Table Of ContentMethods in
Molecular Biology 2268
Sofi a Aires M. Martins
Duarte Miguel F. Prazeres Editors
G Protein-Coupled
Receptor Screening
Assays
Methods and Protocols
Second Edition
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire, UK
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G Protein-Coupled Receptor
Screening Assays
Methods and Protocols
Second Edition
Edited by
fi
So a Aires M. Martins
INESC Microsistemas e Nanotecnologias, Lisbon, Portugal
Duarte Miguel F. Prazeres
IBB—Institute for Bioengineering and Biosciences, Lisbon, Portugal; Department of Bioengineering,
Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
Editors
SofiaAiresM.Martins DuarteMiguelF.Prazeres
INESCMicrosistemas IBB—InstituteforBioengineeringandBiosciences
eNanotecnologias Lisbon,Portugal
Lisbon,Portugal
DepartmentofBioengineering
InstitutoSuperiorTe´cnico
UniversidadedeLisboa
Lisbon,Portugal
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-1220-0 ISBN978-1-0716-1221-7 (eBook)
https://doi.org/10.1007/978-1-0716-1221-7
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CoverIllustrationCaption: RAW264.7cellsundergoingdirectionalmigrationasaresultoflocalizedopticalactivation
oflampreyparapinopsin,aGiproteincoupledreceptor.CellsareexpressingVenus-Parapinopsin(green)andPIP3sensor
PH-Akt-mCh(red).Rectanglesrepresentregionsilluminatedwithbluelight.Photoactivationofparapinopsinwithblue
light triggers activation of native Gi signaling pathways in the cell, as evidenced by increased PIP3 and extending
lamellipodiainthephotoactivatedregion.Authors:N.Gautman,X.Meshik.
ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer
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Preface
Theworldistacklinganunprecedentedcrisis,perhapsthegreatestchallengehumanityhas
faced since the Second World War. A novel coronavirus, unknown to our immune system
emerged in Asia by the end of 2019 and rapidly spread throughout the entire planet. The
COVID-19 pandemic caught the world unprepared and forced societies into a global
lockdown that disrupted the normal practice of economy and governance, as well as our
wayoflife.
As never before, the quest for new therapeutics received a central role in the world’s
agenda.InanattempttocopewiththedevastatingconsequencesofCOVID-19—currently
well beyond a health crisis, but rather expanding toward an economical emergency—
scientific communities and industries are joining efforts worldwide to develop vaccines or
pharmaceuticalcompoundscapableofimprovingthedisease’smanagementandoutcomes.
The second edition of the book G Protein-Coupled Receptor Screening Assays: Methods
and Protocols appears within this context. GPCRs are the largest family of membrane
receptors, the specialized proteins that convert extracellular stimulus into cellular informa-
tion.AmultitudeofligandsareknowntobindGPCRs,includingchemokines,neurotrans-
mitters, metals, peptides, amino acids, steroids, or photons. Ligand binding to a GPCR
causesachangeinthereceptorconformationandactivatesaG-protein.Theactiveformof
theG-proteindissociatesthenintoitsαandβ/γsubunits,activatingspecificeffectorswith
the release of second messengers and producing a signaling cascade that culminates in a
cellular response.
Although GPCRs are not directly involved in viral infection, their role in the so-called
comorbidities or exacerbated immune responses, which are likelyrelated with poorclinical
outcomesinCOVID-19patients,isunquestionable.Infact,GPCRsregulateaplethoraof
physiologicalstates,inwhichmodulationoftheimmuneresponse,metabolism,andhomeo-
stasisrepresentonlylimitedexamples.Theirimportancecanbeemphasizedbythefactthat
more than one third of the current drugs in the market are GPCR-acting compounds.
However, no ligand has yet been identified for over 100 of the approximately 800 known
humanGPCRs.Hence,screeningassaysarecriticaltoolstodiscovernewGPCRtargetsand
explorenewopportunitiesinthepharmacologyarena.
Key Outcomes of This Edition
Thissecondeditionfeaturesover20chaptersdedicatedtothestudyofGPCRsandrelated
screeningassaysthataimatabetterunderstandingofthereceptors’modeofactionoratthe
identification of potential drug candidates. In the book, different methodologies for the
expression and purification of a multitude of GPCRs are described, ranging from the
isolation of messenger RNA, to the development of recombinant constructs targeting
reporter detection systems based on fluorescence and luminescence read-outs. In
Chapter 1, Tiu et al. propose a method for the isolation of lipid rafts—localized domains
in the plasmatic membranes of defined lipidic composition—that serve as anchors for the
organization and interaction dynamics of biomolecules, including GPCRs. Embedded
receptors and respective signaling mechanisms can then be studied in their natural milieu
v
vi Preface
and cellularenvironment.Alternatively, GPCRscan bedirectly isolatedfromprimary cells.
However, functionality ismost often compromised due to losses in their 3-D structure. In
addition,GPCRproteinlevelsaresparseandvalidatedantibodiestoguidespecificisolation
are often not available. Molecular techniques that comprise the isolation and validation of
messenger RNA (mRNA) and cloning in heterologous expression systems constitute two
important methodologies to support subsequent functional studies. In Chapter 2, Sriram
et al. describe a method for the isolation of total mRNA and validation of GPCR-related
sequencesbyRNA-seqandGPCRTaqmanassays.InChapter3,Reevesproposesamethod
for the stable expression of bovine rhodopsin in the HEK293 cell line. The author takes
advantageofbothcytomegaloviruspromoterandcontrolelementsfromtheTn10tetracy-
cline operon present in an E. coli transposon, to generate an inducible gene expression
system. In this view, a stable and strong protein production can be achieved at higher cell
concentrations. In Chapter 4, Yeliseev describes the expression and purification of type II
human cannabinoid receptor CB , in both Escherichia coli and suspension cultures of
2
mammalian cells. To achieve an efficient purification, the CB molecular construct com-
2
prises an His -Tag and two identical Streptag sequences for the subsequent affinity chro-
10
matography purification. The purified receptors are suitable for further applications
includingfunctionalstudies.
Functionalassaysaredefinedasinvivoexperimentsdesignedtoevaluatetheactivityof
the partners involved in a particular cellular pathway. In here, the direct detection of the
ligand-bindingevent,theactivationofG-proteinsorothereffectorenzymes,thereleaseof
secondmessengers,aswellasreceptorinternalizationcanbemonitoredtounveiltheGPCR
activation mechanism and identify potential targets and respective modes of action.
Reportersystemsareinthespotlightofthisbook’sedition,withfluorescenceandbiolumi-
nescenceassays,resonanceenergytransfer(FRET/BRET),opsinsandenzymecomplemen-
tationassaysgainingincreasedmaturitytomonitor nearlyallindividualstepsoftheGPCR
signaling cascade. In Chapter 5, Yasi and Peralta-Yahya use Saccharomyces cerevisiae as the
recombinant host to express the human serotonin receptor 4 (5-HTR ). Whereas the
4
correct expression of 5-HTR in yeasts could be confirmed by recombinant constructs
4
withgreenfluorescentprotein(5-HTR -GFP),aluminescentassaybasedontheNanoLuc
4
luciferase reporter system is used for the fast screening of serotonin receptor-acting com-
pounds. In Chapter 6, Vidic and Hou describe a method for the purification of olfactory
(OR) GPCRs from yeast cells. OR respond to odor molecules and are mainly expressed in
themembraneofolfactorysensoryneurons.Theirintrinsicallyhighsensitivityandselectivity
makesthemidealcandidatesfor thedevelopmentofbiosensorsforthedetectionofvolatile
compounds(e.g.,electronicnoses).
Cell-free(CF)expressionsystemsofferanewtechnologicalplatformforproteinexpres-
sion by mobilizing the cellular translation machinery present in cell lysates for the in vitro
productionofthetargetproteins.InChapter7,Bernhardetal.appliedthetechnologyfor
the purification of the human endothelin B receptor (ETB) and the turkey β1-adrenergic
receptor (β1AR). For further functional studies, however, cell-free systems are particularly
challengingfor membraneproteinsbecausedetergentsarenecessarytodisruptthecellular
membrane and extract the translation machinery. The authors overtook this limitation by
including nanodiscs (ND) in the cell-free reaction mixtures. The GPCR/ND particles are
thenpurifiedbyconventionalaffinitychromatographyandreadyforsubsequentfunctional
assays.
InChapter8,Veiksinaetal.describeamethodbasedonfluorescenceanisotropy(FA)to
characterizetheligand-bindingevent—thefirststepoftheGPCRsignalingcascade—based
Preface vii
onemittinglightpolarizationdifferences.Theauthorsusedbuddedbaculovirusesasagene
expressiontooltoexpresshighlevelsofproteinsininsectcells,andinthisway,overcomethe
limited expression of receptors in native cells. Schihada et al. describe a BRET platform in
Chapter9thatiscompatiblewithHTStomonitorligandbindingtotheGPCRsofinterest.
Thelatteraremodifiedatthefull-lengthoratthetruncatedC-terminuswithNanolucifer-
® ™
ase—NLuc—(donor) and the HaloTag NanoBret Ligand in the third intracellular loop
(acceptor). Upon ligand binding, the receptor conformation changes and concomitantly
modifies the relative distance of the energy partners, which in turn produces a change
inBRET.
LaschetandHansonaddresstheinteractionofGPCRswiththeircognateG-proteinsby
developingafragmentcomplementationassaybasedalsoonnanoluciferaseenzyme.Taking
advantage of the enzyme subunits SmBiI and LgBiT, the authors further modified the
natural peptide regions of the enzyme to increase the affinity between the enzyme units,
which is important to measure the fast and transient interactions of GPCR/G-protein (see
Chapter10).
Bordesetal.(Chapter11)describeaprotocoltoconstructeithertransientorstablecell
linesthatenableFRET-basedbioassays.TheauthorsaddresstheG activationpathway
12/13
byusingconstructswiththesensitiveFRETpairs,pavingthewayforanexpeditescreening
of Rho GTPases activating G proteins. The latter are most often monitored by following
phenotypic responses (e.g., cytoskeleton rearrangements) that occur later, after stimulus
activation. Protocols for cell transduction of EPAC sensors and monitoring of Gi/o-
mediated cAMP response are also described. Lavogina et al. further detail the use of
EPAC sensors to monitor Gs-dependent cAMP cellular release. In their Chapter 12, the
authorsuserecombinantconstructsofEPACproteins,combinedwithsensitiveFRETpairs.
The constructs are amplified in BacMam expression system (a baculovirus vector for gene
deliverytomammaliancells)andusedforthetransductionofMadin-DarbyCanineKidney
cells expressing FSH receptors. Upon ligand binding, the cAMP increase is monitored by
measuring the fluorescence Sensitized Emission (SE) and calculating the acceptor/donor
emissionratio.
The fluorescence detection of intracellular calcium following activation of Gαq/11-
coupledreceptorsconstitutesahallmarkinHTSassaysandarelatedprotocolisdescribedin
Chapter 13. Typically, recombinant cell lines are cultured on microtiter plates (96–1536
wells) and stained with calcium sensitive dyes that exhibit enhanced fluorescence upon
calcium binding. Cell permeable Ca2+ dye formulations are available from several compa-
nies, including no-wash formulations, making these assays compatible with fluorescence
imagingplatereaders(FLIPR).Woszczeketal.describeamethodologyforCa2+thatallows
for the simultaneous concentration-dependent analysis of several receptor agonists and
antagonists.
Protein redistribution assaysfocus onthe latersteps of theGPCRsignaling cascade by
following receptor internalization and recruitment of β-arrestins or by monitoring protein
translocations acrosstheplasma membrane.In Chapter14, MeshikandGautman describe
the use of optically activatable GPCRs—opsins—to detect the translocation of Gβγ
sub-units. The methodology comprises the transient transfection of eukaryotic cells with
opsins, which are then optically activated at specific wavelengths. The resulting cellular
response is then monitored through live cell imaging. Using this approach, it is possible
to detect with high spatial and temporal resolution the activation of different G proteins’
sub-types as well as several effectors, 2nd messengers’ release and cell migration. Alonso-
Gardo´n and Este´vez describe a method to transfect HeLa cells with alternative constructs
viii Preface
targeting the detection of sphingosine 1-phosphate receptor activity by the Split-TEV
method.ThelatterisbasedontobaccoEtchVirus(TEV)proteasefragmentcomplementa-
tionassay.TheauthorsusedtheGVtranscriptionfactor(fusedtotheN-TEVfragment)to
activate a luminescent-based reporter gene system based on Gaussia luciferase (see
Chapter 15). Ma et al. propose a method, compatible with microplate readers, to evaluate
the interaction between Histamine-1 receptor (H R) and β-arrestin via BRET by taking
1
advantage of nanoluciferase (Nluc) activity. Histamine-induced β-arrestin2 recruitment to
theH RbringsthetwoNlucfragmentsinproximity,allowingfunctionalreconstitutionof
1
Nluc activity (see Chapter 16). Dijon et al. explore the same principle of NanoBiT for the
analysis of agonists and antagonist’s pharmacology in Chapter 17. The authors carefully
describe important considerations for the development of the molecular constructs that
enable both endpoint and kinetic analysis. By collecting timecourse data from the ligand’s
concentrationresponsecurvesandbyapplyinganoperationalmodelfor theanalysisofthe
kinetic parameters, the method can infer on potential ligand bias. The latter refers to a
processinwhichdifferentligandsstabilizedifferentreceptorconformations(incontrast,for
example,withtheendogenousligands),therebyactivatingdifferentsignalingpathways.
GPCRs microtiter-based assays or cell imaging techniques are well implemented and
suitable to high-throughput screening assays (HTS). However, cells in their physiological
environment are likely to be exposed to ligand gradients and temporal profiles rather than
being in contact with discrete concentrations of particular compounds. In this view, the
emergence of microfluidic technology has prompted the development of cell-based assays
where fluid flow and shear stress can be fine-tuned to mimic physiological conditions.
Gradient microfluidics are described by Suzuki et al. in Chapter 18. Using the peptide
matingpheromoneα-factorinyeastsasamodelGPCR,theauthorsdescribethefabrication
and operation of a PDMS microfluidic device with a gradient flow generator or pulsating
flow to monitor chemotropic guided cell elongation upon ligand addition. In Chapter 19,
Martinsetal.describeaCa2+mobilizationassaythatisperformedinamicrofluidicsystem.
In here, muscarinic M1 agonist (carbachol) and antagonist (pirenzepine) efficacy can be
determinedwithsimilaraccuracyasinstandardmicroscopycellassays.
Finally, the recent disclosure of several GPCR structures has paved the way for the
rational design of GPCR acting compounds, and in vitro drug design isgaining ground in
drugdiscoveryprograms.Thelastchapterofthiseditionisdedicatedtoinsilicotoolswitha
focus on homology modeling. This methodology uses available 3D structures to build a
model of a target sequence (e.g., binding molecule) based on a given sequence alignment
betweenthetemplateandtarget.Thus,inChapter20,Misztaetal.introducetheGPCRM
web service and detail stepwise instructions on how to use the software to obtain high-
qualityGPCRmodels.
Editors’ Final Remarks
This edition of G Protein-Coupled Receptor Screening Assays was planned considering not
onlytheassaysdirectlyinvolvedinthediscoveryofGPCR-activecompoundsbutalsothose
involved in cell-based experiments designed to study physiological responses. Whether
coming from academia or industry, or being an experienced researcher or a newcomer to
the field, the reader will finda comprehensive list ofmethods and protocolsthat cover the
latest developments on receptor purification, molecular biology, recombinant engineering,
Preface ix
and analytical techniques that enable the real-time monitoring of the complex GPCR
signalingcascadeandidentificationofpotentialdrugtargets.
Wesincerelyhopethatthecontentprovidedherecansomehowcontributetoadvancing
GPCRresearchanddiscoveryandultimatelyleadtotheavailabilityofinnovativeandmore
efficientdrugs.
Acknowledgments
Theguesteditorsaregratefultotheauthorsthatcontributedwiththeir worktothisbook
editionandundersuchunsettledtimes.
Lisbon,Portugal SofiaAiresM.Martins
DuarteMiguelF.Prazeres
