AcademicPressisanimprintofElsevier 225WymanStreet,Waltham,MA02451,USA 525BStreet,Suite1800,SanDiego,CA92101-4495,USA 125LondonWall,London,EC2Y5AS,UK TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UK Firstedition2015 ©2015,ElsevierInc.AllRightsReserved. Nopartofthispublicationmaybereproducedortransmittedinanyformorbyanymeans, electronicormechanical,includingphotocopying,recording,oranyinformationstorageand retrievalsystem,withoutpermissioninwritingfromthepublisher.Detailsonhowtoseek permission,furtherinformationaboutthePublisher’spermissionspoliciesandour arrangementswithorganizationssuchastheCopyrightClearanceCenterandtheCopyright LicensingAgency,canbefoundatourwebsite:www.elsevier.com/permissions. Thisbookandtheindividualcontributionscontainedinitareprotectedundercopyrightby thePublisher(otherthanasmaybenotedherein). Notices Knowledgeandbestpracticeinthisfieldareconstantlychanging.Asnewresearchand experiencebroadenourunderstanding,changesinresearchmethods,professionalpractices, ormedicaltreatmentmaybecomenecessary. Practitionersandresearchersmustalwaysrelyontheirownexperienceandknowledgein evaluatingandusinganyinformation,methods,compounds,orexperimentsdescribed herein.Inusingsuchinformationormethodstheyshouldbemindfuloftheirownsafetyand thesafetyofothers,includingpartiesforwhomtheyhaveaprofessionalresponsibility. Tothefullestextentofthelaw,neitherthePublishernortheauthors,contributors,oreditors, assumeanyliabilityforanyinjuryand/ordamagetopersonsorpropertyasamatterof productsliability,negligenceorotherwise,orfromanyuseoroperationofanymethods, products,instructions,orideascontainedinthematerialherein. ISBN:978-0-12-802939-8 ISSN:1877-1173 ForinformationonallAcademicPresspublications visitourwebsiteatstore.elsevier.com CONTRIBUTORS SanaAlAwabdh INSERMU894,andCentredePsychiatrieetNeurosciences,Universite´ ParisDescartes, SorbonneParisCite´,Paris,France AnnetteG.Beck-Sickinger FacultyofBiosciences,PharmacyandPsychology,InstituteofBiochemistry,Universita¨t Leipzig,Leipzig,Germany ShannaL.Bowman DepartmentofBiologicalSciences,CarnegieMellonUniversity,Pittsburgh,Pennsylvania, USA ChristopherCottingham DepartmentofBiologyandChemistry,MoreheadStateUniversity,Morehead,Kentucky, USA Miche`leDarmon INSERMU894,andCentredePsychiatrieetNeurosciences,Universite´ ParisDescartes, SorbonneParisCite´,Paris,France JasonE.Davis DepartmentofPharmacologyandToxicology,MedicalCollegeofGeorgia,Georgia RegentsUniversity,Augusta,Georgia,USA DenisJ.Dupre´ DepartmentofPharmacology,DalhousieUniversity,Halifax,NovaScotia,Canada Michel-BorisEmerit INSERMU894,andCentredePsychiatrieetNeurosciences,Universite´ ParisDescartes, SorbonneParisCite´,Paris,France CraigJ.Ferryman DepartmentofBiologyandChemistry,MoreheadStateUniversity,Morehead,Kentucky, USA CatalinM.Filipeanu DepartmentofPharmacology,CollegeofMedicine,HowardUniversity,Washington, DistrictofColumbia,USA QinFu DepartmentofPharmacology,SchoolofBasicMedicine,TongjiMedicalCollege, HuazhongUniversityofScienceandTechnology,Wuhan,PRChina EugeniaV.Gurevich DepartmentofPharmacology,VanderbiltUniversity,Nashville,Tennessee,USA VsevolodV.Gurevich DepartmentofPharmacology,VanderbiltUniversity,Nashville,Tennessee,USA ix x Contributors YoshikazuImanishi DepartmentofPharmacology,SchoolofMedicine,CaseWesternReserveUniversity, Cleveland,Ohio,USA JustineE.Kennedy DepartmentofMolecularPharmacologyandTherapeutics,LoyolaUniversityChicago, HealthSciencesDivision,Maywood,Illinois,USA WolfgangKlein Leibniz-Institutfu€rMolekularePharmakologie(FMP),Berlin,Germany AdrianoMarchese DepartmentofMolecularPharmacologyandTherapeutics,LoyolaUniversityChicago, HealthSciencesDivision,Maywood,Illinois,USA JustineMasson INSERMU894,andCentredePsychiatrieetNeurosciences,Universite´ ParisDescartes, SorbonneParisCite´,Paris,France KarinMo€rl FacultyofBiosciences,PharmacyandPsychology,InstituteofBiochemistry,Universita¨t Leipzig,Leipzig,Germany InaNemet DepartmentofPharmacology,SchoolofMedicine,CaseWesternReserveUniversity, Cleveland,Ohio,USA ManojkumarA.Puthenveedu DepartmentofBiologicalSciences,CarnegieMellonUniversity,Pittsburgh,Pennsylvania, USA KausikRay ScientificReviewBranch,NIDCD,NationalInstitutesofHealth,Bethesda,MD,USA PhilipRopelewski DepartmentofPharmacology,SchoolofMedicine,CaseWesternReserveUniversity, Cleveland,Ohio,USA ClaudiaRutz Leibniz-Institutfu€rMolekularePharmakologie(FMP),Berlin,Germany RalfSchu€lein Leibniz-Institutfu€rMolekularePharmakologie(FMP),Berlin,Germany QinWang DepartmentofCell,DevelopmentalandIntegrativeBiology,UniversityofAlabamaat Birmingham,Birmingham,Alabama,USA JaimeWertman DepartmentofMicrobiologyandImmunology,DalhousieUniversity,Halifax,NovaScotia, Canada Contributors xi GuangyuWu DepartmentofPharmacologyandToxicology,MedicalCollegeofGeorgia,Georgia RegentsUniversity,Augusta,Georgia,USA YangK.Xiang DepartmentofPharmacology,UniversityofCalifornia,DavisCalifornia,USA BrentYoung DepartmentofPharmacology,DalhousieUniversity,Halifax,NovaScotia,Canada MaoxiangZhang DepartmentofPharmacologyandToxicology,MedicalCollegeofGeorgia,Georgia RegentsUniversity,Augusta,Georgia,USA PREFACE G protein-coupled receptors (GPCRs) (also known as seven- transmembrane domain receptors or 7TMRs) constitute the largest family ofcellsurfacereceptorsinvolvedinsignalregulationunderdiversephysio- logical and pathological conditions and are drug targets for many diseases. Extensivestudiescarriedoutoverthepast2–3decadeshaveclearlydemon- stratedthatthespatiotemporalregulationofGPCRintracellulartrafficking, includingthecellsurfaceexport,internalization,recycling,anddegradation, is a crucial mechanism that controls receptor transport to the right place which in turn dictates the integrated responses of the cell to hormones anddrugsattherighttime.Addingtothecomplexity,eachofthesetraffick- ing processes is mediated by multiple pathways and is highly regulated by many factors, such as structural determinants, specific motifs, interacting proteins, posttranslational modifications, and transport machineries, alto- gethercoordinatingreceptortransportusingveryspecializedroutes.GPCR trafficking is rapidly evolving and has great potential to translate into new therapeutics. Themainpurposeofthisbookistoreviewourcurrentunderstandingof intracellulartraffickingofsomewell-characterizedGPCRs.Inaddition,this bookwillalsohighlighttherolesoftraffickinginregulatingthefunctionality of the receptors and pinpoint current challenges and future directions in studying GPCR trafficking. The contributors are experts in this area with manyyearsofexperience.Itismyhopethatthisbookwillbeusefultograd- uate students, postdoctoral fellows, and researchers who are interested in general GPCR biology or intracellular trafficking of GPCRs. Iamgratefultoeachofthecontributorsfortheirvaluabletimeandtre- mendouseffortstomakethisbookpossible.Itismygreatpleasuretowork with them to put together a book on this very important topic in GPCR biology. I thank Dr. P. Michael Conn, the Chief Editor of the Progress in Molecular Biology and Translational Science series, for inviting me to edit this volume and always being supportive. I also would like to take this oppor- tunity to thank my former mentor, Dr. Stephen M. Lanier, for leading me into the GPCR field. GUANGYU WU xiii CHAPTER ONE Arrestins: Critical Players in ☆ Trafficking of Many GPCRs Vsevolod V. Gurevich1, Eugenia V. Gurevich DepartmentofPharmacology,VanderbiltUniversity,Nashville,Tennessee,USA 1Correspondingauthor:e-mailaddress:[email protected] Contents 1. ArrestinsandGPCRTrafficking 2 2. Non-visualArrestinsMediateGPCRInternalizationviaCoatedPits 2 3. VisualArrestinsandTraffickingProteins 4 4. UbiquitinationandDeubiquitinationinGPCRCyclingandSignaling 6 5. FasterCyclingPreventsReceptorDownregulation 7 6. ArrestinsinReceptorRecyclingandVesicleTrafficking:Questions WithoutAnswers 8 7. ConclusionsandFutureDirections 9 References 10 Abstract ArrestinsspecificallybindactivephosphorylatedGprotein-coupledreceptors(GPCRs). ReceptorbindinginducesthereleaseofthearrestinC-tail,whichinnon-visualarrestins containshigh-affinitybindingsitesforclathrinanditsadaptorAP2.Thus,servingasa physicallinkbetweenthereceptorandkeycomponentsoftheinternalizationmachin- eryofthecoatedpitisthebest-characterizedfunctionofnon-visualarrestinsinGPCR trafficking.However,arrestinsalsoregulateGPCRtraffickinglessdirectlybyorchestrat- ingtheirubiquitinationanddeubiquitination.Severalreportssuggestthatarrestinsplay additionalrolesinreceptortrafficking.Non-visualarrestinsappeartoberequiredforthe recycling of internalized GPCRs, and the mechanisms of their function in this case remain to be elucidated. Moreover, visual and non-visual arrestins were shown to directlybindN-ethylmaleimide-sensitivefactor,animportantATPaseinvolvedinvesicle trafficking,butneithermoleculardetailsnorthebiologicalroleoftheseinteractionsis clear.Consideringhowmanydifferentproteinsarrestinsappeartobind,wecanconfi- dentlyexpecttheelucidationofadditionaltrafficking-relatedfunctionsoftheseversatile signalingadaptors. ☆Weusesystematicnamesofarrestinproteins:arrestin-1(historicnamesS-antigen,48kDaprotein, visualorrodarrestin),arrestin-2(β-arrestinorβ-arrestin1),arrestin-3(β-arrestin2orhTHY-ARRX), andarrestin-4(coneorX-arrestin;forunclearreasons,its geneiscalled“arrestin3”intheHUGO database). ProgressinMolecularBiologyandTranslationalScience,Volume132 #2015ElsevierInc. 1 ISSN1877-1173 Allrightsreserved. http://dx.doi.org/10.1016/bs.pmbts.2015.02.010 2 VsevolodV.GurevichandEugeniaV.Gurevich ABBREVIATIONS AIP4 atrophin-1-interactingprotein4 AP2 adaptorprotein2 β2AR β -adrenergicreceptor 2 GPCR Gprotein-coupledreceptor GRK Gprotein-coupledreceptorkinase Nedd4 neuralprecursorcellexpresseddevelopmentallydown-regulatedprotein4 1. ARRESTINS AND GPCR TRAFFICKING Preferential binding of arrestins to active phosphorylated receptors was discovered about 30 years ago.1 The finding that arrestin binding sup- pressesreceptorcouplingtocognateGproteinswasmadesoonafterinthe visual system.2 The mechanism turned out to be remarkably simple: direct competition between arrestin and G protein for overlapping sites.3,4 For sometime,itappearedthattheonlyfunctionarrestinshaveistobindactive phosphorylatedGprotein-coupledreceptors(GPCRs),precludingreceptor interactions with G proteins by direct competition.3,4 The first described non-GPCR binding partners of arrestins were trafficking proteins: clathrin in 19965 and clathrin adaptor AP2 a few years later.6 These data demon- stratedthatarrestinsplayanessentialrolenotonlyinGPCRdesensitization7 but also in receptor endocytosis,8 via trafficking signals added by receptor- boundarrestins.Thediscoverythatarrestinsareubiquitinateduponreceptor bindingandregulateubiquitinationofGPCRs9revealedyetanothermech- anism, whereby arrestins regulate receptor trafficking indirectly. Here, we discuss several known mechanisms of arrestin effects on GPCR trafficking and highlight observations that suggest that there are many other mecha- nisms that still remain to be elucidated. 2. NON-VISUAL ARRESTINS MEDIATE GPCR INTERNALIZATION VIA COATED PITS Arrestins promote GPCR internalization by virtue of recruitment of clathrin and AP2 via fairly well-mapped binding sites in the C-tail of non- visual arrestins5,6,10,11 (Fig. 1). Interestingly, the C-tail in the basal confor- mation of all arrestins is anchored to the N-domain,12–16 whereas receptor binding triggers its release.17–19 The expression of separated arrestin C-tail carrying these sites inhibits GPCR internalization, apparently by winning ArrestinsinGPCRTrafficking 3 Figure1 ArrestinsplaymanyrolesinGPCRtrafficking.Arrestins(ARR)bindactivephos- phorylatedGPCRs(shownasaseven-helixbundle).Receptorbindinginducestherelease ofthearrestinC-tail,whichcarriesbindingsitesforclathrin(Clath)andadaptorprotein-2 (AP2).TheinteractionsofthesesiteswithclathrinandAP2promotereceptorinternali- zationviacoatedpits.ArrestinsalsorecruitubiquitinligasesMdfm2,Nedd4,andAIP4to thecomplex,whichfavorsubiquitinationofbothnon-visualarrestinsandatleastsome GPCRs. Arrestins also recruitcertaindeubiquitinationenzymes(USP20and USP33 are shown), facilitating receptor deubiquitination. The role of arrestin interactions with microtubules, centrosome, and N-ethylmaleimide-sensitive factor (NSF) in trafficking ofGPCRsand/orotherproteinsremainstobeelucidated. the competition with the arrestin–receptor complexes for clathrin and AP2.20 This finding provided the first clear evidence of functional signifi- cance of shielding of the arrestin C-tail in the basal conformation and its release upon receptor binding. In free arrestins, the C-tail is anchored to thebodyofthemolecule,whichmakesitinaccessible,preventingitscom- petition with the receptor-bound arrestins for the components of internal- ization machinery (reviewed in Ref. 21). Anotherknownmechanismofarrestinrecruitmenttothecoatedpitisits directbindingtophosphoinositides,whichwasreportedtobenecessaryfor GPCR internalization.22 Since resident coated pit protein AP2 is also rec- ruited to this part of the membrane via phosphoinositide binding,23 one might think that as soon as the arrestin–receptor complex is formed, it hasnochoicebuttomovetothecoatedpit.However,thisdoesnotappear tobethecase.InmuscarinicM2receptor,whichwasamongthefirstshown tobindarrestins,24twoSer/Thrclustersinthethirdcytoplasmicloopwere identified as critical for arrestin binding and receptor desensitization.25 Yet 4 VsevolodV.GurevichandEugeniaV.Gurevich the elimination of these clusters, and even dominant-negative dynamin K44A mutant that blocks the internalization of β2AR in the same cells, did not prevent M2 endocytosis, suggesting that M2 receptor does not usecoatedpitsandinternalizesinanarrestin-independentmanner.25Inter- estingly, overexpression of non-visual arrestins can redirect some M2 to coated pits,25 suggesting that this receptor can use more than one route. ManyotherGPCRswereshowntohavethatchoice.Forexample,chemo- kine receptor CCR5 uses both phosphorylation- and arrestin-dependent and -independent pathways.26 Cysteinyl leukotriene type 1 receptorinter- nalizes normally in mouse embryonic fibroblasts lacking both non-visual arrestins, yet arrestin expression facilitates its internalization,27 apparently directing it to the arrestin-dependent pathway, which is usually not pre- ferred, similar to M2 receptor.25 Metabotropic glutamate receptor mGluR1a constitutively internalizes via arrestin-independent mechanism, whereas its agonist-dependent internalization appears to be mediated by arrestin-2.28 Endogenous and overexpressed serotonin 5HT4 receptor internalizes via arrestin-dependent pathway, but the deletion of Ser/Thr cluster targeted by G protein-coupled receptor kinases (GRKs) redirects it to an alternative pathway and even facilitates its internalization.29 Thus, it appears that the ability of GPCRs to use more than one inter- nalization pathway is a general rule, rather than an exception, likely rep- resenting one of the many backup mechanisms cells usually have. Many receptors have recognizable internalization motifs in their sequence, so arrestinbindingsimplyaddsnewones.Therelativestrengthofthesemotifs, aswellasthearrestinexpressionlevels,likelydeterminesthepathway(s)each receptorchoosesinaparticularcell.Thedominantinternalizationpathway of aparticular receptor isnotnecessarily thesame in differentcell types,or evenatdifferentfunctionalstatesofthesamecell(reviewedinRef.8).Vari- ety,ratherthanuniformity,characterizestheworldofGPCRsignalingand trafficking.30 3. VISUAL ARRESTINS AND TRAFFICKING PROTEINS In vertebraterodphotoreceptors, rhodopsinis localizedon the discs, whicharedetachedfromtheplasmamembrane31andthereforearetopolog- icallyequivalenttovesicleswithinternalizednon-visualGPCRs.Thus,ver- tebrate rhodopsin is not supposed to be internalized. Indeed, arrestin-1, which is the prevalent arrestin isoform in both rods and cones,32 does not haveconventionalclathrin-orAP2-bindingelementsinitsC-tail.33How- ever,sequencecomparisonofarrestin-1andnon-visualsubtypesshowsthat ArrestinsinGPCRTrafficking 5 in the region homologous to AP2-binding motif in arrestin-2 and-3, only one positive charge is missing.34 Therefore, it is hardly surprising that arrestin-1alsobindsAP2,albeitwith(cid:1)30timesloweraffinity.34Constitu- tively active rhodopsin–K296E is a naturally occurring mutant that causes autosomal dominant retinitis pigmentosa in humans, apparently due to constitutive phosphorylation and formation of a stable complex with arrestin-1.35 The concentration of rhodopsin in the outer segment of rods reaches (cid:1)3mM.31 Rods also express roughly 8 arrestin molecules per 10rhodopsins,36–38sotheconcentrationsofbothproteinsandtheircomplex formedinbrightlightareveryhigh.Itturnsoutthatattheseconcentrations evenlowaffinitymatters:thepresenceofWTarrestin-1facilitatesroddeath inanimalsexpressingrhodopsin–K296E,withvisibleaccumulationofAP2 intheoutersegment,whereitisnotobservedinnormalmice.34Incontrast, truncated arrestin-1 lacking the C-tail containing the low-affinity AP2- binding site protects photoreceptors in these animals and preserves their function.34 Thus, in rod and cone photoreceptors, both of which express very high levels of arrestin-1,32 even relatively low-affinity interactions, whichwouldnotmatterinothercells,withsubmicromolarconcentrations of both non-visual arrestins,39,40 can become biologically relevant. Interestingly, the localization of rhodopsin on invaginations of the plasma membrane in flies, in contrast to detached discs in vertebrate rods, is one of the many differences between vertebrate and invertebrate photo- receptors. Another difference directly follows from this localization: Drosophila rhodopsin is internalized, like “normal” vertebrate GPCRs, via clathrin- and AP2-mediated mechanism.41 In fly photoreceptors, arrestin isevenlydistributed,whereasindark-adaptedvertebraterods,itisconcen- tratedintheinnersegment,withfairlysmallfractionintheoutersegment, where rhodopsin resides.36–38 However, in both types of photoreceptors upon illumination, arrestin translocates to rhodopsin-containing mem- branes.36–38,42–45Likenon-visualarrestins,andincontrasttovertebratevisual arrestin,22 visual arrestin in Drosophila has high-affinity phosphoinositide- binding site.43 It was proposed that due to phosphoinositide binding, Drosophilaarrestintranslocatestorhodopsinonphosphoinositide-richvesicles movedwiththehelpofDrosophilamyosinIII(NINAC).42Theparticipation of NINAC in metarhodopsin inactivation in Drosophila was independently confirmed,46 but arrestin translocation was found to be largely driven by its binding to rhodopsin in flies,44 just like in mice.45 Thus, the internalization of invertebrate rhodopsin apparently follows the same rules as many non- visual GPCRs: active receptor recruits arrestin via direct binding,47 which then links it to the key components of the coated pit.5,6,41