# Presentation: 153648

## Efficacy in CB1 Cannabinoid Receptor Signal Transduction

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_**NIDA**_
**November, 2003**** **

**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**

***Frontiers in Addiction Research***

##  Cannabinoid Receptor Subtypes

**CB****1  **
** ****Found in neuronal cells and brain; other non-innervated tissue? **
**Regulates neurotransmitter release               **
**CB****1(A)**
**Splice variant mRNA found in human brain, ****	****	****	****but not predicted in rodent gene****	****	****(Sanofi Recherche)**
**Similar pharmacology and signal transduction as CB****1 **
**CB****2**
**Found in immune tissue (B cells, macrophages, T cells)**
**Activity not fully characterized**
**CB****??  ****or CB?**
**? Antinociceptive effects of anandamide in** **CB****1 ****(-/-) mice  (Martin)**
**? Vascular effects of anandamide not reproduced by other agonists ****	****	****	****	****	****	****	****(Kunos)**

## <footer>

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## CB1 Cannabinoid Receptor, 

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**CB****1**** Cannabinoid Receptor, **
**A G-Protein Coupled Receptor**** **

**EC1**

**EC2**

**EC3**

**IC1**

**IC2**

**IC3**

**3D structure recently determined **
**(*****Biochemistry***** 2002, 41, 11344****)**

**extracellular**

**intracellular**

**Notes:**
The CB1 receptor is shown in 2-D helical net representation.  
The highly conserved amino acid residues are represented in shaded circles. 
Guided by these highly conserved amino acid residues, we built a homology model of the CB1 receptor by using the x-ray structure of bovine rhodopsin as the template.  

## Homology Model of the CB1 Receptor

**Homology Model of the CB****1**** Receptor**

**  *****Biopolymers***** *****(Peptide Sciences)*****, 2003, 71, 169-189**

**E2 loop**
**as a part of **
**binding site**

**extracellular**

**intracellular**

**Notes:**
This is the CB1 receptor homology model viewed from the membrane side, facing TM3, which is colored in yellow. 
Because the second extracellular loop seemed to be involved in ligand binding interaction, 
We extensively searched the conformation of this loop.
White dots represent putative binding sites suggested from a binding site search.

After we constructed a CB1 receptor homology model, we performed docking of various ligands.  
We assumed that hydrophobic interactions is the driving force in the CB1 receptor-ligand binding in the following reasons. 

## Cannabinoid Receptor Agonists

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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)**

## Slide 7

## CB1 Receptor Signal Via Gi/o Proteins

- Signal Transduction Effector   		G protein 	Subunit
- Ion Channels
- 	K**+** currents				Gi (1,2,3? Via cAMP?)
- 	Ca**2+** currents			Gi  or Go      beta-gamma?
- Mitogen-Activated Protein Kinase	Gi (1,2,3?)   beta-gamma?
- 						or Go(1,2)?
- Other?
- 	PLA**2** ?  Ca**2+** mobility?   Focal Adhesion Kinase?   PI3Kinase?
-      NO synthesis?  Sphingomyelin hydrolysis and ceramide?

## Adenylyl Cyclase (types 5,6) Gi (1,2,3?)alpha

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**250  ******

**105 ******

**50 ******

**35 ******

**75 ******

**N18TG2 Cells**

**C6 glioma Cells**

****** ****CB****1 ****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-typeCa2+ currents in differentiated N18 neuroblastoma cells

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***Cannabinoid receptor agonists inhibit N-type******Ca******2+****** currents in differentiated N18 neuroblastoma cells***

**Mackie et al., Mol.Phm.44:498’93**

## C6 Glioma Cells

- <date/time>

-  **-   +**
**Serum**

**WIN55212**

**C6 Glioma Cells**

**Methanan-**
**damide**

**CP55940 **

***Cannabinoid Agonist-induced MAPK Phosphorylation***
***Signal 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

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***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 CB****1**** receptor selective for interaction with            G-proteins**
**Agonists can affect CB****1**** receptor –  G-protein association differentially**
**Speculation on conformational induction & G-protein activation**** **

## Peptides Derived from the Intracellular CB1 Receptor 

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***Peptides Synthesized from the IL3 and C-terminal Domains***

**CB****1****301 begins IL3;3 peptides span the loop**
**CB****1****401 begins at membrane interface, extend beyond cys-palmitoyl anchor**

***Peptides Derived from the Intracellular CB1 Receptor ***

## Peptide CB1401 Disrupts the Association Between CB1 Receptor and Gi3 but not Gi1 or Gi2                  in Rat Brain Membrane Extracts

***Peptide CB******1******401 Disrupts the Association Between CB******1****** 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 CB******1******401 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 CB******1******401 Disrupts the Association Between CB******1****** 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***
**CB****1**** 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.**
**CB****1**** IL3 domain is involved in the association**
** ****with Gi1 & Gi2  but not Gi3.**

## CB1 Receptor Signaling via G-proteins

**The domains of the CB****1**** receptor selective for interaction with            G-proteins**
**Agonists can affect CB****1**** 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

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**Conformational changes in the intracellular surface may direct interaction with selective G proteins**

## Agonist Regulation of Gi/CB1R Association

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***Agonist Regulation of Gi/CB******1******R Association***

## CB1 Receptor Signaling via G-proteins

**The domains of the CB****1**** receptor selective for interaction with            G-proteins**
**Agonists can affect CB****1**** 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**

**blue****/****green****: less lipophilic**
**brown****: more lipophilic**

**Notes:**
Based upon the proposed H-bond between the phenolic OH and the side chain N of K192 in the TM3, the putative binding conformation of CP55244 was determined.
This figure shows that the CP55244 binding site of the CB1 receptor represented by lipophilic potential.
From this docking model, the presumed hydrophobic pocket residues that interacts with the C3 side chain were primarily from TM3.

## WIN55212-2 Binding Model

***aroyl-down1 ***

***aroyl-up1***

**TM2**

**TM3**

**TM5**

**TM7**

**Notes:**
For the unprotonated form,
the aroyl-down conformation showed slightly better Correlation.

## WIN55212-2 and CP55244 Binding to CB1 Receptor

**WIN55212-2 and CP55244 Binding to CB****1 ****Receptor**

**F5.42(278)**

**V3.32(196)**

**E(258) **

**K3.28(192)**

**                 ****CP55244****	****	****WIN55212-2**
**H-bonding: ****	****	****	**

**Y5.39(275)**

**T5.38(274)**

**Notes:**
The nature of binding interactions are different for different ligands.

## 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**

**Notes:**
Rigid-body motion..takes msec or sec....
I plan to MD at high-temp. to induce conformational change.

## Conformational Induction of R-G Complex by A

**Response**

**Phosphorylation by GRK**
**Arrestin association**
**Internalization**

***Conformational Induction of R-G Complex by A***

## Agonist Directed “Trafficking” of Signal Transduction

**A1**

***Response 1***

**A2**

***Response 2***

**A3**

***Response 3***

***InA***

**InARiG**

***Inverse 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.
-  CB**1** 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**