Sponsored by:

Participating Experts:Jeffrey Conn, Ph.D.Vanderbilt University Medical CenterNashville, TN

Michel Bouvier, Ph.D. Institute for Research in Immunology and Cancer (IRIC)University of MontrealMontreal, Canada

Brian Kobilka, M.D. Stanford University School of MedicineStanford, CA

Brought to you by the Science/AAAS

Business Office

Webinar SeriesWebinar SeriesScienceScienceAdvances in GCPR ResearchAdvances in GCPR Research

17 June, 200817 June, 2008

Allosteric

Potentiators

of GPCRs

as a Novel Approach to Treatment of CNS Disorders

P. Jeffrey Conn

Department of PharmacologyVanderbilt Program in Drug Discovery

Vanderbilt University School of MedicineNashville, Tennessee USA

GPCRs

Largest class of cell-surface receptors; three major subclasses.

Critical roles in virtually every organ system

Activated by a diverse range of ligands, (hormones, neurotransmitters, ions, odorants, and photons of light)

Couple to a host of G proteins and activate a wide range of signaling molecules and effector

systems

Involved in multiple human disease states.

Targets of approximately 50% of all modern drugs

Encoded by >1,000 genes yet synthetic ligands

exist for only a small fraction of these

Recent Advances and Directions of GPCR Research

Major advances in understanding of GPCR structure and associated

function

Increasing appreciation of roles of homo-

and heterodimers

Increased understanding of complex signaling pathways, including

G protein-independent signaling mechanisms.

Discovery of drug-like allosteric

modulators that provide high selectivity and novel modes activity at GPCRs.

Schizophrenia

• Positive symptoms: paranoia, hallucinations, delusions, thought disorder.

• Negative symptoms: loss of motivation, blunted affect, withdrawal, anhedonia.

• Cognitive disturbances: impaired attention, executive function, working memory

Activation of mGluR5 for treatment of Schizophrenia

Glu

GluGlu

GluT

(+)

mGluR5M1mAChR

AMPA/KAINATE

NMDA

Glu Glu

Glu GlyGly

GlyT

GlyGlu

(+)

GPCR

GluGlu

GluGlu

GluT

(+)

mGluR5M1mAChR

AMPA/KAINATE

NMDA

GluGlu GluGlu

Glu GlyGly

GlyT

GlyGlu

(+)

GPCR

Allosteric

potentiators

of mGluR5

NN NH

ON

-9 -8 -7 -6 -5 -4

0

20

40

60

80

100

1 μM CDPPB 0.1 μM CDPPB

Vehicle 0.01 μMCDPPB

Log [Glutamate] (M)N

orm

aliz

ed F

luor

esce

nce

(%M

ax_G

lu)

CDPPB is a Novel Systemically Active Allosteric Potentiator of mGluR5

- Highly selective for mGluR5

- Potentiates

effects of endogenous glutamate on in CNS neurons

- Has antipsychotic-like effects in animal models

M u ltip le W e ll A v e r a g e

10 μM DFB plus 300 nM Glutamate

0.67% DMSO plus 300 nM Glutamate

Allosteric

potentiators

of mAChRs

III

III

IV

V

VI

VII

Acetylcholine (orthosteric) binding site

Putative allostericbinding site(s)

III

III

IV

V

VI

VII

Acetylcholine (orthosteric) binding site

Putative allostericbinding site(s)

III

III

IV

V

VI

VII

Acetylcholine (orthosteric) binding site

Putative allostericbinding site(s)

VU10010 is an Allosteric

Potentiator

of the M4 Muscarinic

Receptor

- Increases ACh affinity and efficiency of coupling to G proteins- Selectively potentiates M4 responses at specific synapses- Related compounds have efficacy in animal models of antipsychotic activity.

N S

NH2

HN

O

Cl

rM1

00

20

40

60

80

100

120

vehicle10uM DO12

-12 -11 -10 -9 -8 -7 -6 -5 -4Log [ACh] M

Nor

mal

ized

Flu

ores

cenc

e(%

Max

AC

h R

espo

nse)

rM2-Gqi5

00

20

40

60

80

100

120

-12 -11 -10 -9 -8 -7 -6 -5 -4Log [ACh] M

Nor

mal

ized

Flu

ores

cenc

e(%

Max

AC

h R

espo

nse)

rM3

00

20

40

60

80

100

120

-12 -11 -10 -9 -8 -7 -6 -5 -4Log [ACh] M

Nor

mal

ized

Flu

ores

cenc

e(%

Max

AC

h R

espo

nse)

rM5

00

20

40

60

80

100

120

-12 -11 -10 -9 -8 -7 -6 -5 -4Log [ACh] M

Nor

mal

ized

Flu

ores

cenc

e(%

Max

AC

h R

espo

nse)

rM1 rM2-Gqi5

rM3 rM5

rM1

00

20

40

60

80

100

120

vehicle10uM DO12

-12 -11 -10 -9 -8 -7 -6 -5 -4Log [ACh] M

Nor

mal

ized

Flu

ores

cenc

e(%

Max

AC

h R

espo

nse)

rM2-Gqi5

00

20

40

60

80

100

120

-12 -11 -10 -9 -8 -7 -6 -5 -4Log [ACh] M

Nor

mal

ized

Flu

ores

cenc

e(%

Max

AC

h R

espo

nse)

rM3

00

20

40

60

80

100

120

-12 -11 -10 -9 -8 -7 -6 -5 -4Log [ACh] M

Nor

mal

ized

Flu

ores

cenc

e(%

Max

AC

h R

espo

nse)

rM5

00

20

40

60

80

100

120

-12 -11 -10 -9 -8 -7 -6 -5 -4Log [ACh] M

Nor

mal

ized

Flu

ores

cenc

e(%

Max

AC

h R

espo

nse)

rM1 rM2-Gqi5

rM3 rM5

00

25

50

75

100

125

150

175vehicle

-12 -11 -10 -9 -8 -7 -6 -5 -4

10uM DO12

log [ACh] M

Nor

mal

ized

Flu

ores

cenc

e(%

Max

AC

h R

espo

nse)

rM4-Gqi5

VU10010

00

25

50

75

100

125

150

175vehicle

-12 -11 -10 -9 -8 -7 -6 -5 -4

10uM DO12

log [ACh] M

Nor

mal

ized

Flu

ores

cenc

e(%

Max

AC

h R

espo

nse)

rM4-Gqi5

VU10010

-11 -10 -9 -8 -7 -6 -5 -4-10

10

30

50

70

90

110

130

VU0152099

Vehicle

Log [Carbachol] (M)

% M

ax R

espo

nse

M4

mAChR5HT2A

-11 -10 -9 -8 -7 -6 -5 -4-10

0102030405060708090

100110

VU0152099Vehicle

Log [α-Methyl-5HT] (M)

% M

ax R

espo

nse

5HT2C

-11 -10 -9 -8 -7 -6 -5 -4-10

0102030405060708090

100110120130

VU0152099

Vehicle

Log [α-Methyl-5HT] (M)

% M

ax R

espo

nse

Dopamine D1

-11 -10 -9 -8 -7 -6 -5 -4-10

0102030405060708090

100110120

VU0152099

Vehicle

Log [A68930] (M)

% M

ax R

espo

nse

Adenosine A2B

-11 -10 -9 -8 -7 -6 -5 -4-10

0102030405060708090

100110

VU0152099

Vehicle

Log [NECA] (M)%

Max

Res

pons

e

Histamine H2

-11 -10 -9 -8 -7 -6 -5 -4-10

0102030405060708090

100110

VU0152099

Vehicle

Log [Amthamine] (M)

% M

ax R

espo

nse

Millipore GPCR Profile -

Selectivity Across Family A GPCRs

Family A GPCRs

tested:

Muscarinic

M1 –

M5Adrenergic α1A

, α2A

, α2BDopamine D1, D2, D5Histamine H1

, H2, H3Serotonin 5HT1A

, 5HT2A

, 5HT2B,

5HT2cWeak serotonin 5HT2B

antagonist

Properties of Allosteric

Ligands

•

Allosteric

modulators of multiple GPCRs

have been identified.

•

Different allosteric

modulators can act at distinct sites on a single receptor.

•

Allosteric

modulators can act by regulating agonist affinity or by altering ability to couple to G proteins.

•

Allosteric

modulators can have a range of activities from antagonist to potentiator

and can include neutral ligands.

•

It is possible to develop “partial antagonists”

by targeting allosteric

sites.

•

Allosteric

modulators can differentially regulate coupling of mGluR5 to different signaling pathways.

•

Allosteric

modulators for multiple GPCR have robust activity in vivo.

Vanderbilt Program in Drug DiscoveryP. Jeffrey Conn, Director

HTS LabDave Weaver

Emily DaysDaniel Dorset

Christopher FarmerMichelle LewisKate Lornsen

Dehui MiTasha Nalywajko

Rey RedhaMichael Williams

Carrie Jones*Jennifer AyalaRandy BarrettAshley Brady*

Yelin ChenThomas EkmanAlexis Hammond

Mark GrierDonna Ingram

Paulianda JonesCherry LuoJoy Marlo

Colleen NiswenderNicole MillerKari Johnson

Alice Rodriguez Doug ShefflerJana Shirey*

Analisa ThompsonDaryl Venable

Zixiu XiangMeng Xianzhang

Alex Kane

Pharmacology/Neuroscience Med ChemCraig Lindsley*

Tom BridgesGraeme DennisPhil Kennedy*Evan LeBois

Stacey LindsleyUyen

LeKwangho

KimMatt Mulder

Tomas dePaulisDarren Orton*

Sameer SharmaLyndsey WilliamsRichard Williams

Sandra Zhu

Supported by NINDS, NIMH, MJFF, NARSAD, and Stanley Foundation,NIH MLSCN Network.

DMPKSatyawan

Jadhav*Huiyong

Yin*Usha

Menon*Vanderbilt Metabolic Core

Sponsored by:

Brought to you by the Science/AAAS

Business Office

Webinar SeriesWebinar SeriesScienceScienceAdvances in GCPR ResearchAdvances in GCPR Research

17 June, 200817 June, 2008

Participating Experts:Jeffrey Conn, Ph.D.Vanderbilt University Medical CenterNashville, TN

Michel Bouvier, Ph.D. Institute for Research in Immunology and Cancer (IRIC)University of MontrealMontreal, Canada

Brian Kobilka, M.D. Stanford University School of MedicineStanford, CA

Michel BouvierDepartment of Biochemistry,

Institute for Research inImmunology and Cancer, Drug Discovery Group,Université de Montréal

Detecting GPCR Ligand-Biased Signalling

Conventional GPCR Signaling

N

C

I II III IV V VI VII

α γβ

AdenylateCyclase

Ca++

ChanelPhospholipase C

Etc…

Agonist

InverseAgonist

Antagonist

full

partial

full

partial

Log [ligand]

Act

ivity

0

Ligands Efficacy

GPCR as Signaling Networks or Signalosomes

AdenylateCyclase

Ca++

ChanelPhospholipase C

C

N

IIIIIIIVVVIVII

N

C

I II III IV V VI VII

PDZ NHERF

PDZ cNOS

pPRONck

Grb2

pPROhomer

βarr

PP

ARFRho

SH3

SH3

SH2PLCγ1

T/S-x-V/I/L C

T/S-x-V/I/L

YIPPSHP-2α γβ

etc

β2

AR signalling

to Adenylyl

Cyclase

and MAPK

AC activity

0

20

40

60

80

100

% o

f bas

al a

ctiv

ity(c

AM

P ac

cum

ulat

ion)

-100

-75

-50

-25

0

cAM

P ac

cum

ulat

ion

(% o

f FK

inhi

bitio

n)

Met Bis AtPropBuc Carv

Iso Lab

ERK1/2 activity

Iso Lab Buc Carv Prop Met Bis At Basal

0102030405060708090

100

% o

f is

opro

tere

nol

resp

onse

Galandrin et al. Mol. Pharm. 2006

AC activity

0

20

40

60

80

100

% o

f bas

al a

ctiv

ity(c

AM

P ac

cum

ulat

ion)

-100

-75

-50

-25

0

cAM

P ac

cum

ulat

ion

(% o

f FK

inhi

bitio

n)

Met Bis AtPropBuc Carv

Iso Lab

ERK1/2 activity

Iso Lab Buc Carv Prop Met Bis At Basal

0102030405060708090

100

% o

f is

opro

tere

nol

resp

onse

-75

-50

-25

0

% o

f PM

A in

hibi

tion

Met Bis At

PMA + + + +-Met Bis At

PMA

Galandrin et al. Mol. Pharm. 2006

β2

AR

ERK1/2 activity

Pluridimensional

Efficacy Pattern of the β2

ΑR

AC activity

Iso

AC activity

Two-State Model Theoretical efficacy

AC

Inv- ERKAgo

AC

Ago- ERKInv

AC

Ago- ERKAgo

AC

Inv- ERKInv

ERK1/2 activity

Iso

Lab

Buc

CarvProp

At

MetBis(-)

(+)

(+)

(-)

Pathway Linking β2AR to ERK1/2 Activation

β2AR

Iso

ERK1/2

βarr

c-Src

Gs

βγGi/o

β2AR

ICIProp

ERK1/2

βarr

c-Src

Gs

βγGi/o

Monitoring βarrestin

Recruitment

by BRET

Bio

lum

ines

cenc

e

Wavelength

(nm)400 500

Bio

lum

ines

cenc

e

Wavelength

(nm)400 500

βarrestinRluc

GFP

GFPβarrestinRluc

Activation

0.00

0.02

0.04

0.06

0.08

0.10

BR

ET

rat

io

β2

AR-GFP

* **

**

#

Basal ICI Pro Iso Iso+Pro

Read out X

Read out Z

Read out Y

(3,2,-2)

(a)

(b)

Coordinates of efficacy:

(RE x, RE

y

, RE

z

)

The number of compound classes with distinct efficacy profiles detected (C) increases exponentially with the number of read out considered (n)

such that

C = 2n

Fig.2

Ligand 1 Ligand 3Ligand 2

G protein

dependent

signallingEndocytosis

Gαs

Gαq/11

A

R

C B

R

Gαi/o

(a)

βarr

GRK

AP2

PDZ proteins

Jak

βarr

DynPKCPKA

Clathrin

G protein dependent signalling

Desensitization Endocytosis

G protein independent signalling

A

R

B

R

Endocytosis

Gα12/13

βarr

GIP

G protein

dependent

signallingG protein

independent

signalling

BRET based Biosensor EPAC

RLuc

GFP2

RLuc

GFP2

cAMP

cAMP

binding domain of EPAC fused to Luciferase

and GFP2

δOR

0.29

0.30

0.31

0.32

0.33

- -15 -12 -9 -6 -3∞

Forskolin

(10 uM)PBS

[SNC] (M)

BR

ET

2

V2R

-18 -15 -12 -9 -6 -3

0.3

0.4

0.5

EC50

= 0.025 nM

ForskolinePBS

BR

ET

2

0 5 10 15 20 25 300.18

0.20

0.22

0.24

0.26

0.28

0.30

PBSForskolin

(100 μM)

Time (min)

BR

ET2

[AVP] (M)

CaCa2+2+

Biosensor: Biosensor: ObelinObelin

•

Obelin

is a Ca2+-dependent luciferase, more responsive to variations in Ca2+

levels and less affected by Mg2+

levels than Aequorin.

GABAb

receptorObeline

Ca2+

Ca2+

Ca2+

Coelenterazine

O2

Light

Emission (Fig 1.)

Obelia

Longissima

VéhiculeGABA 1 mM

0 10 20 30 40 50 60 70 80 900

10000

20000

30000

40000

50000No PTU73122U73343

Time (s)

RLU

DeepBlueC 510nmβ1

αi1γ2GFP

hRLuc510 nm

αi1β1

γ2GFP

Luc

IsoBuc Prop

-0.01

0.00

0.01

0.02

0.03

ligan

d-pr

omot

edB

RET

Monitoring Ligand-biased efficacy

Gαi1

-91hRluc/β1

AR-GFP10 Gαi1

-91hRluc/GFP-Gγ2

Liga

nd-p

rom

oted

BR

ET Iso

BucProp

-0

075

-0.050

-0.025

-0.000

0.025

ERK1/2

AC

Iso BucProp

0

20

40

60

80

100

-100

-80

-60

-40

-20cAM

Pac

cum

ulat

in(

Iso Buc Prop0

10

20

30

4050

100

ERK

1/2

activ

atio

n

xCELLigence

technologyN

orm

alis

ed C

IN

orm

alis

ed C

I

Histamine H1 receptor (Gq)

Nor

mal

ised

CI

Dopamine 1 receptor (Gs)

5HT1 receptor (Gi)

SKF 38393

1nMSKF 38393

100nM

SKF 38393 0.1nMCTR

His 30nMHis 30μM

His 30pMCTR

8-OH-DPAT

18nM8-OH-DPAT 100nM

8-OH-DPAT 0.1nM

www.roche-applied-science.com

CTR

Collaborators

Mounia AzziGraciela PineyroPascale CharestSégolène GalandrinGeneviève Oligny LongpréKoji OgawaHélène BoninCéline GalésMireille HogueMartin AudetMonique Lagacé

Bouvier Lab

Guy RousseauSacré-Cœur Hospital Montreal

Duke UniversityTrudy KohoutRobert. J. Lefkowitz

INSERM U388 (Toulouse)Hervé

ParisFunding

Sponsored by:

Webinar SeriesWebinar SeriesScienceScienceAdvances in GCPR ResearchAdvances in GCPR Research

17 June, 200817 June, 2008

Participating Experts:Jeffrey Conn, Ph.D.Vanderbilt University Medical CenterNashville, TN

Michel Bouvier, Ph.D. Institute for Research in Immunology and Cancer (IRIC)University of MontrealMontreal, Canada

Brian Kobilka, M.D. Stanford University School of MedicineStanford, CA

Brought to you by the Science/AAAS

Business Office

Structure and Dynamics of the Human β2

Adrenoceptor

Brian KobilkaStanford University 

•Mediates cadiovascular

and smooth muscle response to adrenaline and noradrenaline

•Signals through Gs, Gi

and arrestin

•Resides in specific plasma membrane microdomains

with other signaling molecules 

•Undergoes agonist‐induced internalization and trafficking to different cellular compartments

•Exists in homo‐

and hetero‐oligomeric

forms in the plasma membrane

•Exhibits a moderate level of basal, agonist independent activity

•Available ligands

exhibit a spectrum of efficacies ranging from full agonists to inverse agonists

The β2

AR is a versatile signaling molecule

Chimeric

receptors and site‐directed mutagenesis

Identify domains involved in ligand binding and G protein coupling specificity

Protein crystallography

Determine the three‐dimensional structure of an inactive state of the β2

AR

Fluorescence spectroscopy

Characterize conformational changes induced by agonists and partial agonists

Characterize the dynamic properties of the receptor

Approaches to characterize the structural basis  for the functional behavior of the β2

AR

Enabling β2

AR Crystallization

Modifications to limit 

heterogeneity

Enabling β2

AR Crystallization

Deglycosylate

Modifications to limit 

heterogeneity

Enabling β2

AR Crystallization

DeglycosylateCarboxyl terminal truncation

Modifications to limit 

heterogeneity

Enabling β2

AR Crystallization

DeglycosylateCarboxyl terminal truncationStabilize dynamic interface

Modifications to limit 

heterogeneity

Enabling β2

AR Crystallization

DeglycosylateCarboxyl terminal truncationStabilize dynamic interface

Modifications to limit 

heterogeneity

Carazolol

(inverse agonist)

Enabling β2

AR Crystallization

DeglycosylateCarboxyl terminal truncationStabilize dynamic interface

Modifications to limit 

heterogeneity

Carazolol

(inverse agonist)

Fab

or T4lysozyme fusion

Enabling β2

AR Crystallization

DeglycosylateCarboxyl terminal truncationStabilize dynamic interface

Modifications to limit 

heterogeneity

β2

AR β2

AR

T4LysozymeFAb5

Carazolol

(inverse agonist)

Fab

or T4lysozyme fusion

β2

AR β2

AR

Enabling β2

AR Crystallization

DeglycosylateCarboxyl terminal truncationStabilize dynamic interface

Modifications to limit 

heterogeneity

T4LysozymeFAb5

Carazolol

(inverse agonist)

Fab

or T4lysozyme fusion

β2

AR‐Fab5 β2

AR‐T4L

2.4Å3.4‐3.7Å

Wild‐type antagonist binding affinity

Wild‐type agonist affinity Increased agonist affinity

Functional Properties

Undergo agonist‐induced conformational changes

β2

ARRhodopsin

ECL2

Comparison of β2

AR and Rhodopsin

Carazolol Retinal

ECL2

β2

ARRhodopsin

ECL2

Comparison of β2

AR and Rhodopsin

Carazolol Retinal

Open accessRestricted 

access ECL2

Rhodopsin

ER

TM3

TM5

TM6

E

Y

Cytoplasmic

face

•Conserved E/DRY sequence                 

Family A GPCRs.  

•Stabilizes inactive state of       

rhodopsin.

Ionic Lock

β2

AR‐T4L

Rhodopsin

E268

R131

TM3

TM5

TM6

D130

Cytoplasmic

face

Ionic Lock – open in β2

AR

ER

E

β2

AR‐Fab

β2

AR‐T4L

Rhodopsin

E268

R131

TM3

TM5

TM6

D130

E268

Cytoplasmic

face

Ionic Lock – open in β2

AR

ER

E

β2

AR‐Fab

β2

AR‐T4L

Rhodopsin

E268

R131

TM3

TM5

TM6

D130

E268

Cytoplasmic

face

Ionic Lock – open in β2

AR

ER

E

•Artifact of crystallography?•Carazolol‐specific conformation?•One of several basal conformations?

Structural determinants of β1

AR and β2

AR  binding specificity

Carazolol

binding pocket

Carazolol

binding pocket

Amino acids 

differences 

between β1

AR 

and β2

AR

Structural determinants of β1

AR and β2

AR  binding specificity

Amino acids 

differences 

between β1

AR 

and β2

AR

Carazolol

binding pocket

Structural determinants of β1

AR and β2

AR  binding specificity

Rhodopsin

β2

AR

W6.48D2.50

N7.49

Y7.53

Role of Conserved 

Water Pocket

P6.50

Signal transduction:coupling agonist 

binding to G protein  activation

W6.48

I2.43

S3.39

D2.50

S7.46

N7.45

N7.49

I6.40

Y7.53

N1.50

Hydrogen bonding network in water pocket

TM7

TM6TM3

TM2

TM2

W6.48

I2.43

S3.39

D2.50

S7.46

N7.45

N7.49

I6.40

Y7.53

N1.50

TM7

TM6TM3

TM2

TM2

Agonist

G protein

Hydrogen bonding network in water pocket

Challenges in obtaining an active structure

Agonists – low affinity, chemically 

unstable

Activating mutations enhance agonist 

affinity, but are often  associated with 

biochemical instability

There may be multiple “active states”

Agonists and partial agonists induce 

conformational heterogeneity

Fully active β2

AR may require G protein

β2

AR:Gs 1:1 or 2:1?

Fluorescence SpectroscopyTrp

quenching of Bimane

Ligand‐induced conformational changes

Bimane

Tryptophan

15 Å

5 6

3

33

6655

Fluorescence SpectroscopyTrp

quenching of Bimane

Ligand‐induced conformational changes

Bimane

Tryptophan

15 Å

5 6

3

33

6655•Site‐specific labeling of single 

reactive cysteines

with Bimane

Fluorescence SpectroscopyTrp

quenching of Bimane

Ligand‐induced conformational changes

Bimane

Tryptophan

15 Å

5 6

3

33

6655 W•Site‐specific labeling of single 

reactive cysteines

with Bimane

•Site‐specific addition of 

tryptophan

Fluorescence SpectroscopyTrp

quenching of Bimane

Ligand‐induced conformational changes

Bimane

Tryptophan

15 Å

33

6655 W•Site‐specific labeling of single 

reactive cysteines

with Bimane

•Site‐specific addition of 

tryptophan

•Tryptophan

quenches bimane

within 5‐15Å

radius

•Conformational changes 

detected by changes in bimane

intensity and lifetime

•Fluorescence lifetime of 

bimane

experiments provides 

information about number and 

distribution of distinct 

conformational states 

33

6655W

Agonist‐induced movement of TM3, 5 and 6

Increase distance

Decrease distance

TM6

TM3

A271

H269

V222

T136

A226

I135

TM5

F223

Fluorescence lifetime 

experiments:1. Provide evidence that 

the β2

AR exists in more 

than one conformational 

state in the absence and 

presence of agonists.

2. Agonists and partial 

agonists induce/stabilize 

distinct conformational 

states.

BimaneTryptophan

Generating diffraction quality crystals of the β2

AR required:

•Structural modifications to minimize heterogeneity

•An antibody or T4Lysozyme insertion to stabilize the receptor and increase polar 

surface area

•Lipid environment (bicelles

and lipidic

cubic phase)

•Microfocus

crystallography

β2

AR crystal structures•Similarities with rhodopsin

•Overall topology•Overlapping binding site•Conserved water pocket

•Notable differences•Second extracellular

loop (access to ligand

binding pocket)

•Open “ionic lock”•Crystal structures do not inform us about active state or unstructured sequences

Fluorescence spectroscopy studies

•Reveals ligand‐specific movement of transmembrane

domains

•Demonstrate multiple conformations in equilibrium.

Summary

Kobilka

LabDan RosenbaumSoren

RasmussenFoon

Sun ThianTong Sun KobilkaJuan Jose FungXiao Jie

YaoMike BokochPeter DaySebastien

Granier

CollaboratorsStanford

Bill WeisHee-Jung Choi

MRC, CambridgeGebhard SchertlerPat Edwards

ID-13 ESRFManfred Burghammer

GM/CA-CAT APSJanet SmithRuslan

SanishviliRobert Fischetti

ScrippsRay StevensVadim CherezovMike Hanson

U. MichiganRoger SunaharaMatthew Whorton

OHSUDavid Farrens

Universitat Autonoma de BarcelonaXavier Deupi

Financial SupportNIH ‐NINDSNIGMS

Gifts from:Lundbeck7TM Pharma

LCP technologyMartin Caffrey

Bicelle technologyN Salem FahamJames Bowie

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