[Contemporary Clinical Neuroscience] The G Protein-Coupled Receptors Handbook Volume 215 || Functional Complementation and the Analysis of GPCR Dimerization

Functional Complementation 267

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From: Contemporary Clinical Neuroscience: The G Protein-Coupled Receptors HandbookEdited by: L. A. Devi © Humana Press Inc., Totowa, NJ

12Functional Complementation and the

Analysis of GPCR Dimerization

Graeme Milligan, Juan J. Carrillo, and Geraldine Pascal

1. INTRODUCTION

Cloning of complementary DNAs (cDNAs), which are predicted to encodeG protein-coupled receptors (GPCRs), required a mechanism to ascertain ifthe single polypeptide encoded by such cDNAs was sufficient to generatethe pharmacology and function anticipated for the receptor in question.Because this generally was the case—and despite evidence of greater com-plexity (reviewed in ref. 1)—it became axiomatic that GPCRs were single,seven-transmembrane-span polypeptides. However, a series ofimmunoblotting studies has suggested that a fraction of cellular GPCRs mightexist as dimers or higher order species in both transfected cell lines and na-tive tissues (2–4). The known propensity of hydrophobic proteins to aggre-gate—particularly when samples were heated prior to separation by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)—meantthat it was possible to disregard these data. However, studies in which differ-entially epitope-tagged forms of a single GPCR could be co-immunoprecipi-tated following their co-expression in heterologous cell lines providedconsiderable evidence for the presence of dimeric or, indeed, higher oligo-meric complexes (2,5). This was generally not observed when the two formsof the GPCR were expressed in separate cell populations that were mixedprior to membrane solubilization and immunoprecipitation. These results in-dicate that co-immunoprecipitation did not result simply from aggregation ofthe hydrophobic transmembrane elements of these polypeptides followingremoval of lipid by treatment with detergents. Equivalent studies then beganto examine the proclivity of different, co-expressed GPCRs to be co-immu-noprecipitated. These studies have produced a large body of evidence sup-

268 Milligan, Carrillo, and Pascal

porting the capacity of GPCRs to exist as heterodimers/-oligomers (for re-view, see refs. 6 and 7). However, studies that have reported a very broadcapacity of particular GPCRs to allow co-immunoprecipitation of co-ex-pressed GPCRs (8) have questioned the relevance of GPCR co-immunopre-cipitation data when this is not accompanied by similar data produced byalternative strategies.

There is also a concern that many “co-immunoprecipitation” studies arepoorly performed and controlled. For example, many studies in this areacentrifuge samples to remove particulate material after detergent treatmentto generate a “soluble” preparation for the immunoprecipitation steps. Often,however, the centrifugation step is performed for only short periods, and thecentrifugal force is too low to achieve this end. This inevitably results insmall membrane fragments being present in the “soluble” fraction and thepossibility that “co-immunoprecipitation” represents nothing more than bothpolypeptides presenting within the same membrane fragment.

These types of issues have resulted in the widespread adoption and use oftechniques that can monitor protein–protein interactions in either membranepreparations or intact living cells. Various forms of resonance energy trans-fer techniques have been employed to explore GPCR dimerization/oligo-merization because of their relative simplicity and exquisite dependence ondistance between the energy donor and acceptor species (9–14). These issuesand the utility of the systems have recently been reviewed (15,16) and arediscussed further within this volume (17). An alternative strategy that canbe used instead of, or in parallel with, resonance energy transfer techniquesis functional complementation produced following co-expression of pairs ofdistinct GPCR or GPCR–G protein fusion mutants.

2. COMPLEMENTATION OF PAIRS OF MUTANT GPCRS

GPCRs can be assembled from co-expressed fragments (12,18). This hasled to the hypothesis that segments of GPCRs can be considered distinctdomains capable of independent folding. Extensions of this idea have alsoallowed the envisaging of models of GPCR dimers that include explicit re-quirement for domain swapping (19,20). Although direct experimental evi-dence in favor of such models is somewhat limited, such evidence wouldpotentially explain marked variations in ligand pharmacology that are some-times observed when two GPCRs capable of heterodimer formation are co-expressed (21) or when chimeric GPCRs are constructed (21,22). Reciprocalchimeric GPCRs between the α2C-adrenoceptor and the m3 muscarinic ace-tylcholine receptor that contained the N-terminus and transmembrane seg-ments I–V of one receptor and transmembrane segments VI–VII and the


C-terminal tail of the second bound neither muscarinic or α2-adrenergicligands. However, the co-expression of the two chimeras resulted in regen-eration of some level of high-affinity binding for both muscarinic and α2-adrenergic antagonists (22). These workers were also able to reconstituteagonist-mediated signal transduction when pairs of distinct, functionally im-paired mutant muscarinic receptors were co-expressed (22). Related studieson the angiotensin II type 1 receptor showed that forms of this GPCR withspecific mutations in either transmembrane region III or transmembrane seg-ment V were unable to bind ligands. However, ligand binding was observedupon co-expression (23). These studies were central in providing evidence infavor of molecular interactions between GPCRs. Despite these conclusions,the number of ligand-binding sites produced following co-expression waslow, and it is unclear if this simply reflects that the individual mutations arevery poorly expressed compared to the wild-type protein, or if the efficiencyof “reconstitution” is poor. One potential explanation is that if the reconstitu-tion of high-affinity ligand binding requires a domain exchange mechanism,then this may be thermodynamically feasible but uncommon compared withdimerization based on linear packing, which would not be anticipated toregenerate ligand binding on co-expression of the two mutants.

The first definitive evidence of GPCR hetero-dimerization was the rec-ognition that the γ-aminobutyric acid (GABA)BR1 polypeptide could bindagonist (although with lower than the anticipated affinity) and antagonistligands but could not generate signals or be trafficked effectively to the cellsurface unless co-expressed with the GABABR2 polypeptide (24,25). Simi-larly to other family C GPCRs, the long extracellular N-terminal extensionbinds the ligand, but the architecture of the prototypic seven-transmembranecore defines the signal transduction unit. Because such GPCRs can be con-sidered bifunctional polypeptides, chimeras between the extracellular re-gion and the transmembrane segments and intracellular elements have beengenerated to aid understanding of the mechanisms of signal propagation,from ligand binding to G protein activation (26).

Several family A GPCRs that respond to large glycoprotein hormonesalso have long extracellular N-termini and, therefore, are amenable to re-lated approaches. One well-studied example is the luteinizing hormone re-ceptor. This GPCR has been used to generate differentially defective mutantpairs—one in the extracellular exodomain that lacks binding of the ligandhuman chorionic gonadotropin and a second that can bind the ligand but isunable to signal because of a mutation in the endodomain (27,28). Their co-expression resulted in the restoration of ligand generation of the secondmessenger cyclic adenosine monophosphate (cAMP). Again, although this


provided a persuasive case for intermolecular interactions among GPCRs,the amplification mechanisms resulting in cAMP generation (which alsooften result in the characteristic of “spare receptors”) do not allow for easyassessment of the effectiveness of the complementation process.

It has been suggested that unlike many other GPCRs, the luteinizing hor-mone receptor does not form stable dimers. Such functional complementa-tion studies can provide evidence for both cis-activation (i.e., ligand boundto the exodomain, causing activation on the same endodomain) or trans-activation, where the ligand activates the partner endodomain within a dimer(29). It is noteworthy that such a model may also explain the complex inter-actions between activated protease-activated receptor (PAR)3 and PAR4GPCRs (30) and, potentially, other complex interactions between PAR sub-types (31). The glycoprotein hormones and PARs are unusual examples offamily A, rhodopsin-like GPCRs. For the bulk of these, the agonist ligandsbind at least partially within the cavity created by the seven-transmembranehelix architecture. We wished to develop a generic functional complemen-tation strategy that would be suitable to study both homo- andheterodimerization of family A GPCRs and that would provide quantitativeassays for the effectiveness of complementation. Therefore, we employedpairs of nonfunctional, but potentially complementary, GPCR–G protein α-subunit fusion proteins.

3. FUNCTIONAL COMPLEMENTATION OF PAIRSOF GPCR–G PROTEIN FUSIONS

3.1. GPCR–G Protein Fusions

The first GPCR–G protein fusion was constructed between the β2-adrenoceptor and the α-subunit of the long isoform of the G protein Gs.When this was expressed in S49 cyc– cells lacking expression of Gαs, the β-adrenoceptor agonist isoprenaline was able to stimulate adenylyl cyclase(32), thus confirming the functionality of both elements of the fusion con-struct. Although the fusion construct had several interesting characteristics(32), it was initially viewed as little more than a curiosity. However, overthe intervening period, a wide range of GPCRs and G proteins have beenused to generate similar fusions (33,34). These have been used to addressissues such as the selectivity of GPCRs in activating different G proteins(35), the role and regulation of posttranslational acylation in the functionand cellular location of GPCRs and G proteins (36–38), as reagents to screenfor agonists at “orphan” GPCRs (39,40) and, most recently, to explore thebasis and selectivity of GPCR dimerization (41).


GPCR–G protein fusions can also be considered as bifunctional polypep-tides because, although they are generated from a single open frame, theycontain the sequence and functionality of both GPCR and G protein. There-fore, mutants that eliminate the ability of agonists to generate a signal corre-sponding to activation of the G protein can be produced by alterations in thesequence of either the GPCR or the G protein element. This allows for theproduction of distinct pairs of fusions that are individually inactive but havethe potential to complement function if they interact. Two obvious regionscan be targeted for the G protein segment of the fusion. All G protein α-subunits have a conserved Val-Gly-Gly-Gln-Arg sequence (Table 1), wheremutation of the second Gly to Ala generates a form of the G protein that isunable to exchange guanosine diphosphate (GDP) for guanosine triphos-phate (GTP) and hence become activated. This provides a simple strategy togenerate GPCR–G protein fusion proteins that are unable to respond to ago-nist ligands even though the GPCRs linked to such mutants are wild-typeand able to bind ligands. Equally, because it is well established that the ex-treme C-terminal region of most G protein α-subunits is a key contact do-main for GPCR-mediated activation (42), judicious mutation in this regioncan produce forms of the G protein that are not responsive to GPCRs. Forexample, because pertussis toxin-catalyzed adenosine diphosphate (ADP)-ribosylation of the Cys residue four amino acids from the C-terminus of allwidely expressed Gi-family G protein α-subunits prevents GPCR-mediatedactivation of these G proteins, Bahia et al. (43) replaced this residue in Gαi1with each of the other naturally occurring amino acids and then assessed theability of each of the mutants to be activated by agonist occupancy of theα2A-adrenoceptor. Substitution of the Cys with more hydrophilic amino ac-ids reduced coupling effectiveness, and there was no significant activationwith either positively or negatively charged amino acids at this position.Similar data have been produced for activation of equivalently mutatedforms of Gαi3 by the 5-HT1A receptor (44). Several of the Gαi1 mutationshave been constructed into fusion proteins with the α2A-adrenoceptor andhave been used to monitor the effects on these alterations on informationtransfer from GPCR to G protein, which was measured as alterations in ago-nist potency and relative efficacy (45,46). Similarly, mutation of the Tyrfour amino acids from the C-terminal of Gα11 resulted in reduction of itsability to be activated by the α1b-adrenoceptor (47). With Asp at this posi-tion, no activation was observed by the agonist-occupied α1b-adrenoceptor,both in co-expression studies and when the modified G protein was con-structed into a fusion with this GPCR (47). Although it has not been exploredin such detail, it is well known that the lack of function of Gαs in the S49


unc cell line reflects an Arg to Pro substitution six amino acids from the C-terminus of this G protein (48). Therefore, although the details of contactsbetween different GPCRs and a specific G protein differ subtly (49), alter-ations in the C-terminal tail of the G protein can produce forms than cannotbe activated by the GPCR of choice.

Table 1A Generic Strategy to Generate Inactive G Protein α-Subunits

G α-subunit Species Sequence

Gs Human VGGQRDERRKGolf Human VGGQRDERRKGi1 Human VGGQRSERKK

Rat VGGQRSERKKGi2 Human VGGQRSERKK

Mouse VGGQRSERKKGi3 Human VGGQRSERKK

Rat VGGQRSERKKGo1 Human VGGQRSERKK

Bovine VGGQRSERKKGo2 Human VGGQRSERKKGz Rat VGGQRSERKK

Bovine VGGQRSERKKTransducin1 Human VGGQRSERKKTransducin2 Rat VGGQRSERKKGustducin Human VGGQRSERKKGq Mouse VGGQRSERKK

Human VGGQRSERKKG11 Rat VGGQRSERKK

Human VGGQRSQRQKG12 Rat VGGQRSQRQK

Human VGGQRSERKRG13 Human VGGQRSERRKG14 Mouse VGGQRSERRKG15 Human VGGQRSERKKG16

G protein α-subunits are highly conserved between mammalian spe-cies, particularly in core domains involved in the binding and hydroly-sis of guanine nucleotides. Alteration of the highlighted glycine residueto alanine results in forms of the G protein that are unable to bind GTP.This provides a generic means to generate G proteins that are unable tobe activated by G protein-coupled receptors (GPCRs) and thus to pro-duce one of the pair of inactive GPCR–G protein fusion proteins used inthe functional complementation strategy.


A range of potential strategies are available to produce inactive GPCR–Gprotein fusions via mutation of the GPCR. However, the most generic (atleast, for many rhodopsin-like class A GPCRs) appears to be via mutation ofone or more highly conserved hydrophobic residues in the second intracel-lular loop of the GPCR to acidic residues. The first of these is frequentlythree amino acids downstream of the highly conserved DRY domain, andthe second is four amino acids further downstream (Table 2).

3.2. Complementation of GPCR–G Protein Fusionsin Cell Membranes

A useful feature of GPCR–G protein fusions is that the defined 1:1 stoichi-ometry ensures that saturation ligand-binding studies define the expressionlevels of the G protein as well as the GPCR. Agonist stimulation of the bind-ing of [35S]GTPγS (50) to such fusion proteins can then provide a proximal,and potentially quantitative, assay of G protein activation which directexperiments have shown to be linear over a good range of membrane amounts(38). It is important to demonstrate that agonist-mediated binding of[35S]GTPγS is truly to the G protein element of the fusion rather than to en-dogenously expressed G proteins. This is an important issue because—at least,for some fusion constructs—it is clear that activation of endogenously ex-pressed G proteins can occur (51,52). However, generally, this seems to be aserious concern only with high-level expression of the GPCR–G protein fu-sion or when a free G protein is also co-expressed at high levels (53). Forfusions incorporating members of the Gi family of G proteins, mutation of thepertussis toxin-sensitive Cys residue to a hydrophobic amino acid, such asIle, prevents pertussis toxin-catalyzed ADP-ribosylation without inhibitingGPCR-mediated activation of the G protein (45,51). Therefore, followingtransfection of cells to express a GPCR–G protein fusion containing such apertussis toxin-resistant mutant, treatment of the cells with pertussis toxinresults in ADP-ribosylation of only the endogenously expressed forms of Gi.Binding of [35S]GTPγS to these expressed forms of Gi hence cannot be stimu-lated by agonist. Therefore, following immunoprecipitation with an anti-Gαiantiserum, agonist-enhanced binding of the nucleotide must reflect loading ofthe G protein element of the fusion protein. For example, membranes pre-pared from pertussis toxin-treated HEK293 cells expressing a δ-opioid pep-tide (DOP)-opioid receptor–Cys351IleGαi1 fusion protein (15 fmol of[3H]antagonist binding sites) were subjected to a [35S]GTPγS binding assayin the absence or presence of the agonist D-ala2, D-leu5-enkephalin (DADLE)and were then subjected to immunoprecipitation. Radioactivity in the pres-ence of the agonist was approximately sixfold higher than in its absence, con-


Table 2A Generic Strategy to Generate GPCR Variants Unable to Activate G Proteins

GPCR Specie G protein Sequence

5-HT1A receptor Human Gi/Go DRYWAITDPID5-HT1B receptor Mouse Gi/Go DRYWAITDAVE5-HT1D receptor Rabbit Gi/Go DRYWAITDALE5-HT2A receptor Rat Gq/G11 DRYVAIQNPIH5-HT2C receptor Rat Gq/G11 DRYVAIRNPIE5-HT4 receptor Mouse Gs DRYYAICCQPL5-HT6 receptor Rat Gs DRYLLILSPLRα1a-adrenoceptor Bovine Gq/G11 DRYIGVSYPLRα1b-adrenoceptor Hamster Gq/G11 DRYIGVRYSLQα2b-adrenoceptor Rat Gi/Go DRYWAVSRALEβ1-adrenoceptor Human Gs DRYLAITSPFRβ2-adrenoceptor Bovine Gs DRYLAITSPFKβ3-adrenoceptor Mouse Gs DRYLAVTNPLRA1 adenosine receptor Human Gi/Go DRYLRVKIPLRA3 adenosine receptor Human Gi/Go DRYLRVKLTVRAcetylcholine M1 receptor Mouse Gq/G11 DRYFSVTRPLSAcetylcholine M2 receptor Human Gi/Go DRYFCVTKPLTAcetylcholine M3 receptor Mouse Gq/G11 DRYFSITRPLTAngiotensin AT1A receptor Human Gq/G11 DRYLAIVHPMKAngiotensin AT1B receptor Rat Gq/G11 DRYLAIVHPMKBradykinin B2 receptor Human Gq/G11 DRYLALVKTMSChemokine CXCR3 Mouse Gi/Go DRYLSIVHATQChemokine CXCR4 Human Gi/Go DRYLAIVHATNDopamine D2 receptor Mouse Gi/Go DRYTAVAMPMLDopamine D3 receptor Rat Gi/Go DRYTAVVMPVHFSH receptor Bovine Gs ERWHTITHAMQGnRH receptor Mouse Gq/G11 DRSLAITQPLAHistamine H1 receptor Mouse Gq/G11 DRYRSVQQPLRHistamine H2 receptor Human Gs DRYCAVMDPLRLH receptor Mouse Gs ERWHTITYAVQMelanocortin 2 receptor Human Gs DRYITIFHALRDOP-opioid receptor Rat Gi/Go, Gz DRYIAVCHPVKKOP-opioid receptor 1 Mouse Gi/Go DRYIAVCHPVKMOP-opioid receptor 1 Rat Gi/Go, Gz DRYIAVCHPVKRhodopsin Bovine Gt ERYVVVCKPMSOxytocin receptor Rat Gq/G11 DRCLAICQPLRP2U purinoceptor 1 Rat Gq/G11 HRCLGVLRPLHProstaglandin D2 receptor Mouse Gs ECWLSLGHPFFProstaglandin E2 receptor Rat Gq/G11 ERCVGVTQPLISomatostatin receptor 2 Human Gi/Go Gq DRYLAVVHPIKThyrotropin-releasing Rat Gq/G11 ERYIAICHPIK

hormone receptor-1

The vast majority of class A, rhodopsin-like G protein-coupled receptors (GPCRs) possess a pairof hydrophobic residues (highlighted) in the second intracellular loop, downstream of the highly

continued


firming the functionality of this construct. When similar experiments wereperformed with a version of this fusion protein that also incorporated aGly203Ala mutation in the G protein, DADLE did not enhance incorporationof the nucleotide. This was also true for a version of the fusion that incorpo-rated Val150Glu and Val154Asp mutations into the second intracellular loop ofthe GPCR element but that was wild-type for Gαi1, other than the pertussistoxin-resistant Cys351Ile mutation. However, when Val150Glu,Val154AspDOP–Cys351IleGαi1 was co-expressed with DOP–Gly203Ala,Cys351IleGαi1membranes prepared after treatment with pertussis toxin and containing 15fmol of DOP-opioid receptor binding sites displayed marked reconstitution of[35S]GTPγS binding in response to DADLE (Fig. 1). Assuming that theintracellular loop 2 mutations do not interfere with DOP–DOP interactions,that GPCRs function as dimers, that dimers bind two ligands, and thatVal150Glu,Val154Asp DOP–Cys351IleGαi1 and DOP–Gly203Ala,Cys351IleGαi1express to equal extents in the cotransfections, simple arithmetic defines thatthe heterodimer (containing one copy of each of Val150Glu,Val154Asp DOP–Cys351IleGαi1 and DOP–Gly203Ala,Cys351IleGαi1) should represent only 50%of the ligand-binding sites. Of the other 50%, 25% would be expected to beVal150Glu,Val154Asp DOP–Cys351IleGαi1 homodimers, and 25% would beexpected to be DOP-Gly203Ala,Cys351IleGαi1 homodimers.

As demonstrated earlier, neither the Val150Glu,Val154Asp DOP–Cys351IleGαi1 nor the DOP–Gly203Ala,Cys351IleGαi1 homodimers bind[35S]GTPγS in response to agonists. Therefore, conceptually, following co-expression, membranes containing 30 fmol of antagonist binding sites couldbe anticipated to bind the same level of [35S]GTPγS in response to agonist asmembranes expressing 15 fmol of the DOP–Cys351Ile Gαi1 fusion protein.Reconstitution was not this effective (Fig. 1). This may imply that the qua-ternary structure of the GPCR–G protein fusion is a higher order oligomerrather than a dimer or that there is a complex mixture of species that mayalso include monomers. Further analyses are required to test these ideas.

Table 2 (From opposite page) conserved ’DRY’ domain at the bottom of transmembraneregion III. In the examples we tested, alteration of one or both of these to acidic residuesresulted in forms of the GPCR that are unable to activate G protein. This provides a poten-tially generic means to form an inactive GPCR–G protein fusion protein. Such mutations donot affect the binding of antagonist ligands. The effect on the binding of agonist ligands ismore complex and generally reflects whether the GPCR in question shows a significant alter-ation in agonist binding in response to addition of guanine nucleotides. For example, manyGq/G11-coupled GPCRs display only a marginal effect on agonist binding affinity; however,for Gs and Gi/Go-coupled receptors, this alteration in affinity can be substantial.


Fig. 1. Functional complementation of agonist-mediated [35S]GTPγS binding byco-expression of pairs of inactive DOP-opioid receptor–Gαi1 fusion proteins. A fu-sion protein was created between the human DOP-opioid receptor and a pertussistoxin-resistant Cys351Ile mutant of Gαi1. Further modifications to this fusion intro-duced either Val150Glu,Val154Asp mutations into the receptor element or Gly203Alainto the G protein. Each of these three fusion proteins was transfected into HEK293cells that were then treated with pertussis toxin to cause ADP-ribosylation ofendogenously expressed forms of Gαi/Gαo. Expression levels of the fusion proteinswere measured by the specific binding of the opioid receptor antagonist[3H]diprenorphine and membrane amounts containing 15 fmol of each fusion pro-tein used in [35S]GTPγS binding studies, which were conducted in the absence orpresence of the synthetic enkephalin DADLE (10 μM). At the termination of assay,samples were immunoprecipitated with an antiserum directed to the C-terminaldecapeptide of Gαi1 and were then counted. Equivalent studies were also performedon membranes of cells into which Val150Glu,Val154Asp DOP-opioid receptor–Cys351IleGαi1 and DOP-opioid receptor–Gly203Ala,Cys351IleGαi1 were co-expressed. Co-expression reconstituted DADLE-stimulated [35S]GTPγS binding, butthis was not produced when membrane preparations individually expressing eitherVal150Glu,Val154Asp DOP-opioid receptor–Cys351IleGαi1 or DOP-opioid receptor–Gly203Ala,Cys351IleGαi1 were mixed.


Importantly, however, co-expression is required to reconstitute function.Membranes prepared from cells each expressing one of the inactive fusionconstructs did not allow agonist-stimulated binding of [35S]GTPγS whenmixed. Equivalent fusion constructs incorporating the μ-opioid peptide(MOP)-opioid receptor have similar characteristics, and reconstitution ofagonist function is again achieved following co-expression of the pair ofindividually inactive fusion constructs. Co-expression of one inactive fusioncontaining the MOP-opioid receptor, with the partner containing the DOP-opioid receptor, also results in reconstitution of function and is consistentwith the formation of a MOP–DOP heterodimer (54,55).

Other G proteins are not targets for pertussis toxin-catalyzed ADP-ribosylation, but are widely expressed. Therefore, other strategies are re-quired to ensure that enhanced binding of [35S]GTPγS truly reflectsactivation of the G protein element of the fusion, rather than endogenous Gprotein. The α1-adrenoceptor agonist phenylephrine elevates binding of[35S]GTPγS following expression of a fusion protein between the α1b-adrenoceptor and Gα11 in HEK293 cells and membrane preparation (53).However, nucleotide binding in the absence of agonist is high, thus limitingthe agonist-induced signal to background. The vast majority of the agonist-independent binding is not to either the G protein within the fusion constructor to endogenously expressed Gα11 and/or Gαq. When samples were immu-noprecipitated with an anti-Gα11/Gαq antiserum, virtually all the agonist-independent binding was lost, but because the agonist-mediated signal wasmaintained, the signal to background ratio was increased greatly (53). Thisdid not establish whether all (or any) of the [35S]GTPγS was bound to the Gprotein element of the fusion protein, because both the fusion construct andthe endogenously expressed G proteins were pulled down. However, fol-lowing expression of an α1b-adrenoceptor–Gly208AlaGα11 fusion protein,virtually no agonist-stimulated binding of [35S]GTPγS was observed in suchimmunoprecipitates (53). These results imply that at the levels of expres-sion achieved, the receptor in this fusion cannot access and activate the endo-genous pools of Gα11/Gαq to any significant degree. Therefore, theagonist-stimulation of [35S]GTPγS binding produced in membranes express-ing the wild-type α1b-adrenoceptor–Gα11 fusion is a direct measure of acti-vation of the fusion protein. A fusion between Leu151Aspα1b-adrenoceptorand wild-type Gα11 also generated very little agonist-dependent [35S]GTPγSbinding (41), defining the poor ability of this mutated receptor to activate Gprotein. Similarly to the opioid receptor constructs discussed earlier, co-expression of Leu151Aspα1b-adrenoceptor–Gα11 and α1b-adrenoceptor–Gly208AlaGα11 resulted in reconstitution of agonist function consistent with


Fig. 2. Detection of α1b-adrenoceptor dimerization by functional complementa-tion of a pair of inactive α1b-adrenoceptor–Gα11 fusion proteins using single-cellCa2+ imaging studies. A fusion protein was constructed between the hamster α1b-adrenoceptor and Gα11. This construct was transfected into a mouse embryo fibro-blast cell line (EF88 cells) derived from a double Gαq/Gα11 knockout mouse alongwith green fluorescent protein (GFP). Imaging of these cells (top panel left) showsonly a single positive cell in the field, and this was the only cell that responded tophenylephrine (basal vs peak [Ca2+]). Because these cells do not express any Gprotein able to induce Ca2+ signaling, these data demonstrate the functionality of


dimerization of the α1b-adrenoceptor bringing the wild-type G protein andwild-type receptor into contact (41). Similar data have been obtained forfusion proteins between the histamine H1 receptor and Gα11.

As mentioned earlier, the binding affinity of both antagonist and agonistligands for the α1b-adrenoceptor–Gα11 fusion protein was unaffected byintroduction of the Leu151Asp mutation that blocked agonist activation. Thismay seem rather surprising, because the affinity of agonist ligands is oftenmodulated by the interaction status of GPCR and G protein, and such effectshave been observed for other GPCR–G protein fusions (56). However, gua-nine nucleotide-induced shifts in agonist affinity are well established to bevery small for α1-adrenoceptors. Significant alterations in agonist affinitymight be observed with the introduction of equivalent mutations in otherGPCR–G protein fusions.

3.3. Complementation Between GPCR–G Protein Fusionsin Intact Cells

When the α1b-adrenoceptor–Gα11 fusion protein was expressed in EF88cells, addition of phenylephrine resulted in elevation of [Ca2+]i (41,47). Thisreflects functionality of the fusion protein because EF88 cells are a line ofmouse embryo fibroblasts derived from an animal in which the genes for bothof the widely expressed Ca2+ mobilizing G proteins, Gα11 and Gαq had beeninactivated (57). Earlier studies demonstrated the requirement for co-expres-sion of both a suitable GPCR and an appropriate G protein in these cells togenerate function (38). Furthermore, only cells that are positively transfectedrespond to the agonist. The poor transfection efficiency of these cells whenusing cationic lipid-based methods means that analysis has generally requiredthe use of single-cell Ca2+ imaging (38,41). However, retrovirally basedinfection or the development of other effective means of transfection wouldallow the use of standard, cuvet-based measurements of Ca2+ levels and easiergeneration of concentration–response curves. As suggested under Subhead-ing 3.2., transfection of these cells with a fusion protein between the wild-type α1b-adrenoceptor and Gly208AlaGα11 failed to generate an elevation in

Fig. 2 (From opposite page) the fusion protein. The detailed time-course of [Ca2+]regulation in response to phenylephrine is shown in the bottom panel (top-mostline). The fusion was modified to introduce a Leu151Asp mutation into the receptorelement (bottom-most line) or a Gly208Ala mutation into the G protein (second linefrom bottom). When expressed in EF88 cells, neither of these mutants responded tophenylephrine; however, agonist-mediated signaling was reconstituted when theywere co-expressed (second line from top).


[Ca2+]i in response to phenylephrine (41). This was also the case forLeu151Aspα1b-adrenoceptor–Gα11. Similarly to the membrane-based assays,co-expression of Leu151Aspα1b-adrenoceptor–Gα11 and α1b-adrenoceptor–Gly208AlaGα1111 resulted in a restoration of phenylephrine-mediated eleva-tion of [Ca2+]i (Fig. 2). Because there are no appropriate endogenous Gproteins in these cells, such data require interactions between the complemen-tary fusions. Similar results were obtained when equivalent fusion proteinsthat incorporated the histamine H1 receptor rather than the α1b-adrenoceptorwere used (41). When Leu151Aspα1b-adrenoceptor–Gα11 and histamine H1–Gly208AlaGα11 were co-expressed, histamine, but not phenylephrine, wasable to elevate [Ca2+]i. The reverse was true when α1b-adrenoceptor–Gly208AlaGα11 was co-expressed with Leu133Asp histamine H1–Gα11 (41).

A weakness of single-cell imaging studies is the impossibility of moni-toring expression levels of each construct. However, when the wild-typehistamine H1 receptor–Gα11 fusion was co-expressed with the isolatedLeu151Aspα1b-adrenoceptor, the capacity of histamine to elevate [Ca2+]i wasreduced. Although these studies are more difficult to interpret than the par-allel experiments performed in membranes of HEK293 cells (in which theexpression levels of both the histamine H1 receptor–Gα11 fusion andLeu151Aspα1b-adrenoceptor could be measured directly by [3H]ligand-bind-ing studies; see Subheading 3.2.), their results are consistent with theLeu151Aspα1b-adrenoceptor forming a nonfunctional heterodimer with thehistamine H1 receptor–Gα11 fusion protein and thus reducing levels of func-tional histamine H1 receptor–Gα11/histamine H1 receptor–Gα11homodimer. This type of strategy should be generally applicable for GPCRsthat couple to the Ca2+ mobilizing G proteins Gq and G11, and may be appro-priate to measure the relative interaction affinities among GPCRs.

4. FUTURE PERSPECTIVES

Potentially, although the subject has yet to be directly explored, studiesakin to those described earlier could be expanded. Many GPCRs can inter-act with the so-called “promiscuous” G proteins G15 and G16 (58). Fusionproteins that incorporate either G15 or G16 with several GPCRs that do notroutinely elevate [Ca2+]i have been generated and have been used to allowligand screening and detailed pharmacological characterization using plat-forms such as the fluorescence imaging plate reader system, which is widelyemployed by the pharmaceutical industry (59–61). Incorporation of aGly211Ala form of Gα16 into a fusion should render it unresponsive to ago-nists and allow equivalent single-cell reconstitution assays, as detailed underSubheading 3.3. Equally, chimeric G proteins containing the backbone of a


Ca2+-mobilizing G protein (with the extreme C-terminal tail modified toprovide interaction with GPCRs that couple selectively to Gi-family or Gs Gproteins) have been widely employed (62–64) to channel signal to a com-mon and easy-to-measure assay endpoint. GPCR–G protein fusions con-taining chimeric G proteins have also been generated and have been shownto be functional (65,66). Selectivity of GPCR interactions can be quantita-tively measured from the extent of reconstitution of [35S]GTPγS binding,with expression of known amounts of two GPCR fusions monitored by satu-ration ligand-binding studies. Equally, the domains and amino acids thatprovide the interfaces of GPCR dimerization should be amenable to analy-sis via mutagenesis studies followed by reconstitution.

ACKNOWLEDGMENTS

Work in the authors’ laboratory in this area is supported by the MedicalResearch Council, the Biotechnology and Biological Sciences ResearchCouncil, and the Wellcome Trust.

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[Contemporary Clinical Neuroscience] The G Protein-Coupled Receptors Handbook Volume 215 || Functional Complementation and the Analysis of GPCR Dimerization