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g kleinau and others GPCR oligomerization 57:1 R59–R80 Review

Oligomerization of GPCRs involved in endocrine regulation

Correspondence Gunnar Kleinau, Anne Müller and Heike Biebermann should be addressed to H Biebermann Institute of Experimental Pediatric Endocrinology (IEPE), Charité-Universitätsmedizin, Berlin, Germany Email [email protected]

Abstract

More than 800 different human membrane-spanning G-protein-coupled receptors Key Words (GPCRs) serve as signal transducers at biological barriers. These receptors are activated by ff G-protein-coupled a wide variety of ligands such as peptides, ions and hormones, and are able to activate receptors a diverse set of intracellular signaling pathways. GPCRs are of central importance in ff homomers endocrine regulation, which underpins the significance of comprehensively studying ff heteromers these receptors and interrelated systems. During the last decade, the capacity for ff homooligomerization multimerization of GPCRs was found to be a common and functionally relevant property. ff heterooligomerization The interaction between GPCR monomers results in higher order complexes such as ff dimers homomers (identical receptor subtype) or heteromers (different receptor subtypes), ff oligomers which may be present in a specific and dynamic monomer/oligomer equilibrium. It is ff endocrine regulation widely accepted that the oligomerization of GPCRs is a mechanism for determining ff endocrine diseases the fine-tuning and expansion of cellular processes by modification of ligand action, ff signaling expression levels, and related signaling outcome. Accordingly, oligomerization provides exciting opportunities to optimize pharmacological treatment with respect to receptor

Journal of Molecular Endocrinology target and tissue selectivity or for the development of diagnostic tools. On the other hand, GPCR heteromerization may be a potential reason for the undesired side effects of pharmacological interventions, faced with numerous and common mutual signaling modifications in heteromeric constellations. Finally, detailed deciphering of the physiological occurrence and relevance of specific GPCR/GPCR–ligand interactions poses a future challenge. This review will tackle the aspects of GPCR oligomerization with specific

emphasis on family A GPCRs involved in endocrine regulation, whereby only a subset of Journal of Molecular these receptors will be discussed in detail. Endocrinology (2016) 57, R59–R80

G-protein-coupled receptors

The importance of GPCRs receptors (GPCRs) (Kristiansen 2004), which are involved in the modulation of almost all physiological processes Physiological functions such as development, growth, (Limbird 2004). The relevance of GPCRs is due to their behavior, learning, emotions, senses and aging are strongly fundamental role as information transducers (Raymond dependent on the control of endocrine circuits. Hormonal 1995, Wess 1998) by serving as hubs for signals transiting signals exert their action via binding to nuclear receptors biological barriers (Rosenbaum et al. 2009). or membrane-bound receptors. The largest superfamily Activation of GPCRs by different ligands, crosstalk of membrane-bound receptors are G-protein-coupled of receptors within the membrane, and their interaction

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10.1530/JME-16-0049 Journal of Molecular Endocrinology & Smith2009, supported byaminoacidsacting as‘micro-switches’(Ahuja 2009, the TMHs relative to one another (Scheerer et al. 2008, activation ischaracterizedby aspatialrearrangementof ( is suggested to occur during ligand-induced activation toggle switching’ mechanism experimental data, a ‘global et al. 2009, ( rich repeat-containingG-protein-coupledreceptors(LGRs) of glycoproteinhormonereceptors(GPHRs)andleucine- ( domain closetothesecondextracellularloop2(ECL2) A GPCRsarebelievedtobindwithinthetransmembrane 2012, ( to improvepharmacologicalapproachesdirectedGPCRs ( structures have been published in the last decade crystal experimental methods (Tate & Schertler 2009), manyGPCR Stevens 2009, structures (reviewedinKobilka&Schertler2008, architecture is confirmed by the inspection of solved GPCR loops (ICLs)andthreeextracellular(ECLs).This helices (TMHs)thatareconnectedbythreeintracellular common structuralarchitecture ofseventransmembrane al.2003, et the familywithmostdifferentmembers(Fredriksson known asfamily1orrhodopsin-likeGPCRs,constitute is subdividedintoseveralfamilies,wherebyfamilyA,also aminoacidmotifs,thesuperfamilyofGPCRs conserved signaling pathways(Kristiansen2004).Basedondifferent glycoprotein hormones,andtheyactivatevarious small peptides,nucleotides,ions,amines,orevenlarge GPCRs areactivatedbyawidevarietyofligandsincluding How doGPCRsfunction? Schlyer &Horuk2006,Masonetal.2012). (Hopkins &Groom2002, approximately 50% of approved drugs modulate GPCRs Due totheirkeyroleintuningphysiologicalprocesses, et al.2009)orionchannelregulation(Veldhuis et al. 2015). mechanisms suchasinductionofgeneexpression(Ho responses (Schwartzetal.2006)andarebasedonvarious overall resultofGPCRactivationarespecificphysiological subsequently induced intracellular signaling pathway. The with cellularorextracellularproteinsdeterminethe Schwartz Svendsen et al. 2008, Surgand etal.2006, Carlsson etal.2011, Zhao &Wu 2012, DOI: 10.1530/JME-16-0049 http://jme.endocrinology-journals.org Review Review Most of the endogenous and synthetic ligands of family Most oftheendogenousandsyntheticligandsfamily Shoichet &Kobilka2012). ). This structural rearrangement is Schertler 2008).Thisstructuralrearrangement is t al.2006, et ). Based on a large amount of Kleinau etal.2013).Basedonalargeamountof ). They share a Fredriksson &Schioth2005).Theysharea Lodowski et al . 2009).Asaresultofadvanced Hofmann etal.2009, Piscitelli et al . 2015)andareusefultools Wichard etal.2011),withtheexception Kontoyianni &Liu2012, Smit etal.2007).Correspondingly, Kleinau & Krause 2009, Tyndall &Sandilya2005, g

Oldham &Hamm2008). bind differentligands(reviewedinGalandrinetal.2007, arrestin), GPCRssharecommon‘read-outs’,evenifthey several G-protein-independentsignalingpathways(e.g.via Due tothecapabilityfor 2002, different signalingactivitystates(Seifert&Wenzel-Seifert Different GPCRconformationsareassumedtoberelated in thesamecelltype(Waldhoer 2012a). Incaseofheterodimerization,GPCRexpression dependent onafunctionalsignificance(Tadagaki could behypothesizedthatarelevantoligomerizationis or (3)aparticularspatialdistancetooneanother. It interactions); (2) direct mutual functional modulation; (Gomes the followingparameters(forfurtherdetails,seealso GPCR–GPCR interrelationsshouldbecharacterizedby ). Such order complexesbetweenGPCRprotomers(Fig. 1 oligomerization isusedfordimeric,tetramericorhigher GPCR-x/GPCR-y (heteromer) constellation. The term term usedtodescribeaGPCR-x/GPCR-x(homomer)or 2001, 2012a), alsoinnativetissue(Albizu has beenreportedfornumerousGPCRs(Tadagaki feature under structural and functional prospective. It Organisation ofGPCRsasoligomersisaninteresting and isthere arelevance foroligomerization? How cananoligomericGPCRconstellationbedefined, mechanism insignaling GPCR oligomerization asaregulatory 2007) andsimultaneousoccurrence( As a hypothetical example, one ligand activates a specific As ahypotheticalexample, one ligandactivatesaspecific biological processes (such as metabolism or reproduction). to balance or coordinate related cellular responsesand ligands toactinasynchronized mannerandthereby particularly provides an opportunity for their respective details). Moreover, theGPCRcapacitytoformheteromers as signalamplificationormodification(seebelow for enlargement andfine-tuningofsignalingoptionssuch the molecularlevelisrelatedtodetermination, significance of both homo- and heteromerization on oligomerization haveinphysiology?To date, thefunctional Ng et al.2012,&Chow2015). B ortastereceptors(Li several GPCRsubfamiliessuchasforthefamilyA, are prerequisites.Oligomerizationhasbeenreportedfor GPCR oligomerization Published byBioscientifica Ltd. Which general functional role may GPCR Which generalfunctionalrolemayGPCR Rozenfeld &Devi2011).Dimerizationisageneral Kobilka &Deupi2007, . 2016)): (1) physicalinteractions (side-chain et al . 2002, et al Downloaded fromBioscientifica.com at10/02/202107:36:32AM activation of G-proteins and activation ofG-proteinsand ). Deupi &Standfuss2011). Harikumar t al.2005, et Gonzalez . 2010a, et al 57 57 : : 1 1 . 2008, et al et al. 2012) Pin Bouvier R60 R60 t al. et t al. et t al. et via freeaccess Journal of Molecular Endocrinology as trace amines(Kleinauetal.2011, to interact with a multitude of diverse GPCR targets such ligandproperty in considerationofthefrequentlyobserved (explained infurtherdetailbelow)becomesmorecomplex with relatedphysiologicalmechanisms.Thisgeneralpicture specific ligandtodirectsignalingindifferentassociations diverse celltypesthatalsobroadensthespectrumofa with otherGPCRsasoligomers,alsoindependencyon counter-regulation. Notably, severalGPCRscaninteract mutual effects,whichisaneffectiveandrapidsystemof GPCR is simultaneously inhibited or downregulated by receptor, whereastheactivityofinteractingheteromeric oligomers, complexeswithdifferent protomerratiosmaybeformedbyGPCR_xandGPCR_y. non-GPCR. (E)GPCR_xmayalsoformhomooligomerswithdifferent interfacesbetweentheprotomers.(F)Assumingtetrameric(orhigherorder)GPCR of thesameGPCRinteractingasahomodimer. (C)DimerizationbetweenGPCR_xwithadifferent GPCR_y. (D)GPCR_xmaypotentiallyinteractwitha Putative constellationsofoligomers.(A)AparticularGPCR(GPCR_x)asamonomer(schematiccylinderindicatestheentirereceptor).(B)Two monomers Figure 1 heterodimers between the D4R and the Furtherexamples includecircadian-controlled 2015). al. dopaminergic neurotransmission (Harmeieret the dopamine-2receptor(D2R) and,inturn,modifies associated receptor1(TAAR1) interacts,forexample, with regulation (Tripathi etal.2015 ). Moreover, thetraceamine- with thechemokinereceptor4controlsbloodpressure receptor (α by previousandrecentfindings,e.g.fortheα in heterooligomericconstellations,isstronglysupported regulation ofphysiologicalprocesses,eitherashomo- or http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0049 Review The involvement of GPCR oligomerization in the The involvementofGPCRoligomerizationinthe 1AR), wherebyheterodimerizationofα g

kleinau © 2016SocietyforEndocrinology Dinter etal.2015a β 1- or andothers Printed inGreatBritain α 1B-adrenergic 1B-adrenergic 1-adrenergic 1-adrenergic ). 1AR 1AR revealed by biophysical or biochemical methods have interface propertycanbe suggested. Severalinterfaces and heterodimers can be assumed, whereby no universal putative interfacesandtypes ofinteractionsforhomo- for differentGPCRs.Inconclusion, may vary interrelations’ or cannot be determined as a standard, and protomers arenotdefinedasobligatefor‘protomer roles forsuchinteractionsorspatialdistancesbetween amino acids or between multitudes of side chains. Specific intermolecular interactionsareconstitutedbetweensingle extracellular N-terminalregion(Uddin 1996, Mancia 2004, reported underinvolvementofTMH4(Carrillo Several GPCR-GPCRprotomerinterfaceshavebeen protomers constitutingoligomeric interfaces? What doweknowaboutinteractionsbetweenGPCR rhythm regulation (Benleulmi-Chaachouaetal.2016). the interactomeofmelatoninreceptorsinvolvedincircadian serotonin andmelatoninsyntheses(Gonzalez receptor, respectively, thatareinvolvedintheregulation of GPCR oligomerization Published byBioscientifica Ltd. Hernanz-Falcon McMillin t al.2008),TMH1,andTMH5-6( Hebert et . 2011, et al . 2004, et al Downloaded fromBioscientifica.com at10/02/202107:36:32AM Yanagawa Guo 57 . 2011)orthe et al . 2005, et al : t al.2012).The et 1 et al . 2012)and R61 2008, t al. et t al. et via freeaccess Journal of Molecular Endocrinology Teichmann et al . 2014),reviewedinLambert2010). ((Hern were foundtointerconvert dynamicallyinanequilibrium mixture ofmonomers and homomers, and the twoforms GPCRs weredescribedtobepotentiallyexpressedas a are mostlikelytobeofdynamiccharacter(Hu ). Furthermore,theGPCR-GPCRinterfaces dimers (Fig. 2 structures ofGPCR been confirmedbydeterminedcrystal previously determined,suchasthosefromthe(A)chemokinereceptorCXCR4(Wu Yanagawa et al.2011,Hu2013),andTMH4–TMH5(Gorinskietal2012).Furthermore,severalcrystalstructuresofdimericGPCRcomplexeswere conditions fordifferent GPCRs attheregionfromICL2–TMH4(Bakkeretal.2004,Guoet al2005Mancia2008),TMH5–TMH6George1998 structural levelbyspecificside-chaininteractionsorclosespatialdistances.Interfacesbetweentheprotomershavebeenfoundunderexperimental Observed GPCRhomodimerinterfacesandputativeheterodimerconstellations.Directprotomerareassumedtobecharacterizedatthe Figure 2 Schrödinger, LLC)wasusedforstructuralrepresentation. heteromers, whichfinallyincludesheteromersofhomomers(Ferre2015 ) (Diiiandv).ThesoftwarePyMOL(MolecularGraphicsSystem,Version 1.3 spatial protomerarrangements(acombinationofdimers)canbespeculated (Diandii).Theidenticalvarietyofcombinationsmayalsobeassumedfor by theseinterfaces,butbetweendiverseprotomers(C)(protomersarecolored differently forvisual separation).Forhomomers,variousscenariosof line withbiophysicaldata,dimerinterfacescanbealsoobservedbetween TMH5-6(asinA).Asaresult,heteromericstatesshouldalsobecharacterized 2012)). ForOPRK1,asalsofordeterminedβ-1ARandopsinstructures,theprotomerinterfaceislocatedatTMH1–TMH2 andhelix8(B),respectively. In DOI: 10.1530/JME-16-0049 http://jme.endocrinology-journals.org Review Review . 2010, et al Kasai . 2011, et al g

kleinau © 2016SocietyforEndocrinology Calebiro andothers Printed inGreatBritain et al. 2013 ). . 2013, et al Oligomerization canhave amajor . 2007, et al spectrum offine-tuning options insignaling(White ( for ageneralsignalingcapacity atfamilyAGPCRs Homo- andheterooligomerizationisnotaprerequisite influenced byoligomerization? Which functionsormolecularproperties ofGPCRsare GPCR oligomerization Whorton Published byBioscientifica Ltd. etal.2010)or(B)theκ-opioidreceptor(OPRK1KOR(Wu t al. 2007), but it defines and widens the et Ciruela . 2010, et al Downloaded fromBioscientifica.com at10/02/202107:36:32AM ). Smith &Milligan2010). 57 57 influence onthe : : 1 1 etal. R62 R62 via freeaccess Journal of Molecular Endocrinology suggested, asindicatedinthe followingexamples: on oligomerization have been identified orhave been scenarios ofreceptorproperty modificationsdependent 3).Severalfunctional heteromeric constellation (Fig. type canfunctionasaprotomer, homomer, orina ( assembly oftheseGPCRsinheterooligomericcomplexes activation mayoccur(Maurice al. 2005). Functional cross-regulation of G-protein et George transduction mechanisms (reviewedinBouvier2001, Lohse 2010),G-proteincouplingselectivity, andsignal et al. 2005) suchas in ligand signaling propertiesofinteractingprotomers(Prinster which mayultimatelyleadtoalterationsinthesignalingpropertiesofcomplex(responseG). (indicated inheterodimercelltype2).Moreover, bivalentligands(illustratedincelltype2)combineparticularfortherespectiveprotomers, other signalingevents(responseCorF).Forhomo-andheterooligomers,ligandbindingpropertiesareknowntodrasticallydiffer tothoseofmonomers interactions shouldleadtotheactivationofsignalingpathwaysthatarepartiallyidenticaleachmonomer, butmayalsoresultintheactivationof Furthermore, theremaybeheterooligomerizationofthereceptors(asdescribedforMC4R),indicatedbyreceptorpairs(mixedcolor).These responses independencyofoligomerizationormonomerizationhavebeenreportedapreviousstudyontheMC4R(Piechowskiet al.2013). oligomerization mayleadtoenhancedsignalingasindicatedforthegreenreceptorincelltype1(boldarrow).Examplesofsuchdifferences insignaling homooligomerization ofthereceptorsmayleadtoaloweredsignaling(slimarrow)comparedwithreceptormonomersignaling.Receptor orphan receptorswithunknownligand(s)mayalsooccurandinfluencethesystemofinteractingproteins(notshown).Incelltype1, receptors incelltype1oroflightbeigecolor2.Theseactivatedifferent signalingpathways(responsesBandE).Ineachcelltype, other downstreamsignalingmolecules)differ. Moreover, ineachcelltype,amultitude ofdifferent GPCRsareexpressed,indicatedasadditionalgreen activation ofintracellulareffectors incelltype1asthesetandamountofparticularintracellularsignalingdeterminants(e.g.G-proteinsubtypesor capable ofactivatingtwodifferent downstreamsignalingeventsafterligandbinding.Thisreceptorhasdifferent propertiesconcerningbindingand intracellular signalingpathwaysindifferent celltypes,indicatedastype1(responseA)and2D).In1,thisreceptoris parameters instrength(arrowsthinorbold)andconstitution(responseboxesA–G)areindicated.Aparticularreceptor(e.g.purple)mayactivatediverse receptor organizationarepotentiallyprobablebuthavenotbeenimplementedhere(e.g.byheterotrimerization).Different intracellularsignaling potential scenariosthatarefeasibleconcerningreceptorsignalingandoligomerizationindifferent celltypes.Furtherdiversescenariosandlevelsof Schematic illustrationofdifferent signalingscenariosthatpotentiallyoccuratGPCRsandGPCRoligomersindifferent celltypes.Thisschemeillustrates Figure 3 Nijmeijer http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0049 Review Specifically, aparticularGPCRinspecific cell . 2002),orforcellsurfaceexpression(Uberti et al et al. 2010). binding (Levoye . 2011b),despitethe et al g

kleinau © 2016SocietyforEndocrinology andothers Printed inGreatBritain et al. 2006a, 3. 2. 1. GPCR oligomerization Published byBioscientifica Ltd. (Levoye etal.2006b),inagivencelltypemay: A heteromer, which may also be an orphan receptor (Durroux2005). monomer and/or bindingcapacities differ comparedwiththe A homomeriscapableofbindingvariousligands, (iii) (ii) Constitution ofhomo-and/orheteromers: (i) et al.2010,Pellissier2011). with theoligomer(Wilson etal.2007, Signaling of the monomer is weaker compared with theoligomer(Piechowskietal.2013). Signaling of the monomer is enhanced compared 2002, Kilpatricketal.2015). that ofhomomersormonomers(Hanyalogluetal. The internalizationofheteromersdiffersfrom Downloaded fromBioscientifica.com at10/02/202107:36:32AM 57 : 1 Magalhaes R63 via freeaccess Journal of Molecular Endocrinology specificity (alsoindicatedasbracketed plussymbol). in vivobackgroundisbyanalyzingallosteric crosstalk.Thelimitationofthismethodistheknowledgeadequateligands, theirdetailedeffects, andtissue methods maybepossible(indicated bybracketedplussymbol).Themostpromisingtechniqueusedtodetect GPCR–GPCRinteractionsinthephysiological availability ofseveralprimaryGPCR antibodiesandthepossibilityoftaggedGPCRexpressioninanimal models,insitudetectionbyantibody-based Dimer detectionusingantibody-based andantibody-independenttechniques.Antibodydetectionprimarily requirestaggingofGPCRs.Duetothe Table 1 Resonance energytransfer(RET) Proximity biotinylation Proximity ligationassays(PLA) Sandwich-ELISA Co-immunolocalization Co-immunoprecipitation (Co-IP) Antibody-based Method is characterizedbystrongdevelopmentandrapid and withregardtotheiraim;however, thisfield and disadvantageswhencomparedwithoneanother Each ofthesetechniquesexhibitsbothadvantages 1). applied todetectGPCR–GPCRinteractions(Table In thelasttwodecades,variousmethodshavebeen How isoligomerizationdetectedexperimentally? Allosteric crosstalk Fluorescence cross-correlationspectroscopy(FCCS) Photoactivated localizationmicroscopy(PALM) Protein complementationassays(PCA) TR-FRET (TR,time-resolved) SRET (S,sequential), BRET (B,bioluminescence), FRET (F, fluorescence),

DOI: 10.1530/JME-16-0049 http://jme.endocrinology-journals.org mostly antibody-independent e.g. basedonfluorescentproteins,enzymes,ubiquitin e.g. viaantibody-basedHTRF - - e.g. via e.g. viaradioligandbinding Review Review based onSNAP-andCLIP-tagtechnology fluorescent ligandsor (iii) (ii) (v) (iv) (i) (Hague etal.2006, activate pathways that differ from both protomers signaling) (AbdAlla et al. 2000, modulate signaling pathways (e.g. strength of et al. 2005); protomer(s) (neutralheterooligomeric)(Prinster activate the same signaling pathways as the 2011, Mohretal.2013Yuan etal.2013). ways suchasbivalentligands(Shonbergetal. bind artificialligandsthatmodifysignalingpath- binding sites)(Durroux2005); bind ligand(s)thatdiffer(e.g.alsoatallosteric 2013); Methods thathavebeenusedtodeterminedi-andoligomerizationofGPCRs. Rashid etal.2007, g

of antibodiesthatexistforaspecificGPCRor Skieterska and areeffectiveinfixedorlysedcells(reviewede.g. are mostlyusedwithdifferentiallyepitope-taggedGPCRs Homo- orheteromerdetectionbyantibody-basedassays limited toin vitro cellculture overexpression conditions. of specificGPCRantibodies,thesetechniquesareoften primarily recognizeepitopetags (Table 1). Due tothelack sandwich-ELISA ortheproximityligationassay, as co-immunoprecipitation, co-immunolocalization, or aninvivomodel.Antibody-basedtechniques,such living, fixedorlysedcells;(iv)invitrocellculture of (ii) dynamicandnondynamicsystems;(iii) analyses follows: (i)antibody-basedandantibody-independent; methodological principlesor experimental conditionsas applied techniquescanbesubdividedbasedontheir ofcommonlyusedapproaches.Currently brief overview particular methodsindetail,butwillmerelyprovidea 2013, al.2007, et of methodsforthedetectionoligomers(e.g.Persani advancement. Becauseofpreviousthoroughreviews GPCR oligomerization Published byBioscientifica Ltd. + + + + + + + + + Gomes t al.2013, et In vitro Kaczor &Selent2011, t al.2016),wewillnotdescribethe et In livingcells + + + + + Application Gomes Downloaded fromBioscientifica.com at10/02/202107:36:32AM t al.2014).Inthecase et In situ (+) (+) + + 57 57 Bonomi &Persani : : 1 1 In vivo (+) R64 R64 via freeaccess

Journal of Molecular Endocrinology regulation Oligomerization ofreceptors inendocrine analyze interactionswiththismethod. (agonist, antagonist, inverse agonist effects) is required to knowledge oftherelevantGPCR-ligandinterdependency of ligandstotheseprotomers.Asaprerequisite,sufficient interacting GPCRsandmayresultincooperativebinding dimerization process may lead to crosstalk between Klein 2012).Thismethodisbasedontheideathat e.g. protein complementationassay(PCA)(forreviewsee the applicationofresonanceenergytransfer(RET)and some antibody-independentmethods.Ofthesemethods, protomers andoligomersinlivingcellsortissuesamplesby refined techniques even allow for the detection of GPCR also possibleinanimalmodels. oligomer, expressionanddetectionoflabeledGPCRsare by hormone/receptorfeedback systems. metabolism or glucose homeostasis are tightly regulated many otherendocrine functions suchasenergy reproduction andadrenal functions.Furthermore, the regulationofgrowth,thyroidfunction,fertility- as it is known forthe hypothalamus and the pituitary which isregulatedinanegativefeedbackmanner to or furthermodulateend-organhormone secretion, an endocrinegland.Thesehormonesexerttheiraction hormones functionattheirendorgans,whichmaybe hormonerelease.Theanterior pituitary to pituitary cells,whichsubsequently leadspecialized pituitary hormones activatetheircognatereceptorson hormones secretedfromthehypothalamus.These circuitsEndocrine regulatory consistofreleasing analysis ofallostericcrosstalk(Durroux2005, and heteromerization in distance limitforinteractionscomparedwithRET. concentration levelorevenassinglemoleculeswithouta monitoring offluorescent-labeledGPCRsatananomolar methodspermitaquantifiedinteraction straightforward Herrick-Davis etal.2013, techniques (FCCS)havebeendeveloped(forreviewsee (PALM) andfluorescencefluctuationspectroscopy homo- orheteromerization. et al.2015)havebeenparticularlyusedtodetectGPCR Bonomi &Persani2013, http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0049 Review Elaborate enhancementofmethodsandconstantly The mostpromisingmethodstodetectGPCRhomo- Moreover, photo-activatedlocalizationmicroscopy Vidi &Watts 2009, Lohse etal.2012, Ciruela etal.2014, Hink &Postma2013).These in vivo systems are via the g

thyroxine (T receptor (TSHR),whichinducestheproduction of activates thethyroidglandviabindingtothyrotropin et al. 2002). properties intermsofβ-arrestinrecruitment(Hanyaloglu which hasbeenfoundtochangetheirfunctional These receptorsubtypesareabletoheterooligomerize, et al. 2007). (Song hormone-dependent receptorphosphorylation but notsignaling(Song&Hinkle2005)andpotentiated 2002). TRHRdimerizationwasfoundtoaffecttrafficking . 2007, et al GPCRs isknownforseveralofGPCRsubfamilies(Young based on in vitro studies. Althougholigomerization of heterooligomers, whereby these findings areprimarily hormones havebeensuggestedtocompriseofhomo-or Many receptorsforreleasinghormonesandtropic in endocrineregulation? What isknownaboutoligomerizationofGPCRsinvolved homooligomerization (Kroeger ligand stimulationissuggestedtofurtherincrease (Kroeger . 1999, et al experiments ( the oligomerconstitution is proposedbasedonBRET but anadditionalroleoftheextracellulardomainon occurs at the transmembrane bundle (Urizar to theTSHR,interactionbetweenreceptorprotomers . 2012, (Zoenen et al et al.2005)orconstitutiveactivationbymutation from TSHstimulation(Bonomi&Persani2013; 2001, been previouslyreportedtohomooligomerize(Latifetal . found invariousareasofthebrain(Cao pituitary, TRHR localization has also been extrapituitary al . ). 2003 In addition to TRHR expression in the et hormone (TRH)bindstotheTRHreceptor(TRHR)(Sun thyrotropin-releasing In specificcells,thepituitary GPCRs ofthepituitary-thyroid axis linked aspects. largest GPCRfamily, familyA,toprovideinsightsinto Chow 2015),wewillfocusonseveralexamplesfromthe a functional relevance of homomers. This is of general respect toligandbindingwas demonstratedandsupports experimental evidencefor negative cooperativitywith GPCR oligomerization Published byBioscientifica Ltd. The secretedhormonethyrotropin(TSH),inturn, There are two different subtypes of TRHR in rodents. Davies etal.2002),whichismostlikelyindependent et al. 2001)intheabsenceofligand;however, Bilek 2000).TheTRHRmayformhomomers Harikumar 4 Urizar ) and triiodothyronine (T et al. 2005).Inthesamestudy, further Biebermann etal.2012b).Inregards . 2008, et al Downloaded fromBioscientifica.com at10/02/202107:36:32AM t al.2001, et Ng 57 . 2012, et al 3 et al. 1998, : ). The TSHR has 1 . 2005), et al Zhu Urizar Heuer R65 Ng & t al. et via freeaccess

Journal of Molecular Endocrinology generation (Osuga receptor fragmentspartially restored ligand-inducedsignal and signaling-inhibited(with diminishedbindingcapacity) expression ofbinding-deficient (withfullsignalingcapacity) oligomerization wereachieved fromstudieswhereco- & Huhtaniemi2000).FirstindicationsforLHCGR play acrucialroleinreproductivephysiology(Themmen yet beenthoroughlyinvestigated(Satakeet al.2013). and secretion of the gonadotropins LH andFSH has not how GnRHRoligomerizationinfluencestheexpression shift fromGstoGisignaling(Sakai between thisorphanGnRHRandanotherparalogdueto a signaling isdecreasedintheheterodimericconstellation (Sakai intracellular Ca2+ elevation and ERK phosphorylation a furtherparticularGnRHRreceptorsubtypeintermsof an orphanreceptorandmodulatesthefunctionalityof Satake axis, fourdifferentsubtypesofGnRHRexist(reviewedin intestinalis, whichlacksahypothalamus-pituitary-gonad al.2004). et of wild-type via a dominant negative effect ( inhibit ligand binding and second messenger production GnRHR dimerizationderivesfrommutantreceptorsthat Cornea (oligomers) duetoGnRHstimulation(Connetal.1982a,b revealed thattheGnRHRisabletoformmicroaggregates its receptor(GnRHR)inthepituitary. Differentstudies binding of gonadotropin-releasing hormone (GnRH) to gonadotropins lutropin(LH)andfollitropin(FSH)is The first step in the production and release of the (HPG) axis The receptors ofthehypothalamic-pituitary-gonadal protomers. be bestexplainedbymutualinterrelationsbetweenTSHR pathogenic mutations(Calebiroetal.2005),whichcan provided bystudiesshowingdominantnegativeeffects support for a functional role of TSHR oligomerization is . 2011 ). Further toactivateGq(IP)(Allenet al necessary two TSHmoleculesboundataTSHRhomodimerare Moreover, that arecentstudyleadstotheobservation that spanoneorderofmagnitude(Biancoetal.2014). true for physiological TSH concentrations in serum hormone concentrations (Urizar with thehighestlevelofsensitivityoccurringatlower at awiderangeofligand(hormone)concentrations importance to further understand endocrine regulation DOI: 10.1530/JME-16-0049 http://jme.endocrinology-journals.org Review Review The receptors for FSH (FSHR) and LH/CG (LHCGR) The receptorsforFSH(FSHR)andLH/CG(LHCGR) t al.2008).Moreover, GnRHR-mediatedcAMP et . 2013).Oneofthereceptorsubtypes(R4)is et al et al. 2001, Interestingly, intheprotochordateCiona et al . 1997).Thiswasthefirstindication Kroeger et al. 2001). Evidence of human g

kleinau © 2016SocietyforEndocrinology t al.2005). This is et . 2012).However,et al andothers Printed inGreatBritain Brothers enhanced, whichgivesrise totheexpressionofa the expressionofpro- agouti-related peptide(AgRP), isrepressed.Moreover, orexigenic peptides,suchas neuropeptideY(NPY)and the hypothalamus(ARC).As aresult,theexpressionof their receptorsonneuronsofthearcuate nucleusin leptin orthepancreaticβ anorexigenic hormones,suchastheadipocyte-derived et al.2012a weight (Cone2005, reaction resultinginthemaintenanceofaconstantbody where informationisintegratedtoanorchestrated communicates peripheralsignalstothehypothalamus, regulation istheleptin–melanocortinpathwaythat maintenance. Themostimportantpathwayofweight and functionasoligomers. regulated and their receptors are reported to constitute cholecystokinin (gut).Actionofthesehormonesistightly adiponectin (adipocytes), ghrelin (stomach), and PYY also expressandsecretehormonessuchasleptin nonendocrinetissues feedback regulation,manyprimary the hypothalamus,pituitary, andend-organs,their regulation comprisingthehormone/receptorsystemsin during granulosacelldifferentiation. LHCGR/FSHR interactionisspeculatedtoplayarole granulosa cells,LHCGRandFSHRareco-expressed (Feng et . al 2013, observed LHCGR co-expressedinvitro cellsystemshavebeen variants (Jonaset al.2015). analysis hasrevealedcontactsofinteractingLHCGR Recently, asophisticatedmethodofsinglemolecule LHCGR oligomerization and receptor–protomer interplay. 2010), whichpointedtoaphysiologicalrelevanceof the homozygousstate,wasproven(Rivero-Mulleretal. a signalingdefectivethatbothsufferfrominfertilityin mice LHCGRmutants,aligandbindingdefectiveand Moreover, the functional rescue of two loss-of-function signaling inoligomericconstellations(Tao of inactiveLHCGRmutantsonwild-typereceptor al.2004, et association of the LHCGR was demonstrated (Tao both simultaneously(Ji one oftwopotentiallyinducedhormonesignals,butnot induce biasedsignalingintermsofthegenerationonly For theFSHR,transactivationwasfoundtomostlikely of afunctionalinterrelationbetweenLHCGRmonomers. GPCR oligomerization Published byBioscientifica Ltd. One prominent example is body weight Besides theclassicalformofendocrinefeedback Furthermore, heteromerizationofFSHand In 2004, constitutive and agonist-dependent self- Fanelli 2007)aswellthenegativeeffects , Farooqi 2014).Fromtheperiphery, Oswal &Yeo 2007, et al. 2004). Downloaded fromBioscientifica.com at10/02/202107:36:32AM opiomelanocortin (POMC) is Mazurkiewicz et al. ). 2015 In -cell-derived leptin,affect 57 57 : : 1 1 Biebermann t al.2004). et R66 R66 via freeaccess

Journal of Molecular Endocrinology The MC4Risalsocapableof interactingwithotherGPCRs kinetic studies of ligand binding (Kopanchuk et al. 2006). support forMC4Roligomerization hasbeenprovidedby MC4R-MC4R variant(Biebermann etal.2003).Further be bestexplainedbyhomomerization oftheMC4Rand the mutantonwildtypewasdetected,whichcan severe early-onsetobesity, adominant-negative effectof heterozygous MC4Rmutationinapatientsufferingfrom al.2015).Investigationofanaturallyoccurring et Biebermann most prominentGPCRinweightregulation(Cone2005 , the nucleusparaventricularis(PVN). and 4 receptors (MC3R and MC4R) in theARCand in (MSHs) thatarecapableofactivatingmelanocortin 3 include variety ofadditionalpeptides.Importantpeptides 2006, ( homodimers andformheteromers(examplesusedhereforthisscheme members ofthefamilyAGPCRshavebeenreportedtoconstitute A complexofGPCRhomo-/heterodimerinteractionnetworks.Many Figure 4 et al. 2008, dopamine-2 receptors;Cb1R,cannabinoidreceptor1. receptor 83;OPRM1,Muopioidreceptor;DRD1/DRD2,dopamine-1and 5-hydroxytryptamine (serotonin)receptor1B;GPR83,G-protein-coupled 5-hydroxytryptamine (serotonin)receptor2C;5HT1B, ghrelin receptor;MC3R,melanocortin-35HT2C, indicate potentiallymoreand/orthusfarunknownpartners.GHSR, constellations assuggestedinFig. 2Diii,ivandv).Dottedarrows monomers (Fig. 2C)orhomodimersFerre2015)(potentiallyin and heteromers,wherebyarrowsindicateinteractionsbetween scheme demonstratesafragmentedsectionofreportedGPCRhomo- Oligomerization KnowledgeBaseProject’(Khelashvilietal.2010).This GPCR oligomerliterature,seealsothe‘TheGProteinCoupledReceptor- cell type.Foracomprehensiveoverviewofavailableexperimentsand properties independencyontheGPCRexpressionpatternaparticular widens theputativemodificationofphysiologicallyrelevantsignaling et al. 2013).Consequently, thespectrumofpotentialinteractionpartners bold letters)(Jiang heteromerize withdifferent GPCRssuchastheghrelinreceptor(GHSR, 2013b Pfeiffer http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0049 Review The MC4R,dominantlyexpressedinthePVN,is Rios )). Moreover, severalofthesofaranalyzedsubjectsareableto et al. 2002, et al. 2006, Vilardaga α - andβ t al.2012a)andappetitecontrol( Garfield et et al. 2006, Ramsay et al. 2008, Decaillot -melanocyte-stimulating hormones et al. 2002, Rediger et al. 2008, Schellekens et al. 2009, Mandrika g

Juhasz kleinau © 2016SocietyforEndocrinology et al. 2013, et al. 2005, et al. 2008, andothers 2011, Printed inGreatBritain Schellekens Muller Ellis Navarro et al. et al. experimental confirmation and discussions(Fig. 4). elucidation ofcombinatorial possibilities willbeamatterof signals thatareinvolvedin energymetabolism.Future likely crucialfortheintegration ofallperipheralandcentral interaction network(e.g.MC3R, GHSRandGPR83)ismost inhibition oftheGHSRsignalingcapacity. 2013a). Interaction between both receptors leads to heteromerization withtheghrelinreceptor(Mulleretal . for heteromerization,ashasbeendemonstratedby GPR83 constituteshomodimersbutalsohasthecapacity compared with lean mice (Muller and circulating adiponectinlevels(DeMoerloozeetal.2000), been previously found to be involved in the control of 1991, in thethymusandhypothalamus(Harriganetal.1989, 2013b).Sofarthisorphanreceptorisexpressede.g. et al. determinant involvedinbodyweightregulation(Muller interacting partnersdifficult. due tomodificationsattheMC3R,MC4R,orfurther makes predictionsofanyphysiological/functionalimpact GPCRs such as GPR83 (Muller et al. b 2013 ( reported tohavefurtherdiverseGPCRinteractionpartners al. 2011). Boththe GHSR and the MC3Rhavebeen et of basal-andghrelin-inducedGHSRsignaling(Rediger of Interaction ofMC3RwithGHSRleadstotheupregulation ). interactions with e.g. the ghrelin receptor (GHSR) (Fig. 4 The MC3RisknowntobeembeddedinanetworkofGPCR and AgRP/NPYneurons(Barshetal.2000, the signalingcapacity. which indicatesalinkbetweenreceptordimerizationand capacity wasincreasedinmonomericMC4Rvariants, MC4R dimerdissociation. Most importantly, thesignaling regions ofthecannabinoid-1receptor, leadtopartial and adjacentregionsofTMH3TMH4,with (Piechowski using thenoninteractingcannabinoid-1receptor oligomerization couldbeachievedbydomainsubstitution constitution of protomer interfaces. Inhibition of MC4R revealed receptordeterminantsthatareinvolvedinthe results arelacking. et al.2009). results haveonlybeenindicatedinvitro studies(Rediger 4),althoughthese that areexpressedinthePVN(Fig. GPCR oligomerization ) and also interact with orphan Borroto-Escuela etal.2014)andalsointeractwithorphan Published byBioscientifica Ltd. α The signaling output of the previously mentioned GPCR The signalingoutputofthepreviouslymentionedGPCR The GPR83wasrecentlyidentifiedasanew In thehypothalamus,MC3RisexpressedonPOMC An interestingstudyonMC4Roligomerization -MSH-stimulated signaling and the downregulation -MSH-stimulated signalingandthedownregulation Gpr83 expressionisdecreasedinobesemicewhen Brezillon etal. 2001). Most importantly, GPR83 has . 2013).SubstitutionsoftheMC4RICL2 et al studies confirming these preliminary In vivostudiesconfirmingthesepreliminary Downloaded fromBioscientifica.com at10/02/202107:36:32AM . 2013b). Moreover,et al 57 ). Rediger etal.2011). : 1 ). This, in turn, ). This, in turn, respective R67 via freeaccess

Journal of Molecular Endocrinology OTR and the V1aR and V2R is established early in the OTR andtheV1aRV2R isestablishedearlyinthe homodimerization and ( which pointstoafunctional significanceofdimerization invitro systems, of oxytocinattheOTRwas observed vivo ( receptors formhomo-andheterodimersinvitro andin 2001). ItwasrecentlyreportedthatOTRandvasopressin including autismandschizophrenia(Gimpl&Fahrenholz conditions areassociatedwiththefunctionofthisreceptor manner (Viero etal.2010).Severaldiseasesorpathogenic role duringlaborandmilkejectioninapositivefeedback gland attheendofpregnancy, andplays animportant uterus andinthemyoepithelialcellsofmammary et al.2013).Intheperiphery, theOTR isexpressedinthe brain development(Gimpl&Fahrenholz2001, Moreover, the OTR is involved in thermoregulation and (e.g. and anxiety, and aggressive, sexual and maternal behaviors andlearning,stressregulation OT areinvolvedinmemory V2R) belongtoasubclassoffamilyAGPCRs.TheOTRand complexity andcommonoccurrenceofoligomerization. Several examples will be provided to pinpoint the 2),ordopaminereceptor(D2R). receptor (OTR)(Table are theprokineticin-receptor2(PROKR2),oxytocin regulation, whichhavebeenreportedtooligomerize, The examples inbracketsareconfirmedtheirinteractionbuthavenotyetbeenfunctionallycharacterized. This overviewrepresents examples ofGPCRsthat are involvedinendocrineregulation,whichhavebeenreportedto comprise homo-orheteromers. MC4R-MC4R MC3R-MC3R Weight and appetiteregulationand/ormetabolism Homomers Table 2 GHSR-GHSR GPR83-GPR83 FSHR-FSHR PROKR2-PROKR2 Reproduction NPY5R-NPY5R NPY1R-NPY1R NPY2R-NPY2R TAAR1-TAAR1 GIPR-GIPR LHCGR-LHCGR GnRHR-GnRHR ). It has been suggested that constitutive Albizu etal.2006).Ithasbeensuggestedthat constitutive DOI: 10.1530/JME-16-0049 http://jme.endocrinology-journals.org Review Review The OTRandvasopressinreceptors(AVPRs: V1aR,V1BR, Further examples of GPCRsinvolvedinendocrine Argiolas 1992, Albizu etal.2010b Examples ofGPCRdimersinvolvedinendocrineregulation. Malik etal.2012, Heteromers MC4R-5HTR1B, GPR7 MC3R-GHSR, NPY2R,MOR GHSR-MC3R, GPR83,SSTR5,DRD1/2,5HTR2C GPR83-GHSR, MC3R FSHR-LHCGR NPY2R-MC3R TAAR1-ADRA2A, D2R GIPR-GLPR LHCGR-FSHR heterodimerization between the heterodimerization between the ). Negative cooperative binding ). Negativecooperativebinding g

kleinau © 2016SocietyforEndocrinology ). Ebner etal.2013). andothers Printed inGreatBritain Kasahara 2012a of ligandtreatment(agonistsorantagonists)(Wrzal etal. modifies ERK1/2-activationallostericallyindependency OTR andβ et al.2013).Heterodimerizationwasalsoreportedfor dopamine receptorinthestriatum(Romero-Fernandez are co-expressed,suchasheterodimerswiththeD2 relevance intissueswheretheaforementionedreceptors 2004). Ofnote,heteromerizationmaybeofphysiological feeding, affect, reward, sleep, attention, memory and feeding, affect, reward, sleep, attention, memory neurons playamajorrolein cognitive functionsincluding for movement and the mesolimbic and mesocortical system (CNS).Thenigrostriatalsystemiscritical nervous insulin release(Murat involved intheregulationofadrenocorticotropinand phosphate production.Vasopressin andCRHareboth accumulation andCRHpotentiatedAVD-induced inositol Moreover, vasopressin potentiatedCRH-inducedcAMP secretion compared with single ligand stimulation. this heterodimermediatesasynergisticcatecholamine recently described(Muratetal.2012).Via costimulation, and thecorticotropin-releasinghormonereceptor1were with and V2Rvasopressinreceptorsdeterminestheinteraction state (Terrillon etal.2003). the plasmamembranedoesnotmodifydimerization endoplasmatic reticulum(ER)andthatligandbindingat GPCR oligomerization Published byBioscientifica Ltd. Four majordopaminergicpathways existinthecentral β , -arrestin andtheirtraffickingpatterns(Terrillon b ). Interestingly, directinteractionsbetween V1b References (Biebermann et al.2003,Tarnow et al.2008, (Rediger et al.2011,2012) (Rediger et al.2011,2012Kern (Muller et al.2013a,b) (Ji et al.2002,2004Tao et al.2004,Feng2013 (Marsango et al.2011) (Dinger et al.2003) (Dinger etal.2003,Redigeret al2009) (Harmeier et al.2015,Dinteretalb) (Schelshorn etal.2012,Ng&Chow2015) (Ji et al.2002,2004Tao et al.2004,Feng2013 (Cornea et al.2001,Kroeger) Rediger et al.2009) Muller et al.2013b) Park et al.2012,Schellekens2013 Mazurkiewicz et al.2015) Mazurkiewicz et al.2015) -2 adrenergic receptors. This interaction -2 adrenergicreceptors.Thisinteraction et al. 2012). Downloaded fromBioscientifica.com at10/02/202107:36:32AM Heterodimerization of V1aR Heterodimerization ofV1aR 57 57 : : 1 1 R68 R68 et al. via freeaccess Journal of Molecular Endocrinology receptor isofinterest. and pathogenic biased signaling modulation at this investigation oftherelationship betweenoligomerization oligomer havebeensuggested ( 2011), andspecificprotomer– also formsconstitutivehomomersinvivo( ( deficits (CPHD)includinggonadotropindeficiency hormone in patientswithidiopathiccombinedpituitary et al. 2005). wild type activates different G-protein subtypes (Chen ( intracellular pathways(biasedsignalingmodulation) have beenidentifiedtocausemodulationofdistinct mutations associatedwithcentralhypogonadism Bonomi etal.2012).PathogenicgermlinePROKR2 of hypogonadotropichypogonadism(Dodeetal.2006, . 2006).ThePROKR2isinvolvedinthepathogenesis et al al.2005, human GnRHdeficiency(Nget PROK2 and PROKR2 as strong candidate genes for morphogenesis andsexualmaturation,suggested bulb revealed theroleofPROK2signalinginolfactory Knockout micemodelsforbothligandandreceptor e al.2007 , Perez et physiological andpathologicalconditions(Maldonado- present inincreasedamountssomeregions. hypersensitive todopamine(Seemanetal.2005, (Li & Ma 2013). In schizophrenia, cortical D2Rs may be and addiction(mesolimbicmesocorticalsystem) as Parkinson’s disease(nigrostriatalsystem),schizophrenia, Dopaminergic pathwaysplayacriticalroleindiseasessuch (Kernetal.2015). hippocampal behaviorandmemory GHSR/D1R interrelationwasfoundtobeinvolvedin (Frederick etal.2015).Recently, invivorelevancefor D1R/D2R heterodimerhasnotyetbeenconfirmed H3 receptor(H3R).Afunctionalrelevanceforthe serotonin 2Areceptor(5HT-2AR), OTR,andthehistamine with the D1R, D4R, D5R, adenosine A2 receptor (A2R), a multitude of heteromers (Perreault D2R compriseshomomers(Guoetal.2003, and hippocampus(Beaulieu&Gainetdinov2011).The tuberclethe striatum,nucleusaccumbens,olfactory cyclase. Regions where the D2R is expressed include receptors (D2,D3andD4subtypes)inactivatetheadenylyl adenylyl cyclaseandincreasecAMPlevels;D2-class D1-class receptors (D1 and D5 subtypes) activate the their signalingcapacities(Beaulieu&Gainetdinov2011): been suggestedtobedividedintotwoclassesrelated learning (Perreaultetal.2014).Dopaminereceptorshave Raivio etal.2012, Libri etal. 2014, http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0049 Review The prokineticinsystemisrelatedtoseveral PROKR2 variantshavebeenfurtherdescribed Sbai etal.2014),wherebythe PROKR2 Negri etal.2007, e al.2012).ThePROKR2 Reynaud et protomer interfacesforthe Sposini et al. 2015).Future g

activate itsreceptor, thecalcitoninreceptor-like receptor factor forcalcitoningene-related peptide(CGRP)to of GPCRs.Itwasinitially identifiedasanobligatory underline anewmodeof thefunctionalregulation (RAMPs), thesingletransmembrane-spanning proteins, Identification ofreceptoractivitymodifyingproteins Interacting transmembraneproteins is resensitized(Whistleret al.2002). 1 (GASP1),whereasintheabsenceofGASP1,receptor interacting protein,theGPCR-associatedsortingprotein receptor occursinthepresenceofanadditionaltype of 2004). Moreover, lysosomal degradation for the decreases FSH-induced cAMP accumulation (Cohen GPCR functionistheadaptorprotein14-3-3tau,which example fornon-PDZ-containingproteinsthatmodulate NHERF1, ADRB2 is degraded (Hall for receptorresensitization,whereasintheabsenceof β-adrenergic receptor2(ADRB2),NHERF1actsasa phospholipase Csignaling(Ardura&Friedman2011).For of theparathyroidhormonereceptor(PTHR)toGq/11 signaling properties fromGs/adenylylcyclase activation factor (NHERF)(Dunn&Ferguson2015),whichchanges group ofproteinssuchastheNa+–Hexchangeregulatory GPCR functions.PDZ-containingproteinsareadiverse which arealargegroupofscaffoldingproteinsmodifying non-PDZ-containing proteins(Walther &Ferguson2015), 95/disc large/zonaoccludens-1)-containingproteins,and be subdividedintoarrestin,PDZ(postsynapticdensity Ferguson 2015).Examplesofintracellularproteinscan in mode ofsignalingandlocalizationtheGPCRs(reviewed Many interactingproteinsexist,modifyingtheefficacyor Intracellular interactingproteins with thetransmembranehelices(Kristiansen2004). or on the transmembrane spanning proteins and interact interfere withthereceptoreitheronintracellularsite (Walther &Ferguson2015).Interactingproteinsmay proteins that link the receptor to downstream effectors et al.2011a).Furthermore,GPCRsalsoactasscaffolding impact traffickingoractasallostericmodulators(Maurice capable ofmodifyingsignalingproperties.Theymay GPCRs are also able to associate with proteins that are GPCRs inhomomericorheteromericconstellations, In additiontotheabove-describedinteractionsbetween GPCRs ininteraction withnon-GPCRproteins GPCR oligomerization Published byBioscientifica Ltd. Ritter &Hall2009, Maurice Downloaded fromBioscientifica.com at10/02/202107:36:32AM t al.2011a, et . 1998). A further et al 57 : 1 Walther & δ-opioid trigger R69 . et al via freeaccess

Journal of Molecular Endocrinology ( or the PTHR, function together with distinct RAMPs VPAC1 receptor;however, otherGPCRs,suchasglucagon All RAMPsinteractwithCL-R,thecalcitoninand members oftheRAMPfamilyhavebeenidentified. to CL-R(McLatchie with RAMP2toCL-Renablesadrenomedullinbind CL-R tofacilitateCGRPbinding;however, association modify ligandspecificitysuchasRAMP1associatedwith (CL-R) (McLatchie to channelsensitizationand activation,whichamplify neurons convergeonmembers oftheTRPfamily, leading versa. Specificsignalsfrom GPCRslocatedonsensory to becloselylinkedthe functionofGPCRsorvice facilitator superfamily (MFS) (Law or substrate transporters, such as members of the major receptor potentialchannels(TRPs)(Veldhuis Channel typessuchasCa2+iontransportingtransient GPCRs andfurthertransmembrane-spanningproteins. a directinteractionandfunctionalassociationbetween 2016). Inturn,thisleadstotheideaofpossibilityfor complex (Doupnik2008, form amacromolecularionchannel/GPCRsignaling channels orvoltage-gatedcalciumchannelCav2.2that reported forGPCRsandionchannelssuchasKIR Direct channels andGPCRs/transporters Existence ofdirect interactions betweenGPCRs/ion regulates energyhomeostasisbyinhibitionofPROKR1. . 2016).Thissuggestsanewpathwaybywhich MRAP2 et al melanocortin GPCRthatis regulated by MRAP2 (Chaly Moreover, prokineticinreceptor-1(PROKR1)isthefirstnon- of theseMCRs(Cooray&Clark2011, hypothalamus, hasbeenidentifiedtoinfluencethefunction protein 2(MRAP2),whichisprimarilyexpressedinthe proteins; however, melanocortinreceptor-associated traffic tothecellsurfaceinabsenceofinteracting receptors (MC1R,MC3R,MC4RandMC5R)areableto escorts theMC2Rtocellsurface.Othermelanocortin et al.2005),asingletransmembrane-spanningproteinthat melanocortin receptor-associated protein (MRAP) (Metherell was impossible.Thischangedwiththeidentificationof inconceivable ascellsurfaceexpressionindifferenttypes ACTH receptor(MC2R,melanocortin2receptor)invitro was Previously, investigationofthefunctionalaspects function isfurtherevidencedbyvariousexamples. Hay DOI: 10.1530/JME-16-0049 http://jme.endocrinology-journals.org Review Review The importance of interacting proteins for GPCR The importanceofinteractingproteinsforGPCR et al. 2006). protein–protein interactions have also been et al. 1998).DifferentmembersofRAMP t al.1998).Sincethen,different et Benleulmi-Chaachoua g

and solidconfirmationvalidation. on functionalproperties.Thishypothesisrequiresfurther importance duetoafastmutualorsynergisticinfluence between differentproteinsandGPCRsshouldbeof Altogether, thisraisesthepossibilitythatanassociation biogenic aminesviatheTAAR1 (Xie or modulationofmonoaminetransportersbycommon (Dehvari ADRB2, GLUT4 and melatonin 1 receptor/Cav 2.2 interplaybetweenthe interactions, e.g.theobserved GPCR/channel (Yekkirala 2013) or GPCR/transporter are fewexamplesthatsupportthenotionofadirect MFS members (e.g. glucose transporter (GLUT)). There for cell metabolism, are transported into the cell by Furthermore, transporter substrates, such as glucose 12-transmembrane helices (e.g. dopamine transporter). transported out of the cell by members of the MFS with . 2015).Ontheotherhand,severalGPCRligandsare et al e.g. pain,itchandneurogenicinflammation(Veldhuis Franco 2010)andcanalsobeaddressed toreceptors oligomerization isacommon propertyforGPCRs(Ferre& (Garland 2013). reported that 365 human GPCRs are potential drug targets with autoantibodies.Interestingly, it was recently dysregulation, virus infections, mutations, or interactions fromhormonal The pathogenictriggersatGPCRsvary in theexcellentreviewbyHengandcoworkers(2013) . cardiovascular diseases,allergiesorcancers,canbefound subdivided intoassociationswithobesity, diabetes, in the pathophysiology of various human diseases, diverse rolesofG-protein-coupledreceptors(GPCRs) ofthe in G-proteins(O’Hayreetal.2013).Anoverview 10% ofcancer-relatedmutationshavebeenidentified tumors harbormutationsinGPCRsandapproximately et al.2004, 2007, (Seifert & Wenzel-Seifert 2002, infertility, andmetabolicneurologicaldisorders including cancer, viralinfections,inflammation, with varioushumandiseases(Hutchingsetal.2010) Malfunctions ofapproximately100GPCRsareassociated pathophysiology? Is there adirect linkbetweenGPCRoligomerizationand dysfunctions GPCR oligomerization inendocrine GPCR oligomerization Published byBioscientifica Ltd. As describedinthenumerous examplesabove, Garcia-Jimenez &Santisteban 2007, t al.2012, et Vassart &Costagliola2011).Almost20%of Benleulmi-Chaachoua Downloaded fromBioscientifica.com at10/02/202107:36:32AM Dorsam & Gutkind 57 57 : : 1 1 t al.2008). et t al.2016), et Schoneberg R70 R70 via freeaccess

Journal of Molecular Endocrinology TSHR equilibrium (Lambert2010 ), definese.g.theLHCGR or homomeric arrangement,even inanoligomer–monomer 2).The and ‘signalosome’ofparticular receptors(Table with themotivationtoinvestigateentire‘interactome’ 2008, . andbehavior(Gonzalez-Maesoet al function inmemory studies andaroleforGHSRinmodulationoftheD1R implicated inpsychosishasbeenidentifiedprevious For example,aserotonin/glutamatereceptorcomplex started to become systematic (e.g. elucidation ofheteromericformshaveonlyrecently has omni-relevanceinphysiologyandmedicine,the pain sensitization(Laffrayetal.2012). dimers, which has an impact on neuropathic obligatory inhibited duetoligand-inducedmonomerizationofthe Moreover, for theGABABreceptors,signalingactivityis normotensive pregnantwomen(AbdAllaetal.2001 ). from pre-eclampticwomenthanonplatelets tobemoreabundantonplatelets heteromer wasobserved to be functionally correlated with preeclampsia, as this receptor-2 (B2R).TheAT1R–B2R heterodimer is supposed case forangiotensinreceptorII(AT1R) andbradykinin relevance ofoligomerizationwasindicatedinaspecific (Pei et al. ). 2010 Furthermore, the pathophysiological postmortem striatum of patientsthan in normal subjects D1R–D2R heterodimer was detectedathigherlevelsinthe to beimplicatedindepression(Rashid dopamine receptorsubtypes,D1RandD2R,arelikely al. 2000, et are oneofthebeststudiedsubjectsinthisfield(Gomes GPCRs, inrelationtoneurophysiologicalprocesses, 2011, al. cases ofpathophysiologicalimportance(Tadagaki et several examples(Overton&Blumer2000)including GPCR oligomerizationinvivohasbeenconfirmedby findings andpotentiallyartificialdata,therelevanceof protomer interfacesorintheligandbindingsites. modified oligomericproperties,e.g.bymutantsinthe that adysfunctionofGPCRsignalingcanalsobedueto likely protomer/protomer arrangement,makesitvery the correctfunctionofGPCRscorrelateswithaspecific described heresofar. Bycombiningthesetwofacts,that have beenrendered more preciselyforallthe GPCRs is offunctionalrelevance,althoughithas/couldnot 2).Secondly, oligomerization LHCGR orMC4R(Table involved inendocrineregulationsuchastheTSHR, http://jme.endocrinology-journals.org DOI: 10.1530/JME-16-0049 Review For GPCRsinvolvedinendocrineregulation,which Despite the huge and increasing amount of per seascomplexes.To date,therehavebeenno Kern 2012b, 2013, t al.2015).Heteromerization between the et Tschische etal.2011, Prinster t al. 2005, et g

kleinau © 2016SocietyforEndocrinology Rediger Kern Fuxe et al. ). 2012 andothers t al.2007).The et Printed inGreatBritain t al.2015). et t al. 2009), et in vitro MC4R (Biebermann 2005), GnRHR(Grosseetal.1997, 2004), TSHR(Calebiro 1998), CCR5(Benkiraneetal.1997LHCGRTao rhodopsin (Kurada&O’Tousa 1995),AVPR2 (Zhu&Wess fornumerousGPCRssuchas have beenobserved receptor splicevariants.Suchdominantnegativeeffects effect ofreportedheterozygotemutantsordistinct and loss-of-functionphenotypes:thedominant negative potentially highlightsadirectlinkbetweenoligomerization exceptions (Zoenenet al.2012). explanations ofactivationorinactivation,withfew this propertyintocurrentandpreviousmechanistical difficulties inadequatelycombiningorincorporating 2012, Tao 2006, (Themmen &Huhtaniemi2000, patients withhypo-orhyperfunctionhavebeendescribed the occurrenceofloss-of-orgain-of-functionmutantsin modified andnonfunctionalreceptorvariant. to thewild-typereceptorbymultimerizationwith that thenegativeeffectofreceptorvariantsistransferred For theaforementionedexamples,itisgenerallyassumed and symptoms of neurodegeneration (AbdAlla et al. 2009). negative AT2R receptoroligomersinduceG-proteinarrest the prostacyclinreceptor(Ibrahimetal.2010).Dominant confirmed (Zoenen modification ofoligomeric stateshasnotyetbeen proven on their impact on oligomerization and a direct variants, althoughrare.CAMs oftheTSHRhavebeen or hypogonadismmaybeassociatedwithinactivating LHCGR, male-limited precocious puberty (by CAMs) or nonautoimmunehyperthyroidism,whereasinthe diseased conditionssuchascongenitalhypothyroidism binding capacity. InTSHR,pathogenicmutations leadto such asdecreasedexpressionlevelorlossofligand individual orlinkedparametersatthe accompanied byaloss-of-functionmayinfluencemany (Smits etal.2003)isalsofeasible.Inactivatingmutants side-chain substitutionsuchasthatreportedfortheFSHR combination ofbothmechanismscausedbyonesingle ligand selectivityhavebeendescribed.Theinterrelated mutations (CAMs)orsubstitutionsleadingtoalossof In caseofgain-of-function,constitutivelyactivating level (excellentlyreviewedinVassart &Costagliola2011). different mechanismsatthereceptoronmolecular Kleinau 2014).Inparticular, themutationsmaycause GPCR oligomerization Published byBioscientifica Ltd. In theGPCRsdescribedhere,oneparticularfeature For theGHSR,MC3R,MC4R,PROKR2orGPHRs, Troppmann etal.2013, 2010, Vassart & Costagliola 2011, . 2012, et al t al.2003, et et al. 2005),MC1R(Beaumontet al. Downloaded fromBioscientifica.com at10/02/202107:36:32AM Libri Biebermann Tarnow etal.2008),or Schoneberg Brothers . 2014, et al 57 receptor orligand : 1 . 2012b). et al Rediger et al. . 2004), et al . 2004, et al Vassart & etal. R71 via freeaccess

Journal of Molecular Endocrinology . 2012, et al current measurement methods do not simultaneously current measurement methods donotsimultaneously different heteromersmay indeed exist.Unfortunately, such astransporters,itmust beconsideredthatmany 2008) ormanyothertransmembrane spanningproteins (likely torangebetween10and100 the cellsurfacewithahighnumberofexpressedGPCRs assembly states (Song definitive answersforthefunctionalityofdifferent present inamonomer/oligomerequilibriumwilllead to homodimers, andthoseofwild-typereceptor‘mixtures’ achieve. Onlyacomparisonofthepropertiesmonomers, oligomerization ofGPCRs,whichiscurrentlydifficult to oligomers. Thisrequiresforced monomerizationand of functionaleffectsinducedbyeithermonomersor such ascelltypeordevelopmentalstage. analyses), whichisdependentonphysiologicalparameters the particularGPCRinteractionpartners(interactome is ofimportance.Thisincludestheidentification consequences, including the physiological relevance, oligomerization andrelatedstructural–functional or newtherapeuticagents.Therefore,studyingGPCR of newperspectivesonthemechanismsestablished are bothchallengingandexcitingfortheidentification andthetargetingofhomo-orheteromers drug discovery oligomeric receptormodelsintostrategiesforGPCR The incorporation of pharmacological interventions. fascinating avenuesforpotentialdirectedandselective and inendocrineregulation.Thisperspectiveopens such as pathogenic dysfunctions and clinical disorders, physiological roles,andtoexplaininterrelatedprocesses consideration incomprehensivelyunderstandingtheir to take GPCR oligomers into Therefore, it is necessary and bydirectedinvivosearches forGPCRoligomers. will furtherincrease with theuseofadvanced methods of examplesforrelevantoligomershavebeenreportedand signaling regulationandpharmacology. Acertainnumber GPCR function regarding transport, signal transduction, Oligomerization iswidelyacceptedtobeapivotalaspectof Open aspectsandfuture perspectives in futurestudies. function should be considered and systematically proven or aspecificentity, interfereswithaproper oligomer Moreover, theopportunitythatinactivatingmutations, in otherreceptorsmayindeedmodulateoligomerization. This doesnotexcludethepossibilitythatnaturalmutations DOI: 10.1530/JME-16-0049 http://jme.endocrinology-journals.org Review Review A further major effort in future studies is the dissection A furthermajoreffortinfuturestudiesisthedissection 2014, Calebiro et al. 2007 , ). Of specific note, at . 2013).Ofspecificnote,at et al Lambert 2010, g

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