Heterodimerization of c-aminobutyric acid B receptor subunits asrevealed by the yeast two-hybrid system

Julia H. White,a,* Alan Wise,b and Fiona H. Marshallc

a Pathway Discovery, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UKb Systems Research, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK

c Molecular Pharmacology, Millennium Pharmaceuticals Ltd., Granta Park, Great Abington, Cambridge, CB1 6ET, UK

Accepted 4 June 2002

Abstract

Several lines of evidence suggested that the first c-aminobutyric acid B receptor to be cloned required an additional factor forfunctional expression. GABAB1 was retained within the endoplasmic reticulum and failed to couple to signal transduction pathways

on stimulation with agonists. In radioligand binding experiments it was found that although the affinity of antagonists showed a

close agreement between rat brain membranes and membranes expressing the cloned receptor, agonist ligands were significantly

weaker at recombinant receptors. Using the C-terminal tail as bait, a yeast two-hybrid screen was run against a human brain cDNA

library and identified a second receptor, GABAB2, as a major interacting protein. This interaction was confirmed by coimmuno-

precipitation as well as extensive colocalization studies. Coexpression of the two seven-transmembrane proteins generated a fully

functional receptor, which was expressed at the cell surface confirming the importance of receptor heterodimerization for GABABreceptor activity. � 2002 Elsevier Science (USA). All rights reserved.

Keywords: c-Aminobutyric acid B receptor; Coimmunoprecipitation; Glycosylation; Yeast two-hybrid system; GTPcS binding

1. Introduction

It is now well established that G-protein-coupled re-ceptors (GPCRs) are able to oligomerize into both ho-modimers and heterodimers and that this can result inchanges in the ligand specificity, pharmacology, andsignaling of receptors [1,2]. This has important impli-cations for understanding and characterizing receptorfunction and also for developing new drugs acting onthis protein superfamily. The c-aminobutyric acid BðGABABÞ heterodimeric receptor is, to date, the onlyknown example where two seven-transmembrane re-ceptors must come together to form an obligate hete-rodimer, where neither of the receptor subunits isfunctional when expressed alone. A similar situation,however, exists with the CGRP and adrenomedullinreceptors where a GPCR must heterodimerize withsingle-transmembrane proteins known as RAMPs toform fully functional receptors [3]. Within the human

genome more than 700 GPCRs have been identified,around half of which remain orphan receptors despiteextensive screening against large numbers of candidateligands. One possibility is that some of these orphanreceptors like the GABAB receptor require anotherpartner to be functional. By considering the techniquesthat led to the recognition that the GABAB receptorrequires an additional ‘‘factor’’ to be functional, we canbegin to assess whether other orphan receptors may becandidates for heterodimerization or RAMP-like ac-cessory proteins.The GABAB receptor was described [4] many years

prior to cloning and its pharmacology, localization, andsignaling mechanisms were well characterized usingnative tissue. The first GABAB receptor (GABAB1) wasexpression cloned in 1997 [5]. However, from the outset,there were clearly discrepancies between the recombi-nant receptor and the endogenous receptor, as charac-terized from brain membranes. First, in radioligandbinding studies, agonist affinities were found to bearound 100-fold weaker in membranes prepared fromcells expressing GABAB1 compared with those prepared

Methods 27 (2002) 301–310

www.academicpress.com

*Corresponding author.

E-mail address: julia.h.white@gsk.com (J.H. White).

1046-2023/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.PII: S1046 -2023 (02 )00087-7

from rat brain. Second, only very weak functional re-sponses in an adenylyl cyclase assay were described. Inour hands GABAB1 failed to respond to GABA or otherknown GABAB agonists using a variety of differentfunctional readouts. Assuming there are no errors in thecDNA construct, the most likely reason a receptor failsto respond to agonists as expected is either a low level ofprotein expression or a lack of receptor expression at thecell surface. Both of these possibilities can be studiedusing Western blotting or flow cytometry comparingintact and permeabilized cells. This can be done usingantibodies raised to the native protein or, more com-monly, using antibodies to N-terminal epitope tags.In the case of the GABAB receptor, GABAB1 was not

expressed at the cell surface and further studies exam-ining the glycosylation status of the protein suggestedthat it was retained within the endoplasmic reticulum(ER) [7,8]. This suggested that an associated protein wasrequired to traffic the receptor to the plasma membrane.To identify such receptor-associated cofactors, a num-ber of approaches can be employed, each with its ad-vantages and drawbacks. These include affinitypurification, when high-affinity antisera or ligands areavailable, and expression-cloning approaches, where li-braries are screened to generate receptor function (e.g.,[3]). However, one of the most powerful approaches tothe selection and identification of associated proteins isthat of the yeast two-hybrid (Y2H) system (see [8]). Thisrelies on the functional assembly of a transcriptionfactor in the yeast nucleus as a result of two proteinsinteracting. Y2H possesses the advantage that it is arelatively easy technique in vivo for the discovery ofunknown protein interactions within the context of theliving cell, unlike other techniques in vitro such as geloverlay assays and affinity pulldowns.The use of Y2H led to the identification of a strong

interaction between GABAB1 and a second relatedGPCR, called GABAB2 [6,9]. The interaction was me-diated through coiled-coil domains located within theC-terminal tails of both receptors [6,9,10]. However,subsequent publications have suggested that the extra-cellular and transmembrane domains are also involved[11,12]. The initial observation, suggesting receptorheterodimerization, was confirmed biochemically usingcoimmunoprecipitation. Coexpression of GABAB2 withGABAB1 enabled GABAB1 to be expressed at the cellsurface as a mature glycoprotein, which responded toGABAB agonists in functional assays [6,13,14]. Thisobservation of heterodimerization has subsequentlybeen confirmed in vivo by other techniques includingcoimmunoprecipitation of native receptors and exten-sive distribution studies using immunohistochemistryand in situ hybridization techniques. Moreover, themechanism by which GABAB2 trafficks GABAB1 to thecell surface has also been elucidated [11,15,16] and isfurther described by Margeta-Mitrovic in this issue.

2. Methods

2.1. The yeast two-hybrid system

The yeast two-hybrid or Interaction trap system is apowerful genetic approach using engineered strains ofthe budding yeast Saccharomyces cerevisiae for the dis-covery and characterization of protein–protein interac-tions. The method was first developed over a decade ago[17] and has been extensively used for many proteintargets [8,18]. In principle, the system relies on themodular nature of transcription factors, with distinctfunctional domains. One domain, the DNA bindingdomain (BD), is responsible for binding the transcrip-tion factor to a target DNA sequence within a promoterwhile a second domain, the activation domain (AD),recruits the transcriptional machinery to promote genetranscription. The two-hybrid system exploits the factthat a binding domain is incapable of activating tran-scription unless it is physically, but not necessarilycovalently, attached to an activation domain. Therefore,to establish a two-hybrid interaction, the protein of in-terest, generally referred to as the bait, is expressed via ayeast expression vector as a fusion with the BD of atranscription factor (Fig. 1). Commonly, the yeastGAL4 BD or Escherichia coli lexA BD is used in con-junction with the appropriate promoter recognition se-quences. The expression plasmid, once transformed intothe yeast host, expresses the desired BD–bait fusion inthe nucleus. The yeast host cell is engineered to possesssensitive reporter genes responsive to the particular BDby the appropriate activating specific sequences beingpresent within the promoter. The BD binds to thispromoter, and if the bait fusion protein fails to activatetranscription in the absence of other expressed proteins,then it is suitable for Y2H analysis.To identify unknown associated protein partners

against particular baits, the bait must be exposed in theyeast cell to a relevant cDNA library of proteins ex-pressed as activation domain (AD) fusions. Typical ADsemployed in many of the Y2H systems are S. cerevisiaeGAL4, herpes simplex virus VP16, and E. coli B42.Proteins that associate with the bait bring the BD andAD into juxtaposition, creating a functional transcrip-tion factor. This hybrid transcription factor thus resultsin expression from the reporter gene, allowing selectionof yeast expressing the interacting protein partners.One of the main advantages of the Y2H system resides

in its sensitive and selectable reporter genes that permitinteracting proteins to be selected rapidly from themultiple genes present within a cDNA library. Generally,there are two types of reporter genes: those encoding anutritional requirement, such as the HIS3 or LEU2 geneproducts, and those encoding genes with an enzymaticactivity that can be measured quantitatively. Reportersencoding a nutritional gene allow positive interactors to

302 J.H. White et al. / Methods 27 (2002) 301–310

be selected by colony growth in the absence of the ex-ogenous supplement, as the yeast host has the genomiccopy of the gene deleted and is thus unable to grow in theabsence of a functional Y2H interaction. This results in apowerful positive selection pressure for the preferentialgrowth of a clonal population expressing the interactinglibrary insert. Moreover, use of the HIS3-encoded imi-dazoleglycerol-phosphate dehydratase reporter gene inthe presence of the competitive inhibitor 3-AT 3-amino-

1,2,4-triazole (3-AT) allows only those yeast cells ex-pressing sufficient levels of strongly associated two-hy-brid interactors to overcome the 3-AT levels and to begrowth-selected. This allows the stringency of a screen tobe set according to the level of 3-AT added.Typically the LacZ gene, encoding b-galactosidase, is

used for the latter type of ‘‘enzymatic activity’’ reporter.b-Galactosidase has the advantage of being extremelystable in yeast and can be readily detected using ex-tremely sensitive chromagenic substrates such as 5-bro-mo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal)and o-nitrophenyl-b-galactopyranoside (ONPG). Themain disadvantage is that these substrates are non-cell-permeable and require a lysis step in the assay. A per-meable b-galactosidase substrate is now available(X-a-GAL), which enables b-galactosidase activity to bescored in living cells.Once an interaction between two proteins is positively

scored from both reporter genes, then polymerase chainreaction (PCR) and/or plasmid rescue and amplificationin E. coli can be used to recover cDNA library insertsfor sequence analysis. However, some proteins are well-known promiscuous interactors and either have no ob-vious relevance to the bait or interact with multipleunrelated baits in a nonspecific manner (otherwiseknown as false positives). To eliminate these, associa-tions should be confirmed against the original bait, aswell as against a panel of unrelated baits to checkspecificity. Although the specificity checks will eliminatemany of false positives, any interactions discovered bythe Y2H system need to be verified through independentbiochemical and functional assays. Indeed, like alltechniques, Y2H possesses several advantages and lim-itations (Table 1).

2.2. Yeast two-hybrid screening

2.2.1. Yeast strains, plasmids, and mediaSaccharomyces cerevisiae Y190 [MATa, gal4 gal80,

ade2-101, his3, trp1-901, ura3-52, leu2-3, 112, UR-A3::GAL1-lacZ, LYS2::GAL1-HIS3, cyhR] was used forall described yeast two-hybrid work [22,23] Gal4BD fu-sion vectors were either pYTH9 [19] or pYTH16, anepisomal version of pYTH9, and the Gal4AD vectorused was pACT2 [22].A total Human Brain MATCHMAKER library

(HL4004AH) in pACT2 was purchased from ClontechLaboratories (Palo Alto, CA) and amplified accordingto the manufacturer’s instructions. All yeast weremaintained on standard yeast media [24].

2.2.2. Construction of bait vectorsThe GABAB1 C-terminal domain was amplified from

the full-length clone, using PCR primers 50-GTTGTCCCCATGGTGCCCAAGATGCGCAGGCTGATCACCand 50-GTCCTGCGGCCGCGGATCCTCACTTATA

Fig. 1. The yeast two-hybrid system. (a) Schematic representation of

the Y2H system. Bait protein is expressed in yeast as a fusion with a

binding domain such as GAL4BD. Similarly, the potential interactor

‘‘prey’’ protein is expressed as a fusion with an activation domain, such

as GAL4AD. Both fusion proteins translocate to the yeast nucleus,

where the BD is able to bind its recognition sequence with the pro-

moter of a reporter construct. The BD fusion is unable to activate

transcription alone but when the bait and prey proteins interact, a

functional transcription factor is formed that activates transcription

from the reporter gene. (b) Library screening by Y2H. Plasmids ex-

pressing bait–BD fusions and a library of AD–cDNA fusions are co-

transformed into a Y2H engineered yeast strain. By means of the

activation of selectable nutritional reporter genes, only those yeast

transformants coexpressing a functional transcription factor through a

bait-and-prey interaction grow to form colonies on selective agar. A

second reporter gene encoding b-galactosidase is likewise activated sothe yeast colonies will turn blue in the presence of the X-Gal chro-

magenic substrate. Those yeast cells expressing nonassociating library

proteins will not produce a functional transcription factor, will not

activate transcription, and therefore will not grow into colonies on the

selective agar.

J.H. White et al. / Methods 27 (2002) 301–310 303

AAGCAAATGCACTCG. PCR conditions were forTaq polymerase (Perkin–Elmer, Norwalk, CT) and runusing a Perkin–Elmer, 9600 PCR cycler for an initialheating of 95 �C 3min; followed by 25 cycles of 95 �C1min, 55 �C 1min, and 72 �C 3min; followed by 72 �Cfor 10min and storage at 4 �C. PCR product was size-fractionated on 0.8% agarose, gel purified, and restrictedwith NcoI and NotI restriction enzymes for direct sub-cloning into pYTH9 polylinker. This construct containsthe C-terminal 108 amino acids from the base of TM7.The cloned PCR product was sequenced and confirmedas error free and fused in-frame to yeast GAL4BD.

2.2.3. Construction of screening yeast host strain andlibrary transformationThe GAL4BD–GABAB1 C-terminal fusion in pYTH9

was stable integrated into the genome of yeast Y190 atthe trp1 locus by targeted homologous recombination(19). Y190 cells expressing the GAL4BD–GABAB1 C-terminal fusion were selected and a high-efficiency yeasttransformation protocol was adopted to transform in thebrain cDNA library. Briefly, approximately 1� 1010cells were grown to mid-log phase in YEPD at 30 �C,washed both in sterile water and in 0.1M lithium acetate/1� TE (pH 7.5), and finally resuspended in 4.0ml 0.1Mlithium acetate/TE solution. One hundred micrograms oflibrary plasmid, together with 4.0mg sheared salmonsperm DNA, was added to the cells and 24ml of 0.1Mlithium acetate/40% polyethylene glycol (PEG) 3350added. Cells were incubated at 30 �C for 30min, and thenheat-shocked at 42 �C for 15min. Cells were harvested,

resuspended in 10ml sterile water, and plated onto 10Nunc bioassay dishes containing selective agar plus20mM 3-AT. Plates were incubated for several days at30 �C and positive clones, which grew under selectionand expressed LacZ, were selected for further analysis.

2.2.4. Freeze-fracture assaysYeast colonies were transferred by replica plating onto

Whatman No. 54 filter papers and rapidly lysed by beingdipped twice into liquid nitrogen and allowed to thawinbetween. Each filter was incubated in 2ml of Z-buffer(60mM Na2HPO4 � 7H2O, 40mM NaH2PO4 �H2O,10mM KCl, 0.1mMMgSO4 � 7H2O, pH 7.0) containing1mg/ml X-Gal and 0.27% (v/v) 2-mercaptoethanol. Fil-ters are incubated at 37 �C until a blue color.

2.2.5. Quantification of b-galactosidase activityApproximately 2� 108 cells, grown to logarithmic

phase, were harvested, resuspended in 200ll 0.1M Tris–Cl, pH 7.5/0.05% Triton X-100, and lysed by twice snap-freezing in liquid nitrogen. An aliquot of each sample(V) depending on lacZ activity was added to 200ll of4mg/ml o-nitrophenyl-b-galactopyranosides (ONPG)/Z-buffer/0.27% (v/v) 2-mercaptoethanol and incubatedat 37 �C until a light straw color was observed. The re-action was stopped by addition of 500ll 1M Na2CO3and A420 values were measured. Cell concentration wasestimated by resuspending 20ll of lysed cell debris in1ml H2O and measuring A600 (W). b–Gal activity (units,U) is measured as U ¼ 100ðA420Þ=½A600 � V � T �, whereT is time of incubation for the assay.

Table 1

Yeast two-hybrid system

Advantages

• Y2H is an extremely inexpensive technique, requiring no specialized items of equipment.

• Yeast cells have excellent molecular genetic technologies established and are easy to use.

• Y2H is a eukaryotic in vivo technology, as opposed to in vitro interaction assays.

• Y2H is extremely sensitive and can detect weak and/or transient interactions.

• Several Y2H kits are commercially available together with many premade cDNA libraries from a variety of hosts and tissues.

• Versions of the Y2H system are available that express from regulated promoters, thus allowing toxic proteins to be examined.

• Baits can be made up from whole proteins or subdomains. Empirically, subdomains often work better.

• Y2H allows for rapid and sensitive cDNA library screening for unknown interacting partners. The approach generates information on the

interaction domain and yields the cDNA clone as part of the result.

• Many modifications of the Y2H system have been developed including one-and trihybrid systems.

Limitations

• Yeast is a lower eukaryotic cell and does not mirror the cellular context of higher eukaryotic cells.

• Bait proteins are transported to the yeast nucleus and this may not be the correct cellular compartment for the type of protein under investigation.

• Y2H is an overexpression system, with baits expressed as fusion proteins in the (often) foreign context of the yeast nucleus; therefore, it is prone to

generate ‘‘false positive.’’ Thereby all Y2H interactions need to be validated by some independent experimentation. Y2H systems exist that use

low-copy vectors to try to minimize numbers of false positives.

• Bait protein must be correctly folded into its native confirmation when expressed as a BD fusion in the nucleus. The position of the fusion

junction can markedly influence protein folding and thereby the ability to interact with a protein partner.

• Certain classes of proteins are generally not amenable to Y2H. This includes transcription factors, proteins spanning the membrane several times

over, extracellular domains, and those baits that are able to promote promiscuous transcription.

• Y2H will not detect associations dependent on certain posttranslational modifications such as glycosylation. However phospho-dependent

interactions can be detected by trihybrid screening.

304 J.H. White et al. / Methods 27 (2002) 301–310

2.3. Y2H screening of the GABAb R1 C terminus (Fig. 2)

Approximately 4� 106 human brain library cDNAswere transformed against the C terminus of GABAB1‘‘bait’’ and grown on selective medium in the presence of20mM 3-AT for 3–7 days at 30 �C. Yeast showing ac-tivation of both HIS3 and LacZ reporter genes wereselected; the library plasmid was recovered from theyeast and sequenced. In total, two main interactingproteins were recovered several times, each being con-firmed as a strong specific interactor in the confirmatoryand specificity assays. The first interacting library cDNAencoded the C terminus of the related GABAB2 receptorand all clones recovered expressed the coiled-coil do-main but not the seventh transmembrane (TM) domainof the receptor, as expected since TM domains do notwork well in Y2H. Subsequent assays for the LacZ re-porter proved that the GABAB1 and GABAB2 C terminiassociated as a heterodimer but homodimerizationcould not be detected (Fig. 2). Interestingly, the secondassociated protein, recovered in greater abundance thanGABAB2 C terminus, was the CREB2 or ATF4 tran-scription factor [20,21].

2.4. Confirmation of yeast two-hybrid interaction follow-ing expression in mammalian cells: use of coimmunopre-cipitation

Based on the identification of the putative interac-tion using Y2H we next confirmed the interaction byimmunoprecipitation studies. Myc-tagged GABAB1band or hemagglutinin (HA)-tagged GABAB2 wastransiently expressed in HEK293T cells. Immunopre-cipitation of Myc-GABAB1b from detergent-solubilizedcell fractions with Myc antisera led to immunodetec-tion of HA-GABAB2 within immune complexes usingHA as the primary antibody, but only on receptorcoexpression (Fig. 3, lanes 1–3). GABAB1 andGABAB2 association was also confirmed by coim-munodetection of Myc–GABAB1b from immune com-plexes captured using the anti-HA antibody, when thetwo receptor forms were coexpressed (Fig. 3, lanes4–6). Transfection and immunoprecipitation proce-dures are outlined below.

Cell culture and transfectionHEK293T cells were maintained in Dulbecco’s

modified Eagle’s medium (DMEM) containing 10% (v/v) fetal calf serum and 2mM glutamine. Cells wereseeded in 60-mm culture dishes and grown to 60–80%confluency (18–24 h) prior to transfection withpCDNA3 containing the relevant DNA species usingLipofectAMINE reagent. For transfection, 3lg ofDNA was mixed with 10ll of LipofectAMINE in 0.2mlof Opti-MEM (Life Technologies) and was incubated atroom temperature for 30min prior to the addition of1.6ml of Opti-MEM. Cells were exposed to the Lipo-fectAMINE/DNA mixture for 5 h and 2ml of 20% (v/v)newborn calf serum in DMEM was then added. Cellswere harvested 48–72 h after transfection.

Fig. 2. Y2H demonstrates herodimerization but not homodimerization

between GABAB1 and GABAB2 C termini. b-Galactosidase activitywas measured in yeast strain Y190 expressing GABAB1 or GABAB2 C

termini or with empty vector as controls, either by (a) freeze-fracture

assay or (b) quantified using ONPG relative to cell numbers. (Copy-

right permission from Nature, http://www.nature.com/.) Of each

combination of proteins expressed, the first named represents the

GAL4BD fusion and the second is the Gal4AD fusion. GAL4BD and

GAL4AD denote empty-vector controls.

Fig. 3. Coimmunoprecipitation studies of the GABAB heterodimer in

HEK293T cells. Lanes 1, 4: immunoprecipitates of cells transfected

with Myc–GABAB R1b only; lanes 2, 5: HA–GABAB R2 only; lanes 3,

6: immunoprecipitates of cells transfected with Myc–GABAB R1b

together with HA–GABAB R2. Lanes 1–3: lysates immunoprecipitated

with 9E10 (Myc) and blotted to 12CA5 (HA); lanes 4–6: lysates im-

munoprecipitated with 12CA5 (HA) and blotted with 9E10 (Myc).

(Copyright permission from Nature, http://www.nature.com/.)

J.H. White et al. / Methods 27 (2002) 301–310 305

2.4.1. Immunoprecipitation proceduresTransiently transfected HEK293T cells were har-

vested as described above. Cells from each dish wereresuspended in 1ml of 50mM Tris–HCl, 150mM NaCl,1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate,pH 7.5 (lysis buffer), supplemented with Complete pro-tease inhibitor cocktail tablets (1 tablet/25ml) (RocheDiagnostics). Cell lysis and membrane protein solubili-zation were achieved by homogenization for 20 s with aPolytron homogenizer followed by gentle mixing for30min at 4 �C. Insoluble debris was removed by mi-crocentrifugation at 16,000g for 15min at 4 �C and thesupernatant was precleared by incubating with 50ll ofprotein A–agarose (Roche Diagnostics) for 3 h at 4 �Con a helical wheel to reduce background caused bynonspecific adsorption of cellular proteins. The solubi-lized supernatant was then divided into 2� 500-ll ali-quots and 20ll of either HA or Myc antiserum wasadded to each. Immunoprecipitation was allowed toproceed for 1 h at 4 �C on a helical wheel prior to theaddition of 50ll of protein A–agarose suspension.Capture of immune complexes progressed overnight at4 �C on a helical wheel. Complexes were then collectedby microcentrifugation 12,000g for 1min at 4 �C andsupernatant was discarded. Beads were then washed bygentle resuspension and agitation sequentially in 1ml of50mM Tris–HCl, pH 7.5, 500mM NaCl, 0.1% (v/v)Nonidet P-40, and 0.05% (w/v) sodium deoxycholatefollowed by 1ml of 50mM Tris–HCl, pH 7.5, 0.1% (v/v)Nonidet P-40, and 0.05% (w/v) sodium deoxycholate.Immunoprecipitated proteins were released from proteinA–agarose by incubation in 30ll of SDS–PAGE samplebuffer at 70 �C for 10min and analyzed by SDS–PAGEfollowed by immunoblotting.

2.4.2. Cell harvesting and preparation of membranes for35[S]GTPcS binding and immunoblottingTransfected cells transiently expressing GABAB re-

ceptor subunits R1 and R2 were harvested simply byscraping from the culture dish in phosphate buffered-sa-line (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4,1.8mM KH2PO4, pH 7.2) (PBS). Cells were then recov-ered by centrifugation at 2000g for 2min in a microcen-trifuge at 4 �C.Cellsmust be thoroughlywashed in PBS toensure complete removal of any residual serum as this willact as a G-protein stimulator and may interfere withdownstream functional assays. Harvested cells may bestored as a pellet at )80 �C for up to 2 months or can beused directly for the production of plasma membranes.Plasma membrane-containing P2 particulate frac-

tions were then prepared from the above cell pastes. Allprocedures were carried out at 4 �C. Cell pellets wereresuspended in 1ml of 10mM Tris–HCl and 0.1mMEDTA, pH 7.5 (buffer A), and rupture of the cells wasachieved by homogenization for 20 s with a Polytronhomogenizer followed by passage (five times) through a

25-gauge needle. Cell lysates were centrifuged at 1000gfor 10min in a microcentrifuge to pellet the nuclei andunbroken cells, and P2 particulate fractions were thenrecovered by microcentrifugation at 16,000g for 30min.P2 particulate fractions were resuspended in buffer Aand stored at )80 �C until required. Protein concentra-tions were determined using the bicinchoninic acid(BCA) procedure [25] using BSA as a standard. Itshould be noted that most protocols detailing isolationof plasma membranes stipulate a centrifugation step inexcess of 40,000g to ensure membrane recovery. How-ever, we have found equivalent yields can be achievedusing 16,000g for 30min which has the advantage ofobviating the need for an ultracentrifuge, thus expedit-ing isolation of membranes.

2.5. 35[S]GTPcS binding

Activated receptors increase the rate of exchange ofGDP for GTP on the G-protein a subunit and therebyincrease the rate of GTP hydrolysis [26,27]. Members ofthe Gi family of G-proteins possess the highest rates ofguanine nucleotide exchange of all the heterotrimeric G-proteins [27]. This property exclusively allows thefunction of only Gi G-proteins in a complex membranemixture activated by specific agonists to be assessed bymeasuring the exchange of GDP for GTP and/or thesubsequent hydrolysis of GTP. Agonist-stimulatedbinding of an 35S-labeled form of the poorly hydrolyz-able analog of GTP, GTPcS ([35S]GTPcS) [28], lendsitself perfectly as a suitable functional assay to measureactivation of the GABAB receptor. This assay can beperformed using two techniques which differ only in themeans by which bound nucleotide is separated fromfree: (1) using wheat germ agglutinnin scintillationproximity assay (SPA) bead technology; (2) using tra-ditional separation of bound from free nucleotide byfiltration. The assay using SPA technology is moreconvenient than the filtration assay and is detailedherein with particular relevance to the GABAB receptor.Assays were performed in 96-well format using a

method modified from that described in Weiland andJakobs [28]. Membranes (10lg per point) were dilutedto 0.083mg/ml in assay buffer (20mM Hepes, 100mMNaCl, 10mM MgCl2, pH7.4) supplemented with sapo-nin (10mg/liter) and preincubated with 10lM GDP.Various concentrations of GABA were then added fol-lowed by [35S]GTPcS (1170Ci/mmol, Amersham) at0.3 nM (total volume of 100ll) and binding was allowedto proceed at room temperature for 30min. Nonspecificbinding was determined by the inclusion of 0.6mMGTP. Wheat germ agglutinin SPA beads (Amersham)(0.5mg) in 25 ll assay buffer were added and the wholewas incubated at room temperature for 30min withagitation. Plates were centrifuged at 1500g for 5min and[35S]GTPcS bound was determined by scintillation

306 J.H. White et al. / Methods 27 (2002) 301–310

Fig. 4. Coexpression of GABAB R1 and GABAB R2 receptors in HEK293T cells leads to GABA-mediated stimulation of [35S]GTPcS binding

activity. [35S]GTPcS binding activity was measured from membranes expressing Go1a together with GABAB receptor subunits. (A) [35S]GTPcSbinding in the absence (open bars) or presence (filled bars) of GABA (10mM). (B) The ability of varying concentrations of GABA to stimulate the

binding of [35S]GTPcS in membranes expressing either Go1a and HA-GABAB2 alone (open circles) or in combination with either GABAB1a (closedsquares) or GABAB1b (closed triangles). The data shown are the meansSD of triplicate measurements and are representative of three independentexperiments. (Copyright permission from Nature, http://www.nature.com/.)

J.H. White et al. / Methods 27 (2002) 301–310 307

counting on a Wallac 1450 Microbeta Trilux scintilla-tion counter. Fig. 4 demonstrates such an assay usingmembranes from HEK293T cells transiently transfectedto express Go1a in combination with either GABAB1 orGABAB2 or with both receptor subunits (Fig. 4A). Thefigure shows that coexpression of both subunits is re-quired to provide an agonist concentration-dependentstimulation of [35S]GTPcS binding ðEC50 ¼ 7:80:4� 105MÞ (Fig. 4B). These values are equivalent tothose of GABA-mediated stimulation of [35S]GTPcSbinding to rat brain membranes (5:9 0:4� 105 M)(data not shown). Hence, coexpression of GABAB1 andGABAB2 resulted in the generation of a functionalGABAB receptor.Note: To generate a significant agonist-mediated re-

sponse in transient systems it is often necessary to co-transfect receptor with additional Gi=Go G-protein.

2.6. FACS analysis

Flow-cytometry analysis (FACS) is a powerful toolthat can be used to study the cellular distribution ofimmunotagged proteins. Hence, this technique wasemployed to study the cellular localization of GABABreceptor subunits expressed in isolation and in combi-nation. HEK293T cells were transiently transfected withcDNA as described. Forty-eight to seventy-two hoursfollowing transfection cells were recovered and washedtwice in PBS supplemented with 0.1% (w/v) NaN3 and2.5% (v/v) fetal calf serum (FACS buffer). Cells wereresuspended in FACS buffer and incubated with primaryantibody 9E10 (c-myc) or HA for 15min at room tem-perature. Following three further washes with PBS, cellswere incubated with secondary antibody (sheep anti-mouse Fab2 coupled with fluorescein isothiocyanate)

diluted 1:30 for 15min at room temperature. For per-meabilized cells, the Fix and Perm Kit (Caltag) wasused. FACS analysis was performed on a Coulter EliteFACS cytometer. Thirty thousand cells were analyzed ineach experiment. Fig. 5 shows that in intact cells ex-pressing Myc–GABAB1b alone, no cell surface anti-Mycimmunoreactivity was detected. In contrast, immu-noreactivity toward Myc was detected in 35% of per-meabilized cells, reflecting intracellular expression ofGABAB1b. However, when GABAB2 was cotransfectedwith Myc–GABAB1b, 20% of intact cells displayed cellsurface anti-Myc immunoreactivity. Hence, these dataindicate that, in the presence of GABAB2, GABAB1b isefficiently moved to the cell surface. Interestingly, 14%of HEK293T cells transfected with HA–GABAB2showed surface immunoreactivity (Fig. 5c), suggestingthat this receptor can be efficiently trafficked to the cellsurface in the absence of GABAB R1.

2.7. Immunological studies and receptor glycosylation

Endoglycosidases F and H can be used to differentiatebetween immature, core glycosylated, and terminallyglycosylated N-linked glycoproteins that have passedthrough the Golgi apparatus [3]. Therefore, these en-zymes were used to examine the glycosylation status ofboth GABAB1 and GABAB2. Membranes from trans-fected cells were treated with either endoglycosidase F orH and expressedGABAB receptors were characterized byimmunoblotting to compare relative electrophoreticmobilities of the receptors (Fig. 6). Cell membranes ex-pressing either GABAB1a or GABAB1b produced distinctbands ofMr 130K and 100K, respectively (Fig. 6, lanes 1and 4), which, following endoglycosidase F treatment,decreased in size to single immunoreactive species of Mr

Fig. 5. Cell surface localization of GABAB R1 receptor is dependent on coexpression with GABAB R2. (A) FACS analysis using anti-c-Myc as

primary antibody on intact cells: (a) mock transfected; (b) GABAB1b; (c) GABAB1b þGABAB2. (B) FACS analysis using anti-c-Myc as primaryantibody on permeabilized cells: (a) mock transfected; (b) GABAB1b; (c) GABAB1b þGABAB2. (C) FACS analysis using anti-HA as primary an-tibody: (a) mock transfected; (b) GABAB2. (Copyright permission from Nature, http://www.nature.com/.)

308 J.H. White et al. / Methods 27 (2002) 301–310

110K and 80K (Fig. 6, lanes 2 and 5). This showed thatrecombinant GABAB1a and GABAB1b are glycoproteins,in agreement with the observations of Kaupmann et al.[5]. However, both forms were also sensitive to endogly-cosidase H treatment, indicating that the expressed pro-teins were only core glycosylated (lanes 3 and 6) andlacked terminal glycosylation. This observation, togetherwith that of the FACS analysis, suggested that the pro-teins were immaturely glycosylated and retained on in-ternal membranes. Significantly, when either GABAB1a(lanes 7–9) or GABAB1b (lanes 10–12) was coexpressedwith HA–GABAB2, a component of GABAB1 was resis-tant to endoglycosidaseHdigestion, suggesting thatwhencoexpressed with GABAB R2, a significant fraction ofGABAB1 was now amature glycoprotein (lanes 9 and 12).Similar studies with HA–GABAB1 gave an immuno-

reactive species ofMr 120K (Fig. 6, lanes 13, 16, 19) whichwas sensitive to endoglycosidase F (lanes 14, 17, 20) butresistant to endoglycosidase H (lanes 15, 18, 21) treat-ment, whether expressed alone or in combination withGABAB1. Thus, these data indicated that expressed HA–GABAB2 was a mature glycoprotein whose glycosylationstatus was not affected by coexpression with GABAB R1.Hence, the deglycosylation studies suggested that hete-rodimerization, possibly occurring in the Golgi complex,could be a prerequisite for maturation and transport of

GABAB R1 to the plasma membrane. Immunoblottingand deglycosylation protocols are described below.Antiserum 501 was raised against a synthetic peptide

corresponding to the C-terminal 15 amino acids of theGABAB1 receptor and was produced in a sheep, using aconjugate of this peptide and keyhole limpet hemocya-nin (Calbiochem) as antigen. Membrane samples (30–60lg) were resolved by SDS–PAGE using 10% (w/v)acrylamide. Following electrophoresis, proteins weresubsequently transferred to nitrocellulose (Hybond-ECL, Amersham), probed with antiserum 501 at 1:1000dilution, and visualized by enhanced chemiluminescence(ECL, Amersham). Epitope tags were visualized byimmunoblotting with anti-Myc (1:100 dilution) or anti-HA (1:500) monoclonal antibodies.Enzymatic removal of asparagine-linked (N-linked)

carbohydrate moieties with endoglycosidases F and Hwas performed essentially according to the manufac-turer’s (Roche Diagnostics) instructions using 50lg ofmembrane protein per enzyme reaction. GABAB re-ceptor glycosylation status was then studied followingSDS–PAGE/immunoblotting of samples.

3. Conclusions

The techniques described here enabled the observa-tion that the fully functional GABAB receptor was anobligate heterodimer, comprising two closely relatedand interacting GPCRs. Since these initial experimentaldata, many more GPCRs have been reported to di-merize [1,2], and indeed, the idea of other GPCR-asso-ciated proteins is now coming to the fore [29]. However,sensitive biochemical studies in mammalian recombi-nant systems, which generate compelling data suggestingdimerization between receptors in various combinations,should be used with care as misleading data can be easilygenerated. For example with immunoprecipitationstrategies, due to the hydrophobic nature of the seven-transmembrane helices of GPCRs, stringent controlsshould be put in place to exclude nonspecific interac-tions between receptor pairs that may result followingdetergent dissolution of cellular membranes. This maybe a significant problem when overexpression systemsare employed to study dimerization. Indeed, more recentstudies have used various forms of time-resolved fluo-rescence resonance energy transfer and bioluminescenceresonance energy transfer to study GPCR homo- andheterooligomerization in living cells [30–32, this issue]. Itis critically important to go back to native tissue andconfirm that the observed GPCR dimerization is ofphysiological significance. Clearly for receptors to formheterodimers, the two partnering GPCRs should becoexpressed in the same cell and indeed in the samesubcellular compartment for some point during their lifecycle. Colocalization at a cellular level is therefore

Fig. 6. Coexpression of GABAB1 variants with GABAB2 receptors in

HEK293T cells results in terminal glycosylation of GABAB1. Mem-

brane fractions from cells transfected with either GABAB1a (lanes 1–3),

GABAB1b (lanes 4–6), or HA-GABAB2 (lanes 13–15), or with 1lg eachof HA-GABAB2 in combination with 1lg of either GABAB1a (lanes 7–9, 16–18) or GABAB1b (lanes 10–12, 19–21). Glycosylation status of

transfected receptors was assessed following treatment with either ve-

hicle (lanes 1, 4, 7, 10, 13, 16, 19), endoglycosidase F (lanes 2, 5, 8, 11,

14, 17, 20), or endoglycosidase H (lanes 3, 6, 9, 12, 15, 18, 21), Upper:

Antiserum 501 was used as primary reagent to allow identification of

both GABAB1a and GABAB1b. Lower: Anti-HA antiserum was em-

ployed to permit identification of HA-epitope-tagged GABAB2.

(Copyright permission from Nature, http://www.nature.com/.)

J.H. White et al. / Methods 27 (2002) 301–310 309

necessary for in vivo confirmation, using either in situhybridization or preferably immunohistochemistry.Coimmunoprecipitation from native tissue also providessuperior evidence for a physiological interaction. Re-ceptors that are capable of heterodimerization in over-expressed cell systems with resulting alterations to theirpharmacological profile may not actually do so in realtissue. For orphan receptors coexpression data maythemselves provide the initial evidence for receptor di-merization. In the case of the GABAB receptor conclu-sive evidence for the importance of heterodimerizationin vivo has been demonstrated by the GABAB1 receptorknockout which loses all responses to GABAB agonistsdespite expressing GABAB2 [33,34].

References

[1] M. Bouvier, Nat. Rev. Neurosci. 2 (2001) 274–286.

[2] G. Milligan, J. Cell Sci. 114 (2001) 1265–1271.

[3] L.M. McLatchie, N.J. Fraser, M.J. Main, A. Wise, J. Brown, N.

Thompson, R. Solari, M.G. Lee, S.M. Foord, Nature 393 (1998)

333–339.

[4] N.G. Bowery, Annu. Rev. Pharmacol. Toxicol. 33 (1993) 109–147.

[5] K. Kaupmann, K. Huggel, J. Heid, P.J. Flor, S. Bischoff, S.J.

Mickel, G. McMaster, C. Angst, H. Bittiger, W. Froestl, B.

Bettler, Nature 386 (1997) 239–246.

[6] J.H. White, A. Wise, M. Main, A. Green, N.J. Fraser, G.H.

Disney, A.A. Barnes, P. Emson, S.M. Foord, F.H. Marshall,

Nature 396 (1998) 679–682.

[7] A. Couve, A.K. Filippov, C.N. Connolly, B. Bettler, D.A. Brown,

S.J. Moss, J. Biol. Chem. 273 (1998) 26361–26367.

[8] P.L. Bartel, S. Fields, The Yeast Two Hybrid System, Oxford

Univ. Press, London, 1997.

[9] R. Kuner, G. Kohr, S. Grunewald, G. Eisenhardt, A. Bach, H.C.

Kornau, Science 283 74–77.

[10] R.A. Kammerer, S. Frank, T. Schulthess, R. Landwehr,

A. Lustig, J. Engel, Biochemistry 38 (1999) 13263–13269.

[11] M. Margeta-Mitrovic, Y.N. Jan, L.Y. Jan, Neuron 27 (2000) 97–

106.

[12] D.A. Schwarz, G. Barry, S.D. Eliasof, R.E. Petroski, P.J.Conlon,

R.A. Maki, J. Biol. Chem. 275 32174–32181.

[13] K.A. Jones, B. Borowsky, J.A. Tamm, D.A. Craig, M.M. Durkin,

M. Dai, W.J. Yao, M. Johnson, C. Gunwaldsen, L.Y. Huang, C.

Tang, Q.R. Shen, J.A. Salon, K. Morse, T. Laz, K.E. Smith, D.

Nagarathnam, S.A. Noble, T.A. Branchek, C. Gerald, Nature 396

(1998) 674–679.

[14] K. Kaupmann, B. Malitschek, V. Schuler, J. Heid, W. Froestl,

P. Beck, J. Mosbacher, S. Bischoff, A. Kulik, R. Shigemoto,

A. Karschin, B. Bettler, Nature 396 (1998a) 683–687.

[15] A. Pagano, G. Rovelli, J. Mosbacher, T. Lohmann, B. Duthey,

D. Stauffer, D. Ristig, V. Schuler, I. Meigel, C. Lampert, T. Stein,

L. Prezeau, J. Blahos, J. Pin, W. Froestl, R. Kuhn, J. Heid,

K. Kaupmann, B. Bettler, J. Neurosci. 21 (2000) 1189–1202.

[16] A.R. Calver, M.J. Robbins, C. Cosio, S.Q. Rice, A.J. Babbs,

W.D. Hirst, I. Boyfield, M.D. Wood, R.B. Russell, G.W. Price, A.

Couve, S.J. Moss, M.N. Pangalos, J. Neurosci. 21 (2001) 1203–

1210.

[17] S. Fields, O. Song, Nature 340 (1989) 245–246.

[18] G.G. Toby, E.A. Golemis, Methods 24 (2001) 201–217.

[19] K.J. Fuller, M.A. Morse, J.H. White, S.A. Dowell, M.J. Sims,

Biotechniques 25 (1998) 85–92.

[20] J.H. White, R.A. McIllhinney, A. Wise, F. Ciruela, W.Y. Chan,

P.C. Emson, A. Billinton, F.H. Marshall, Proc. Natl. Acad. Sci.

USA 97 (2000) 13967–13972.

[21] R.B. Nehring, H.P. Horikawa, O. El Far, M. Kneussel, J.H.

Brandstatter, S. Stamm, E. Wischmeyer, H. Betz, A. Karschin,

J. Biol. Chem. 275 (2001) 35185–35191.

[22] T. Durfee, K. Becherer, P. Chen, S. Yeh, Y. Yang, A. Kilburn,

W. Lee, S.J. Elledge, Genes Dev. 7 (1993) 555–569.

[23] J.W. Harper, G.R. Adami, N. Wei, K. Keyomarsi, S.J. Elledge,

Cell 75 (1993) 805–816.

[24] F.M. Ausubel, R. Brent, R.E. Kingston, J. Moore, G. Seidman,

J.A. Smith, K. Sruhl (Eds.), Current Protocols in Molecular

Biology, Wiley, New York, 1994.

[25] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H.

Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J.

Olson, D.C. Klenk, Anal. Biochem. 150 (1985) 76–85.

[26] H.R. Bourne, D.A. Sanders, F. McCormick, Nature 348 (1990)

125–132.

[27] A.G. Gilman, Annu. Rev. Biochem. 56 (1987) 615–649.

[28] T. Weiland, K.H. Jakobs, Methods Enzymol. 237 (1994) 3–

13.

[29] G. Milligan, J.H. White, Trends Pharmacol. Sci. 10 (2001) 513–

518.

[30] S. Angers, A. Salahpour, E. Joly, D. Chelsky, M. Dennis,

M. Bouvier, Proc. Natl. Acad. Sci. USA 97 (2000) 3684–3689.

[31] M. Rocheville, D.C. Lange, U. Kumar, R. Sasi, R.C. Patel, Y.C.

Patel, Science 288 (2000) 154–157.

[32] M. McVey, D. Ramsay, E. Kellett, S. Rees, S. Wilson, A.J. Pope,

G. Milligan, J. Biol. Chem. 276 (2001) 14092–14099.

[33] V. Schuler, C. Luscher, C. Blanchet, N. Klix, G. Sansig, K.

Klebs, M. Schmutz, J. Heid, C. Gentry, L. Urban, A. Fox, W.

Spooren, A.L. Jaton, J. Vigouret, M. Pozza, P.H. Kelly, J.

Mosbacher, W. Froestl, E. Kaslin, R. Korn, S. Bischoff, K.

Kaupmann, H. van derPutten, B. Settler, Neuron 31 (1) (2001)

47–58.

[34] H.M. Prosser, C.H. Gill, W.D. Hirst, E. Grau, M. Robbins, A.

Calver, E.M. Soffin, C.E. Farmer, J. Gray, E. Schenck, B.S.

Warmerdam, C. Clapham, C. Reavill, D.C. Rogers, T. Stean,

N. Upton, K. Humphreys, A. Randall, M. Geppert, C.H.

Davies, M.N. Pangalos, Mol. Cell. Neurosci. 17 (2001) 1059–

1070.

310 J.H. White et al. / Methods 27 (2002) 301–310