibit N-typeCa2+ currents in differentiated N18 neuroblastoma cells
Mackie et al., Mol.Phm.44:498’93
- + Serum
WIN55212
C6 Glioma Cells
Methanan-damide
CP55940
Cannabinoid Agonist-induced MAPK PhosphorylationSignal Transduction via Gi/o
+ - + - + - Pertussis Toxin
N18TG2 Neuroblastoma Cells
- - - + + + Pertussis Toxin- + Serum
MA WIN CP MA WIN CP
CP55940 and Methanandamide induce Nitric Oxide (NO) production in N18TG2 neuroblastoma cells
Control CP55940
Methanandamide L-NNA + Methanandamide
CB1 Receptor Signaling via G-proteins
• The domains of the CB1 receptor selective for interaction with G-proteins
• Agonists can affect CB1 receptor – G-protein association differentially
• Speculation on conformational induction & G-protein activation
Peptides Synthesized from the IL3 and C-terminal Domains
CB1301 begins IL3;3 peptides span the loop
CB1401 begins at membrane interface, extend beyond cys-palmitoyl anchor
Peptides Derived from the Intracellular CB1 Receptor
GK
ST
HS
I II
K
DEC3
I7.31(375)
F
L
V
L
KI
C
N
F
L
T
CS
K
A
LM
N
F
M
AV
I
V
G
KG
LM
D
WL
IY
CPM
H5.34(270)
T
I
I
G
D
F
F
Y
S
D
V
H
P
WL
C
E
TI
VSQ
L
L
IV
V
P
K
T
L
I
G
AL
VI
NS
YP
T
IV
TL
V
R
I
KV
L
IA
LI
DL
S
VL
V
MY
FL
YA
IL
YA
L
AV
C
RL
I
KW
A
QT
V
RT
QV
P
RA
D
Q
M
COOH
R6.28(336)
LRA
S
K
DAL M
FR FRH
S SP
EC
GT
A
W
H
E
W
K
G
A
C
V
V
N
I
I
T
F
S
M
R
P
R
H
Q
K
V
G
S
A
A
K
I
Y
S
F
S
C2
V
D
R
S
V
L
T
S
M
A
D
F
L
L
R
T
R
N
V
K
A
L
I
T
F
F
A
Y
R
I
P
R
H
L
I
HH
V
F
L
F
S
F
F
A
S
D
Y
D
I
NH2
V
IY
A
S
I
F
V
SV
P
H
S
CR
VA
T
R
Q
T
N
I
F
L
C
QP
C1
S
H
A
S
T
V
R
VN
S
V
I
L
LLLG
LELLL
L
GL
LG
G
GL
E1
E2E3
Extracellular side
Cytoplasmic side
TM1 TM3TM5
TM7N1.28(112)
S1.60(144)
H2.68(181)
R2.37(150)
D3.20(184)
H3.55(219)T4.38(229)
L4.62(253)
I5.61(297)S7.57(401)
Gi1 & Gi2 Gi3 & Go
G6.61(369)
Peptide CB1401 Disrupts the Association Between CB1 Receptor and Gi3 but not Gi1 or Gi2 in Rat Brain Membrane Extracts
Peptide 401: - + - + - +
Gi1 Gi2 Gi3
Peptide CB1401 Disrupts the CB1 Receptor Association with Go but not Gi1/2 in Rat Brain Membrane Extracts
Peptide CB1401 Disrupts the Association Between CB1 Receptor and Gi3 but not Gi1 or Gi2 in Rat Brain Membrane Extracts
Peptide 401: - + - + - +
Gi1 Gi2 Gi3
Peptides from IL3 Disrupt the CB1R Association with Gi1 & 2 but not Gi3 in N18TG2 membrane extracts
IL3 peptides: - + - + - +
CB1R
Gi alpha
Conclusions•CB1 Receptor-G alpha complexes exist in the absence of agonists, but can be disrupted by pertussis toxin or GTP analogs.•The juxtamembrane C-terminal domain is involved in the association with Go & Gi3, but not Gi1 & G2.•CB1 IL3 domain is involved in the association
with Gi1 & Gi2 but not Gi3.
CB1 Receptor Signaling via G-proteins
• The domains of the CB1 receptor selective for interaction with G-proteins
• Agonists can affect CB1 receptor – G-protein association differentially
• Speculation on conformational induction & G-protein activation
Conformational changes in the intracellular surface may direct interaction with selective G proteins
0
20
40
60
80
100
10 100 1000
i1
Log [WIN 55212-2] (nM)
Rel
ativ
e D
ensi
tyG
i/C
B1R
i3 i2
0
20
40
60
80
100
10 100 1000
i2
i1
i3
Log [DALN] (nM)
Rel
ativ
e D
ensi
tyG
i/C
B1R
0
20
40
60
80
100
10 100 1000
i3
i2 i1
Log [Methanandamide] (nM)
Rel
ativ
e D
ensi
tyG
i/C
B1R
Agonist Regulation of Gi/CB1R Association
CB1 Receptor Signaling via G-proteins
• The domains of the CB1 receptor selective for interaction with G-proteins
• Agonists can affect CB1 receptor – G-protein association differentially
• Speculation on conformational induction & G-protein activation
CP55244 Binding Model Biopolymers (Peptide Sciences), 2003, 71, 169
Assumption: H-bonding between K3.28(192) and phenolic OH
OH
OH
HO +H3NK3.28(192)
blue/green: less lipophilicbrown: more lipophilic
WIN55212-2 Binding Model
aroyl-down1
aroyl-up1
TM2
TM3
TM5
TM7
WIN55212-2 and CP55244 Binding to CB1 Receptor
F5.42(278)V3.32(196)
E(258)
K3.28(192)
CP55244 WIN55212-2
H-bonding:
Y5.39(275)
T5.38(274)
G-protein Activation Mechanism by Receptor Conformational Change
By breaking H-bonding network
By breaking hydrophobic interaction
By breaking H-bonding network
CP55244
WIN55212-2
Response
Phosphorylation by GRK
Arrestin association
Internalization
Conformational Induction of R-G Complex by A
A1
Response 1
A2
Response 2
A3
Response 3
InAInARiGInverse Agonist Response
Agonist Directed “Trafficking” of Signal Transduction
`• Signal transduction pathways will depend
upon the G-proteins and effector pathways present in the cell.
• Domain specificity for G-proteins suggests that induction or selection of different conformations of the CB1 receptor can direct selective signal transduction pathways.
• CB1 receptor signaling through a given pathway may be directed by agonist-specific conformational changes in the receptor.
Prospectus
• Few CB receptor subtypes limits use of pharmacophoric distinctions in ligand affinities to separate therapeutic from untoward effects.
• Can we develop agonists that induce receptor conformations that activate specific G proteins ?
• Manipulation of G protein coupling may promote signal transduction pathways limited to cell types that regulate therapeutic responses.
Collaborators & Acknowledgements
• JLC-BBRI at NCCU
Derek Norford, Skyla Carney, Abdel-Azim Assi
John Joong-Youn Shim
Somnath Mukhopadhyay
CMDNJ-RWJMS William Welsh
J Nehru Univ, Delhi Sudha Cowsik
• $$ National Institute on Drug Abuse