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1、782Biochemistry2005,44,782-789RoleoftheN-TerminalDomainoftheCalcitoninReceptor-likeReceptorinLigandBinding²MadhuChauhan,³KrishnaRajarathnam,*,§andChandraYallampalli*,³DepartmentsofObstetricsandGynecologyandHumanBiologicalChemistryandGenetics,SealyCenterforStructuralBiology,Un
2、iVersityofTexasMedicalBranchatGalVeston,GalVeston,Texas77555ReceiVedApril27,2004;ReVisedManuscriptReceiVedAugust26,2004ABSTRACT:Calcitoninreceptor-likereceptor(CRLR)isaseven-transmembrane(7-TM)domainclassBGprotein-coupledreceptor(GPCR)whichrequirescoexpressionofdifferentrecep
3、toractivitymodifyingproteins(RAMP)tobecomeafunctionalcalcitoningene-relatedpeptide(CGRP)receptororanadrenomedullin(AM)receptor.TheN-terminal(Nt)extracellularregionofclassBGPCRsinligandbindinghasbeenreportedforreceptorssuchasglucagonandparathyroidhormone.WehypothesizethattheNt
4、-domainofCRLR(Nt-CRLR)isanautonomouslyfoldedunitpossessingawell-definedstructureandisinvolvedinligandbindingandspecificity.ToobtainstructuralandfunctionalinformationontheNt-CRLR,weclonedandexpressedtheNt-CRLRasafusionproteininEscherichiacoli.Overexpressedproteinformedaninclus
5、ionbody,whichwasrefoldedandpurified,resultinginasolublemonomericprotein.Far-UVCDandfluorescencespectraofNt-CRLRshowedcharacteristicsofafoldedprotein.TheabilityofNt-CRLRtobindCGRPandAMindependentofRAMPswasdeterminedbystudyinginhibitionof125I-CGRPand125I-AMbindingtopregnantratu
6、terinemembraneinthepresenceofNt-CRLRprotein.WeobservethatNt-CRLRinhibits125I-CGRPand125I-AMbindingtoratuterusinadose-dependentfashion(IC50)0.25and0.29íM,respectively).Takentogether,ourdataprovideevidencethatNt-CRLRisstructuredandfurtherthatasignificantpartofthebindingaffinity
7、comesfrombindingtotheNt-domain.Calcitoningene-relatedpeptide(CGRP),1adrenomedullinwithRAMP1resultsinCGRPfunction,whereasassociation(AM),amylin,andcalcitonin(CT)belongtothecalcitoninwithRAMP2orRAMP3resultsinAMfunction(20).CTfamilyofpeptides,distributedinperipheraltissuesandrec
8、eptorbindsCTintheabsenceofRAMPsandbindsnervoussystem,andinduceawidev
