Báo cáo khoa học: C-terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity ppt

with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity Chandramouli Reddy Chillakuri, Christoph Reinhart and Hartmut Michel Department of Molecul

with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity

Chandramouli Reddy Chillakuri, Christoph Reinhart and Hartmut Michel

Department of Molecular Membrane Biology, Max-Planck-Institute for Biophysics, Frankfurt ⁄ Main, Germany

Cannabinoid receptors belong to the seven

transmem-brane G protein coupled receptor (GPCR) family

Two different subtypes have been reported in humans,

namely cannabinoid receptor 1 (CB1) [1] and

cannabi-noid receptor 2 (CB2) [2] A splice variant of CB1,

called CB1a, has also been identified to be expressed in

low levels in rodent brain [3] Cannabinoid receptors

form the site of action for the active ingredients of

marijuana (D9-tetrahydrocannabinol, D9THC),

ananda-mide being the important endocannabinoid CB1 is

present predominantly in the nerve axons of the

cen-tral nervous system, is known to be neuroprotective in

its function and thus forms an important target in the

pharmaceutical industry [4] CB1 is coupled to the

Gi⁄ o family of heterotrimeric G proteins Activation

of the CB1-mediated Gi⁄ o proteins inhibits adenylyl cyclases to reduce cAMP production, inhibit calcium channels and increase inwardly rectifying potassium currents The modulation of ion channels has been shown to be independent of cAMP production, indicating that G proteins, especially Gbc, may inter-act directly with the effector molecules [5,6]

An earlier concept states that agonist-bound GPCR alone can couple to G proteins to transduce the signal However, this rather historical hypothesis is opposed today because of several reports confirming

Keywords

cannabinoid receptor; G protein coupled

receptor; G proteins; membrane proteins;

signal transduction

Correspondence

H Michel, Department of Molecular

Membrane Biology, Max-Planck-Institute for

Biophysics, Max-von-Laue Str.3,

D-60438 Frankfurt ⁄ Main, Germany

Fax: +49 69 6303 1002

Tel: +49 69 6303 1001

E-mail: Hartmut.Michel@mpibp-frankfurt.

mpg.de

(Received 23 July 2007, revised 15

Septem-ber 2007, accepted 8 OctoSeptem-ber 2007)

doi:10.1111/j.1742-4658.2007.06132.x

We have investigated the existence of a precoupled form of the distal C-ter-minal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric

G proteins in a heterologous insect cell expression system CB1-417 showed higher production levels than the full-length receptor The production lev-els obtained in our expression system were double the values reported in the literature We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization, as was predicted in the literature Using fluorescence resonance energy transfer, we found that CB1-417 and Gai1b1c2 proteins were colocalized in the cells GTPcS bind-ing assays with the Sf9 cell membranes containbind-ing CB1-417 and the G pro-tein trimer showed that the receptor could constitutively activate the Gai1

protein in the absence of agonists A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor We found that the CB1-417⁄ Gai1b1c2 complex could be solubilized from Sf9 cell mem-branes and coimmunoprecipitated In this study, we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells, and that the protein complex is stable in detergent solution Thus, our sys-tem can be used to produce sufficient quantities of the protein complex to start structural studies

Abbreviations

Bmax, maximum binding capacity; CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; CFP, cyan fluorescent protein; CHS, cholesterol hemisuccinate; DM, decylmaltoside; FRET, fluorescence resonance energy transfer; GPCR, G protein coupled receptor; K d , equilibrium dissociation constant; m.o.i., multiplicity of infection; D 9 THC, D 9 -tetrahydrocannabinol; YFP, yellow fluorescent protein.

the precoupled form of GPCR and G proteins In a

recent study, using fluorescence resonance energy

trans-fer (FRET), several receptors were identified, such as

the muscarinic receptor M4, the adrenergic receptor

a2A, the adenosine receptor A1 and the dopamine

receptor D2, in a precoupled form to the G protein

tri-mer (Gaob1c2) [7] It was suggested that GPCR dimers

and the G protein heterotrimer are present in cell

mem-branes in a resting state as a pentameric complex in the

absence of agonists However, it may not be true that

this whole complex is resting and inactive, and can only

be activated by agonists Reports constantly support

the constitutive activity of GPCRs, indicating that

GPCRs have some basal activity in the cell even in the

absence of agonists Inverse agonists (antagonists) to

several GPCRs have shown that these ligands can

reverse this basal activation of GPCRs, demonstrating

the existence of constitutively active receptors [8]

Solubilization of the cannabinoid receptor from rat

brain and GTP binding studies initiated the concept of

the presence of a stable CB1⁄ G protein complex [9]

The development of the CB1-specific antagonist

SR 141716A led to the identification of constitutively

active receptors [10] The existence of such an active

form was tested, and it was found that CB1 can

sequester G proteins from a common pool, making

them unavailable to other GPCRs present in the cell

[11] Meanwhile, it was found that a CB1 C-terminal

peptide (CB1-401–417) was able to activate the specific

G proteins in brain [12] Further, a C-terminal trun-cated CB1 (CB1-417) was found to have enhanced ability to sequester G proteins and exhibited increased constitutive activity [13] Recently, it has been reported that different subtypes of Gai coimmunoprecipitate with CB1 from N18TG2 cell membranes in the absence of exogenously added agonists [14] Most of the results discussed above were either performed on native tissue or mammalian cells In this work, for the first time, we investigated the existence of a precoupled form of truncated CB1 in a heterologous Sf9 insect cell expression system Insect cell expression systems are cheaper than mammalian expression systems, and are therefore more feasible for the large-scale production

of receptor required for structural determination

Results Functional production of CB1 The C-terminal truncated CB1 (CB1-417) was pro-duced in a functional form in Sf9 insect cells (the gene constructs used in this study are shown in Fig 1) Analysis of the protein produced, by immunoblotting with antiflag M2 IgG, showed (Fig 2) a monomeric band at 47 kDa and an oligomeric band specific to the cannabinoid receptor at a size of approximately

Strep-tagII CB1

His Flag

P PH Melittin

Tev

1 to 472 aa

Strep-tagII CB1

His Flag

P PH Melittin

Tev

1 to 417 aa

YFP CB1

His Flag

P PH Melittin

Tev

1 to 417 aa

G?i1 CFP

G?i1

P PH

Strep-tagII CB1

His Flag

P PH Melittin

Tev

1 to 472 aa

Strep-tagII CB1

His Flag

P PH Melittin

Tev

1 to 417 aa

YFP CB1

His Flag

P PH Melittin

Tev

1 to 417 aa

CFP

P PH

Fig 1 Gene constructs used for the expression in insect cells The four gene constructs were prepared using the basic pVL baculovirus transfer vector CFP, cyan fluorescent protein; Flag, flag epitope used for immunoblotting; His, decahistidine tag; PPH, polyhedrin promoter; Strep-tagII, used for the purification of the receptor; YFP, yellow fluorescent protein The names of each construct mentioned in this work are given before each graphic representation.

160 kDa A similar higher oligomeric band was

reported for full-length CB1 between 160 and 200 kDa

Wager–Miller et al [15] took this oligomeric form of

the cannabinoid receptor as an example to explain the dimerization of GPCRs Radioligand binding assay showed saturation binding on the cell membranes (Fig 3) It was found that the use of 10 multiplicity of infection (m.o.i.) virus and incubation for 72 h resulted

in the best production levels (data not shown) The production levels of both constructs of this receptor were much higher than the values reported so far in the literature The full-length receptor (FHTCB1StII) gave a maximum binding capacity (Bmax) of 39.7 ± 1.3 pmolÆmg)1, and the C-terminal truncated version [FHTCB1(417)StII] gave a Bmax value of 52 ± 3.09 pmolÆmg)1 The equilibrium dissociation constants (Kd) observed were 2.5 and 3.6 nm, which fall within the range of the values reported earlier The best value reported so far is 24.5 pmolÆmg)1 for N-terminal histi-dine-tagged CB1 in the Sf21 cell line [16] Therefore, the gene constructs used in this report with a StrepII tag on the C-terminus gave a two-fold increase in pro-duction levels compared with the constructs used in the literature The truncated receptor showed a 30% higher production than the full-length receptor

Determining the colocalization of the CB1⁄ G protein complex by FRET

The cannabinoid receptor exists in a precoupled form

to G proteins The receptor R can exist in an RGGDP

175

83

62

47.5

32.5

KDa M 1 2

Fig 2 Immunoblotting of length and truncated CB1 The

full-length and distal C-terminal truncated CB1 were produced in Sf9

cells Thirty micrograms of cell membrane were used to run

SDS ⁄ PAGE, and were immunoblotted using antiflag M2 IgG The

lower band is the monomeric band and the upper band is the

oligomeric band (presumably tetrameric) Lane 1, marker in kDa;

lane 2, full-length CB1; lane 3, truncated construct of CB1

(CB1-417).

0 0.5x10 4 1x10 4 1.5x10 4 2.0x10 4 2.5x10 4

CB1

[SR 141716A]

40

30

20

10

0

0 0.2x10 4 0.6x10 4 1.0x10 4 1.4x10 4

CB1(417)

[SR 141716A]

0 0.5x10 4 1x10 4 1.5x10 4 2.0x10 4 2.5x10 4

40

35

30

25

20

15

10

5

0

CB1

[SR 141716A]

50

0 0.2x10 4 0.6x10 4 1.0x10 4 1.4x10 4

CB1(417)

[SR 141716A]

Fig 3 Saturation binding curves of full-length and truncated CB1 One microgram of cell membrane containing either full-length or truncated CB1 was used to determine the production level of the protein Eight concentrations (200 p M to 25 n M ) of the radioactive cannabinoid ligand

SR 141716A were used Each point is the specific binding calculated from the mean of triplicates of the positive reaction and duplicates of the negative reaction The x-axis represents the concentration of the radioactive ligand in picomoles The y-axis represents the specific bind-ing in pmolÆmg)1of total cell membranes The full-length receptor gave a Bmaxvalue of 39.7 ± 1.3 pmolÆmg)1and Kdvalue of 2.5 n M The truncated receptor gave a Bmaxvalue of 52 ± 3.09 pmolÆmg)1and Kdvalue of 3.6 n M

(GDP-bound form) or RG– (nucleotide-lacking form)

in addition to the free R form [17] These G

protein-bound forms of the receptor are believed to be

respon-sible for the sequestration and constitutive activity

mechanisms of the receptor In this study, we

investi-gated the presence of such a precoupled complex in

heterologous Sf9 cells using the FRET technique

FRET occurs when a donor (cyan fluorescent protein,

CFP) transfers its energy obtained on excitation to an

acceptor (yellow fluorescent protein, YFP) by dipole–

dipole interactions This phenomenon occurs when

FRET partners specifically interact and are present

within a close proximity of less than 100 A˚

In this study, we used FHTCB1(417)-YFP

(accep-tor) and Gi1-CFP (donor) fusion proteins Sf9 cells

coexpressing FHTCB1(417)-YFP, Gi1-CFP and b1c2

were imaged using a laser scanning confocal

micro-scope No cannabinoid ligands were included in the

cell cultures or buffers Nevertheless, both proteins

were found to be colocalized in the cell membrane

(Fig 4) The fluorescence energy transfer between the

proteins was investigated by acceptor bleaching and

donor dequenching experiments The conditions used

for bleaching, with a 515 nm laser, were optimal for

> 90% acceptor bleaching and minimal donor

bleaching When the acceptor protein was bleached,

there was an increase in donor fluorescence The

increase in donor fluorescence calculated for 10 cells

was 7% ± 2% As a negative control CB1-CFP and

histamine1 receptor-YFP were coexpressed to almost

equal levels and the experiment was repeated in the

same way as above These two GPCRs have not been

reported to form a dimer complex An increase of

less than 2% was detected, which could be a result of

the random collision of fluorescent molecules in the

membrane This was considered as the background signal

Constitutive activity of the truncated cannabinoid receptor

The cannabinoid receptor has been shown to exhibit constitutive activity, thereby transducing the biological signal, even in the absence of ligand [18] Truncation

of the distal C-terminal tail has been shown to enhance the constitutive activity and sequestration ability of the cannabinoid receptor [13] Using the patch clamp tech-nique on neurones, it has been shown that the C-termi-nal distal tail constrains the receptor from interacting with G proteins In this study, we investigated the con-stitutive activity of the truncated cannabinoid receptor

in heterologous Sf9 cell membranes We used fluores-cent and radioactive GTPcS binding assays to observe the constitutive activity In the fluorescence experi-ment, the cell membranes containing CB1-417 were incubated with purified Gai1 or GasL proteins

CB1-417 enhanced the fluorescent GTPcS binding to the

Gai1 protein, whereas no effect was seen when GasL protein was used, which is not a physiological partner

to the cannabinoid receptor (Fig 5A) This increase in GTPcS binding was observed even in the absence of agonist Wild-type Sf9 cell membranes were included

in the reactions to monitor the basal activity of the

Ga proteins used Purified Gai1 protein showed an increased activity in the presence of cell membranes, although the reason was unclear However, this increase in GTPcS binding was not additive with increasing wild-type membrane concentration, unlike the CB1-417 membrane, which showed an additive effect on GTPcS binding (Fig 5B) GDP (10 lm) was

Fig 4 Confocal images showing the

colo-calization of the receptor and G protein Sf9

cells producing the CB1-417-YFP fusion

pro-tein and Gi1-CFP fusion protein were imaged

using a laser scanning confocal microscope.

CFP was excited with a 458 nm laser and

YFP with a 515 nm laser Images were

col-lected using the filters 475–525 nm (CFP)

and > 530 nm (YFP) Overlap images show

the colocalized receptor and the G protein.

YFP was bleached using a 515 nm laser in a

donor dequenching experiment Donor

de-quenching gave a 7% increase in acceptor

fluorescence The white rectangle in the

images shows the area bleached using the

laser.

used in the reactions in order to reduce the basal

activ-ity of the purified G proteins

Sf9 cell membranes containing heterotrimeric G

pro-teins alone or together with the cannabinoid receptor

were used for the radioactive GTPcS binding assay

The production of all subunits was confirmed by

immunoblotting against each subunit There was a

sig-nificant increase in GTP binding when the receptor

was coexpressed together with the G proteins (Fig 6)

This increase in the absence of ligand shows the

constitutive activity of the receptor The antagonist

AM251 inhibited this GTP binding to G proteins The

agonist WIN 55,212–2 increased GTP binding to a

lesser extent These ligand-dependent effects were not seen in the membranes lacking the receptor Similar results have been reported in [19], where cannabinoid receptors produced in Sf9 cell membranes and G pro-teins purified from brain were reconstituted In the present study, a defined receptor and G protein complex was used rather than a whole pool of Ga subunits

Coimmunoprecipitation of CB1-417 and the

G protein complex Sf9 cell membranes containing the Gai1b1c2 protein complex together with FHTCB1(417)StII were used for coimmunoprecipitation (Fig 7) The membranes

Time (min)

Time (min)

24

A

B

23

21

19

17

15

13

11

34

32

28

24

20

16

12

Fig 5 Fluorescent Bodipy GTPcS binding assay (A) Specific

increase in GTPcS binding to the G i1 (r) protein and not the G sL

(j) protein in the presence of membranes containing the truncated

cannabinoid receptor Symbols s and d represent GTPcS binding

to pure G i1 and G sL in the absence of cell membranes Symbols e

and h represent GTPcS binding to Gi1 and GsLin the presence of

Sf9 cell membranes (B) Increase in GTPcS binding to Gi1is

addi-tive because of CB1 and not just because of the cell membranes.

Doubling the concentration of cell membranes with CB1 (r) adds

to the GTPcS binding, whereas Sf9 cell membranes (e) do not

show a similar effect Symbols n and m represent the binding of

GTPcS to G i1 protein in the presence of a 1· concentration of Sf9

membranes or CB1 membranes, respectively (The bullets used in

this figure are not data points, but are used to distinguish between

the different spectra.)

0 10000 20000 30000 40000 50000 60000 70000 80000 90000

Fig 6 Radioactive GTPcS binding assay Thirty micrograms of cell membrane containing G protein trimer only (G) or G protein trimer coexpressed with truncated CB1 (R) were used to estimate GTPcS binding Coexpression of the receptor together with G proteins increased GTPcS binding The cannabinoid receptor agonist WIN 55,212–2 (A) further increased GTPcS binding This binding was inhibited by the cannabinoid selective antagonist AM251 (IA).

containing only G proteins or receptor + G proteins

were solubilized using a 1% decylmaltoside

(DM) + 0.2% cholesterol hemisuccinate (CHS)

mix-ture for 1 h The presence of CHS during the

solubili-zation and purification of GPCRs has been

demonstrated to be crucial in retaining the

functional-ity of the receptor [20] We have had a similar

experi-ence with other GPCRs in our laboratory (C R

Chillakuri et al., unpublished data) In a recent report,

CHS was used in the purification of CB2 [21]

Mukho-padhyay & Howlett [14] used Chaps detergent to

solu-bilize the CB1⁄ G protein complex from N18TG2

neuroblastoma cell membranes Immunoprecipitation

of the receptor⁄ G protein complex was performed as

described in Experimental procedures The eluted

pro-tein from the antiflag M2 agarose matrix was analysed

using different antibodies The immunoblot with

antiflag M2 IgG showed the CB1 band at 47 kDa

Anti-histidine tag immunoblot showed the receptor

band as well as the bc dimer (c subunit has the

histidine tag on the N-terminus) at  34 kDa The

immunoprecipitated sample from the membranes

containing only G proteins did not show any specific band in either immunoblot Anti-Gi1 immunoblot showed a faint band in the negative control, indicating

a nonspecific interaction of Gai1with the matrix How-ever, the signal in the positive control was higher, and therefore it was concluded that the Gai1 protein was specifically bound to the receptor The presence of bc subunits only in the positive reaction supports this conclusion

Discussion One of the prime limiting factors for structural studies

of GPCRs is the availability of material Obtaining sufficient quantities of pure and active receptor is in itself a challenge for many GPCRs, including CB1 In this study, we focused on the overproduction of CB1 and the investigation of the precoupled form of this receptor with G proteins Structural studies of the receptor⁄ G protein complex are needed to understand the mechanism of interaction between the partners Instead of producing the subunits separately and using them for cocrystallization, it may be worthwhile to isolate the ternary complex for structural studies Another proposal behind the choice of the recep-tor⁄ G protein complex for three-dimensional crystalli-zation is that the G protein trimer increases the hydrophilic portion of the complex, and thus enhances the chances of crystallization of GPCR, an integral membrane protein, which has been a challenge for crystallographers

We used a heterologous insect cell expression system for the overproduction of CB1 We obtained two-fold higher production levels for this receptor than those reported in the literature [16] Truncation of the distal C-terminal tail of CB1 has been reported to increase the constitutive activity and sequestration tendency of the receptor [13] In this study, we observed that this truncation also increases the production levels of the receptor in Sf9 insect cells The truncated receptor was produced in insect cell culture at up to 500 lgÆL)1 ( 52 pmolÆmg)1 of 47 kDa protein) These moder-ately higher production levels provide better scope for producing more protein required for structural studies Truncation of the receptor did not hinder ligand bind-ing to the receptor, indicatbind-ing that the receptor was functional An important observation from the immunoblot of the truncated receptor was that this truncated receptor also exists as an oligomer, as does full-length CB1 Wager-Miller et al [15] reported that the C-terminal tail may be important in the assembly

of the oligomer Our results show that at least the distal C-terminal tail (418–472) is not involved in

CB1(417)

Anti-Flag M2 IgG

Anti-His tag IgG

Anti-His tag IgG

Anti-G i1 /G i2 IgG

Receptor

Receptor

G

G i1

Fig 7 Coimmunoprecipitation Sf9 cell membranes containing

CB1-417 and the Ga i1 b1c2trimer complex were solubilized using a

mixture of DM and CHS The complex was immunoprecipitated

using antiflag M2 IgG agarose matrix The matrix was washed

thrice and the bound protein was eluted by denaturation with SDS

gel loading buffer Immunoprecipitated samples: lane 1, cell

membrane containing G protein trimer only; lane 2, cell membrane

containing both receptor and G protein trimer The antibodies used

to identify the different subunits of the complex are denoted on

the left side of the image The subunit that was identified is

men-tioned on the right side of the immunoblot image The anti-G i1 ⁄ G i2

IgG immunoblot showed that the Ga subunit exhibits nonspecific

binding to the matrix However, the intensity of the Ga

subunit was much higher when the receptor was present in the

solubilizate.

oligomerization, as the truncated protein was observed

to oligomerize

According to the classical hypothesis, the presence

of agonist is necessary for receptor⁄ G protein complex

formation and activation In contradiction to this

hypothesis, some receptors, such as the j-opioid

recep-tor [22], dopamine receprecep-tor D2, adrenergic receprecep-tor

a2A, muscarinic receptor M4 and adenosine receptor

A1, have been found to exist as complexes with their

corresponding G proteins prior to ligand activation [7]

CB1 was found to exist in a precoupled form in

N18TG2 cells, even in the absence of ligands [14] In

this study, we investigated the existence of a

precou-pled form of truncated CB1 in Sf9 insect cells A

FRET experiment on Sf9 cells producing the truncated

cannabinoid receptor and the G protein heterotrimer

showed that these proteins were colocalized The

fluo-rescent and radioactive GTPcS binding experiments

showed that the truncated receptor produced in Sf9

cells retained the ability to activate G proteins in the

absence of ligands The fluorescent GTPcS binding

experiment showed the specificity of the cannabinoid

receptor to Gai1 protein and not GasL protein The

radioactive GTPcS binding experiment showed that

the receptor was constitutively active and the

antago-nist AM251 inhibited the basal activity of the receptor

Similar results have been reported previously by Glass

& Northup [19] using the G proteins purified from

bovine brain The relatively small increase in GTPcS

binding to G protein on addition of the agonist WIN

55,212–2 in our experiments can be explained by the

presence of low levels of the receptor conformational

state recognized by the agonist We observed that the

maximum binding of antagonist (AM251) to the

recep-tor was five times higher than the maximum binding

of agonist (CP-55 940) (data not shown) This shows

that most of the receptor produced in insect cells is in

a conformation not recognized by the full agonist A

similar result was reported by Xu et al [16] Another

reason may be that the agonist-activated

conforma-tional state found in Sf9 cell membranes prefers other

subtypes of Gai⁄ o protein than the Gai1 used In this

study, we also investigated the possibility of the

solubi-lization and isolation of the whole receptor⁄ G protein

complex produced in insect cells The

coimmunopre-cipitation experiment showed that the complex could

be solubilized using the mild nonionic detergent DM

in combination with CHS These mild conditions are

necessary to retain the function of the receptor and the

G protein complex

Taken together, our results confirm that the distal

C-terminal truncated CB1 can be produced in a

func-tional form in Sf9 insect cells in higher yields than

those obtained previously This receptor exhibits con-stitutive activity, and it is also possible to coimmuno-precipitate the whole receptor⁄ G protein complex in the absence of any ligands This paves the way for fur-ther investigations to determine the possibility of stabi-lizing and purifying this complex to homogeneity, so that it can be used for crystallization, in order to obtain a better understanding of the interaction between the partners, and the mechanism of signal transduction

Experimental procedures Chemicals and reagents

The general laboratory chemicals used were of analytical grade and were purchased from Roth (Carl Roth & Co

KG, Karlsruhe, Germany), Merck (Merck KGaA, Darms-tadt, Germany) and Fluka⁄ Sigma (Sigma-Aldrich Chemie GmbH, Diesenhofen, Germany) Bodipy FL-GTPcS was purchased from Molecular Probes (Eugene, OR, USA) Radioactive cannabinoid agonist [3H]CP-55 940 was obtained from Perkin Elmer LAS, (Deutschland) GmbH⁄ Rodgan-Ju¨gesheim, Germany, and antagonist [3H]SR 141716A was purchased from Amersham Biosciences GTPc[S35] was obtained from Perkin Elmer Life Sciences Unlabelled cannabinoid ligands WIN 55,212–2 mesylate and AM251 were purchased from Tocris (Bristol, UK)

DM was purchased from Glycon Biochemicals,

Luckenwal-de, Germany The protease inhibitors used were obtained from Biomol Feinchemikalien GmbH, Hamburg, Germany Antiflag M2 IgG conjugated to alkaline phosphatase and anti-polyhistidine antibody conjugated to alkaline phospha-tase were obtained from Sigma-Aldrich Chemie GmbH (Munich, Germany) Anti-Gai1⁄ Gai2 IgG was purchased from Calbiochem (Merck KGaA, Darmstadt, Germany) Anti-flag M2 IgG agarose was obtained from Sigma-Aldrich Chemie GmbH

Cloning

The gene encoding CB1 was cloned into modified pVL1393 baculovirus transfer vector with the mellitin signal sequence The forward primer for the CB1 gene with the BamHI restriction site was 5¢-GC G GAT CC G ACC ATG GCG AAG TCG ATC CTA GAT GGC-3¢ The reverse primer for the full-length CB1 gene, with EcoRI and NotI restriction sites, was 5¢-GAA T GC GGC CGC TCA CTT TTC GAA TTG AGG GTG CGA CCA GAA TTC AGC CTC GGC AGA CGT GTC TGT GGA-3¢, which contains the StrepII tag between the EcoRI and NotI sites The reverse primer for the truncated receptor with the EcoRI site was 5¢-CCA GAA TTC GCC TTC ACA AGA GGG AAA CAT-3¢ The full-length receptor gene (1–472 amino

acids) was cloned between the BamHI and NotI sites of the

vector to prepare pVLMelFHTevCB1StrepII The truncated

receptor (1–417 amino acids) was cloned into the BamHI

and EcoRI sites of the above vector to prepare

pVLMelFHTevCB1(417)StrepII In the CB1-417-YFP

con-struct, the YFP gene was cloned between the EcoRI and

NotI sites of the truncated construct Gi1-CFP was prepared

by introducing CFP between the NcoI and XbaI sites

These restriction sites (ACC ATG GTG TCT AGA) were

generated in the Gi1 protein between amino acids Ser62

(TCA) and Glu63 (GAA) by overlap PCR The Gi1-CFP

gene was cloned into the pVL vector (no mellitin sequence)

using BamHI and EcoRI Digestion of DNA and ligation

were performed according to the protocols in the New

England Biolabs GmbH, Frankfurt am Main, Germany

The DH5a strain of Escherichia coli was used to amplify

and clone the DNA constructs Gai1and GasL genes were

cloned into the pDEST14 vector using Gateway cloning

technology (Invitrogen), for protein production in E coli

Recombinant virus production and selection

Recombinant virus for the DNA constructs was prepared

according to the protocols given in Invitrogen’s baculovirus

expression system catalogue Three to five positive plaques

(according to X-gal selection) for each gene were selected,

and virus was produced by infecting Sf9 cells The virus

from these clones was used to infect the 2· 106 cells in a

tissue culture plate After 4 days of incubation, the cells

were harvested by spinning in a centrifuge; 2· 105

cells from each clone were lysed using 1% SDS in a buffer

con-taining protease inhibitors and DNase This lysate was used

for analysis on SDS⁄ PAGE The gel was immunoblotted

using anti-flag M2 IgG to confirm recombinant protein

production The clone showing the best expression profile

was selected, and the corresponding virus was amplified

and stored at 4C The titre for the virus was calculated

using the 96-well plate end point dilution assay [23] Virus

for human Gai1and bovine Gb1c2was obtained from

for-mer laboratory members [24]

Cell culture and cell membrane preparation

Sf9 cells were grown in TNMFH medium (C.C pro

GmbH, Germany) containing 5% fetal bovine serum (PAA

Laboratories GmbH, Germany) The cells were maintained

in a tissue culture flask For suspension culture, cells from

the tissue culture flask were added to the medium

contain-ing 0.1% Pluronic F68 at a density of 1· 105 cellsÆmL)1

The conical flask was incubated in a shaker at 27C and

125 r.p.m Cells were grown to a density of 2· 106

cell-sÆmL)1, and harvested using sterile centrifuge tubes by

cen-trifugation at 1000 g Old medium was removed and an

equal volume of fresh medium was added to the cells and

resuspended gently using a pipette Virus was used at 10

m.o.i for protein production Cells were incubated for

3 days before harvesting Sf9 cells were harvested by centri-fugation The pellet was resuspended in ice-cold lysis buffer (20 mm Tris⁄ HCl pH 8.0, 100 mm NaCl, 5 mm MgCl2,

250 mm sucrose) containing protease inhibitors (1 lm E64,

5 lgÆmL)1 leupeptin, 2 lgÆmL)1 pepstatin A, 10 lgÆmL)1 aprotonin) The cells were broken in a Parr Bomb for 1 h

at 35 kg⁄ cm2 The suspension was collected and centrifuged

at 2000 g to remove the unbroken cells The turbid super-natant was ultracentrifuged at 100 000 g to sediment the cell membranes The membrane pellet was resuspended in resuspension buffer (20 mm Tris⁄ HCl pH 8.0, 100 mm NaCl, 5 mm MgCl2, 10% glycerol) and homogenized using

a potter The homogenate was aliquoted and stored at ) 80 C in a freezer

Radioactive cannabinoid ligand binding

The total protein concentration was estimated by a bicinch-oninic acid protein assay kit (Pierce, Rockford, IL, USA);

1 lg of Sf9 cell membranes was used for each reaction in a saturation binding assay Membranes were added to bind-ing assay buffer A (20 mm Tris⁄ HCl, 5 mm MgCl2, 1 mm EDTA, 1% BSA) Eight concentrations of [3H]SR 141716A were used in the saturation binding assay Triplicates of positive reactions (radioactive ligand only) and duplicates

of negative reactions (radioactive ligand + 10 lm cold ligand AM251) were set up in 1.5 mL Eppendorf tubes for each radioactive ligand concentration The reactions (250 lL) were incubated for 1 h at 30C and filtered over glass fibre filters (GF-B) from Whatmann GmbH (Dassel, Germany) The filters were washed three times with warm (30C) binding assay buffer The filters were collected in

5 mL radioactivity counting tubes and 4.5 mL of scintillant (Roth) was added The radioactivity was measured in terms

of disintegrations per minute (d.p.m.) The specific d.p.m (mean positive reactions) mean negative reactions) was used to calculate the receptor concentration in pmolÆmg)1

A Kaleida graph (Synergy software) was used to plot the receptor binding sites versus radioactive ligand concentra-tion in a nonlinear regression curve using the following for-mula: specific binding Y¼ (M1· M0)⁄ (M2+ M0); M1¼ 1; M2¼ 1 M1is Bmaxand M2is Kd

Determination of the colocalization of receptor and G protein by FRET

Cells were allowed to attach to the glass surface before the images were taken An LSM-510 Meta confocal microscope (Carl-Zeiss AG, Oberkochen, Germany), fitted with a · 60 oil objective, was used to collect the images CFP was excited with a 458 nm laser and images were obtained using

a bandpass filter from 475 to 525 nm YFP was excited with a 515 nm laser and images were obtained using a longpass filter above 530 nm The acceptor bleaching

experiment was performed by sequential scanning A region

of the cell along the surface was selected manually using

the software provided with the microscope, and was

bleached using a 515 nm laser at 80% laser power

Pre-bleach and postPre-bleach images were taken for both proteins

using their respective filters The difference in the donor

flu-orescence was calculated from the background subtracted

images The percentage FRET was calculated using the

fol-lowing formula: (intensity of postbleach image) intensity

of prebleach image)⁄ intensity of prebleach image Ten cells

were observed and the mean value was calculated

Cross-talk between the channels was corrected using the cells

pro-ducing only CFP or only YFP The laser power, pinhole

size and detector gain were chosen optimally to avoid

satu-ration of the images

Fluorescent Bodipy FL-GTPcS binding assay

To measure the basal G protein activation by the

cannabi-noid receptor, a fluorescent Bodipy FL-GTPcS binding

assay was performed Sf9 cell membranes coexpressing

CB1-417 and Gb1c2were used Gai1and GasLwere produced in

E coli and purified The cell membrane concentration was

chosen to contain 20 nm receptor (as estimated by

radioli-gand binding) in the final reaction Purified G protein in

buffer B (20 mm Tris⁄ HCl pH 8.0, 100 mm NaCl, 5 mm

MgCl2, 10 lm GDP, 0.25 mm dithiothreitol) was used at a

five-fold molar excess (100 nm) to the receptor A 20 lL

membrane⁄ G protein reaction mixture (10·) was made and

incubated at 25C for 45 min This reaction mixture was

diluted in assay buffer (buffer B + 500 nm Bodipy

FL-GTPcS), and the fluorescence was monitored immediately

using a time drive program for 5 min in an LSM-50

lumi-nescence spectrophotometer (Perkin Elmer Life Sciences)

The fluorescent ligand was excited at 485 nm and the

emis-sion was monitored at 520 nn In a negative control

experi-ment, wild-type Sf9 cell membranes were used

Radioactive GTPc[S35] binding assay

Radioactive binding assay was used as a complementary

experiment to observe the constitutive activity of the

recep-tor Sf9 cell membranes containing CB1-417 and Gai1b1c2

were used Cell membranes containing only G protein

sub-units were used as a negative control Thirty micrograms of

total cell membrane were used for each reaction Cell

mem-branes were diluted in buffer C (buffer B containing 0.5%

BSA) to prepare a reaction volume of 200 lL Agonist or

antagonist (4 lm) dissolved in dimethylsulfoxide was used

as required in the reactions containing ligands Radioactive

GTPcS was diluted in buffer C and added to each reaction

to obtain a final concentration of 4 nm The reactions were

incubated at 30C for 1 h The reactions were filtered

under vacuum, over glass fibre filters wetted with buffer C

The filters were washed thrice with buffer C The

radio-activity on these filters was counted using a b-counter calibrated with the14C isotope

Coimmunoprecipitation

Cell membranes containing the receptor and G proteins were solubilized using a mixture of 1% DM and 0.2% CHS at 4C for 1 h The supernatant was clarified by ul-tracentrifugation and incubated with 50 lL of anti-flag M2 IgG agarose (rinsed with buffer B) at 4C for 1 h The supernatant was removed and the antibody matrix was washed three times with buffer B containing 0.2% DM and 0.04% CHS The matrix was resuspended in SDS gel load-ing buffer to elute the protein bound to the antibody by denaturation This eluate was used to run an SDS gel and analysed by immunoblotting Anti-M2 IgG was used to identify the receptor Anti-polyhistidine tag IgG was used

to detect both the receptor and the Gbc dimer (histidine tag on the C-terminus of the c subunit) Gai1⁄ i2 antibody was used to detect Gai1protein

Acknowledgements

We would like to thank the UMR cDNA resource centre for the kind donation of the cDNA for CB1

We thank Heinz Schewe (Bio-zentrum, University of Frankfurt, Germany) for his help in using the laser scanning confocal microscope This work was funded

by the Max-Planck-Gesselschaft and Sanofi-Aventis

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