Efficacy in CB1 Cannabinoid Receptor Signal Transduction

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Efficacy in CB1 Cannabinoid Receptor Signal
Transduction

• Allyn Howlett, Ph.D.
• Neuroscience/Drug Abuse Research Program
• J. L. Chambers Biomedical/Biotechnology Research
  Institute
• North Carolina Central University
• Supported by Natl Institute on Drug Abuse

NIDA November, 2003
Frontiers in Addiction Research
.
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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)

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CB1 Cannabinoid Receptor, A G-Protein Coupled
Receptor
EC2
EC3
EC1
extracellular
intracellular
IC1
IC2
IC3
3D structure recently determined (Biochemistry
2002, 41, 11344)
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Homology Model of the CB1 Receptor
extracellular
E2 loop as a part of binding site
intracellular
Biopolymers (Peptide Sciences), 2003, 71,
169-189
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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)

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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?

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Adenylyl Cyclase (types 5,6) Gi (1,2,3?)alpha
(types 1,3,8 to inhibit? Or types 2,4,7
to stimulate?)
250 ?
N18TG2 Cells
C6 glioma Cells
105 ?
75 ?
? CB1 R
50 ?
35 ?
CP52444 CP55940
D9-THC
CBN
CBD ()isomers
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Cannabinoid receptor agonists inhibit N-typeCa2
currents in differentiated N18 neuroblastoma cells
Mackie et al., Mol.Phm.4449893
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Cannabinoid Agonist-induced MAPK
Phosphorylation Signal Transduction via Gi/o
N18TG2 Neuroblastoma Cells
- - - Pertussis
Toxin
- Serum
MA WIN CP MA WIN CP
C6 Glioma Cells
- - - Pertussis
Toxin
- Serum
WIN55212
Methanan- damide
CP55940
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CP55940 and Methanandamide induce Nitric Oxide
(NO) production in N18TG2 neuroblastoma cells
Control
CP55940
Methanandamide
L-NNA Methanandamide
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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

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Peptides Derived from the Intracellular CB1
Receptor
Peptides Synthesized from the IL3 and C-terminal
Domains
CB1301 begins IL33 peptides span the loop CB1401
begins at membrane interface, extend beyond
cys-palmitoyl anchor
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Peptide CB1401 Disrupts the Association Between
CB1 Receptor and Gi3 but not Gi1 or Gi2
in Rat Brain Membrane Extracts
Gi1 Gi2
Gi3
Peptide 401 -
- -
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Peptide CB1401 Disrupts the CB1 Receptor
Association with Go but not Gi1/2
in Rat Brain Membrane Extracts
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Peptide CB1401 Disrupts the Association Between
CB1 Receptor and Gi3 but not Gi1 or Gi2
in Rat Brain Membrane Extracts
Gi1 Gi2
Gi3
Peptide 401 -
- -
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Peptides from IL3 Disrupt the CB1R Association
with Gi1 2 but not Gi3 in N18TG2 membrane
extracts
CB1R Gi alpha
IL3 peptides - -
-

• 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.

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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

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Conformational changes in the intracellular
surface may direct interaction with selective G
proteins
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Agonist Regulation of Gi/CB1R Association
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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

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CP55244 Binding Model Biopolymers (Peptide
Sciences), 2003, 71, 169
Assumption H-bonding between K3.28(192) and
phenolic OH
blue/green less lipophilic brown more lipophilic
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WIN55212-2 Binding Model
aroyl-up1
TM5
TM3
TM2
aroyl-down1
TM7
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WIN55212-2 and CP55244 Binding to CB1 Receptor
E(258)
K3.28(192)
Y5.39(275)
T5.38(274)
F5.42(278)
V3.32(196)
CP55244 WIN55212-2 H-bonding

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G-protein Activation Mechanism by Receptor
Conformational Change
By breaking H-bonding network
By breaking H-bonding network
WIN55212-2
CP55244
By breaking hydrophobic interaction
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Conformational Induction of R-G Complex by A
Response
Phosphorylation by GRK Arrestin
association Internalization
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Agonist Directed Trafficking of Signal
Transduction
Response 1
Response 2
A1
A2
Response 3
Inverse Agonist Response
A3
InA
InARiG
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• 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.

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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.

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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