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Opposite Effects of the Melanocortin-2 Receptor Accessory Protein MRAP on MC2 andMC5 Receptor Dimerization and Trafficking*

Julien A. Sebag and Patricia M. HinkleFrom the Department of Pharmacology and Physiology

University of Rochester Medical Center, Rochester, New York 14642Running title: MRAP Regulation of MC5 Receptor Dimerization and Trafficking

Address correspondence to: Patricia M. Hinkle, Dept. of Pharmacology and Physiology,University of Rochester Medical Center, Rochester, NY 14642. Tel: 585-275-4933. Fax: 585-273-2652. E-mail: Patricia_Hinkle@urmc.rochester.edu

MC2 (ACTH) receptors requireMC2 receptor accessory protein (MRAP)to reach the cell surface. In this study, weshow that MRAP has the opposite effecton the closely related MC5 receptor. InELISA and microscopy experiments,MC2 receptor was retained in the ER inthe absence of MRAP and targeted to theplasma membrane with MRAP. MC5receptor was at the plasma membrane inthe absence of MRAP, but trappedintracellularly when expressed withMRAP. Using bimolecular fluorescencecomplementation, where one fragment ofyellow fluorescent protein (YFP) wasfused to receptors and another to MRAP,we showed that MC2 receptor-MRAPdimers were present at the plasmamembrane, whereas MC5 receptor-MRAP dimers were intracellular. BothMC2 and MC5 receptors co-precipitatedwith MRAP. MRAP did not alterexpression of β2-adrenergic receptors orco-precipitate with them. To determine ifMRAP affects formation of receptoroligomers, we co-expressed MC2receptors fused to YFP fragments in thepresence or absence of MRAP. YFPfluorescence, reporting MC2 receptorhomodimers, was readily detectable withor without MRAP. In contrast, MC5receptor homodimers were visible in theabsence of MRAP, but little fluorescencewas observed by microscopic analysiswhen MRAP was co-expressed. Co-precipitation of differentially taggedreceptors confirmed that MRAP blocksMC5 receptor dimerization. The regionsof MRAP required for its effects on MC2and MC5 receptors differed. These resultsestablish that MRAP forms stable

complexes with two differentmelanocortin receptors, facilitatingsurface expression of MC2 receptor butdisrupting dimerization and surfacelocalization of MC5 receptor.

In mammals, the five members ofthe melanocortin receptor family playdiverse physiological roles. MC1 receptors(melanocyte stimulating hormone receptors)control pigmentation in many animals, MC2receptors (ACTH receptors) regulate adrenalcorticosteroid synthesis, MC3 and MC4receptors in brain influence food intake andenergy expenditure, and MC5 receptorscontrol exocrine gland secretion (1).Melanocortin receptors (MC1 through MC5)are structurally related G protein-coupledreceptors that respond to agonists with anincrease in cAMP. The receptors differ intheir affinity for physiological agonists (α−,β− and γ- melanocyte stimulating hormone,ACTH) and antagonists (agouti, agouti-related protein).

Unlike other melanocortin receptors,MC2 receptors are selectively regulated byACTH. The MC2 receptor is also unusual inits requirement for an accessory protein, theMC2 receptor accessory protein (MRAP)(2). MRAP must be expressed with the MC2receptor in order for the receptor to undergoglycosylation, traffic to the plasmamembrane, bind ACTH and stimulateadenylyl cyclase (2-4). Individuals withinactivating mutations of either the MC2receptor or MRAP suffer from ACTHresistance and severe glucocorticoiddeficiency (2).

MRAP is a small protein with aconserved amino terminus, singlemembrane-spanning domain and

http://www.jbc.org/cgi/doi/10.1074/jbc.M109.022400The latest version is at JBC Papers in Press. Published on June 17, 2009 as Manuscript M109.022400

Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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nonconserved carboxyl terminus that canalso differ among splice variants. MRAPforms antiparallel homodimers, which areexceedingly rare structures, and it is thesedimers that co-precipitate with the MC2receptor (3). Mutagenic analysis hasrevealed that the C-terminal region ofMRAP is not essential, but the amino-terminal and transmembrane regions arenecessary for function (3-5). Deletion oralanine-substitution of a critical four aminoacid segment, LDYI, at residues 18-21 ofthe mouse sequence, results in an MRAPmolecule that facilitates expression of theMC2 receptor on the plasma membrane butdoes not allow the receptor to bind agonistor signal (4). MRAP is not required for cellsurface expression of other melanocortinreceptors and can inhibit expression orsignaling in some cases (3,6).

The MC5 receptor was identified onthe basis of its homology to othermelanocortin receptors and is thought to bethe ancestral ACTH (MC2) receptor (7).Analysis of MC5 receptor knockout micerevealed that the MC5 receptor is importantin controlling exocrine gland secretion (8)and behavioral responses depending onpheromones secreted by the preputial gland(9). MC2 and MC5 receptors are closelyrelated, with 46% identity and 67%homology at the amino acid level. MC2 andMC5 receptors are both found on 3T3-L1adipocytes and in some adipose tissues inanimals (10,11). Furthermore, MC2 andMC5 receptors are both expressed in adrenalcortex during embryonic development, whenthe MC5 receptor appears before the MC2receptor (12) . Mammalian MC2 and MC5receptors are activated by different pro-opiomelanocortin peptides, respondingphysiologically to ACTH and melanocytestimulating hormone, respectively. Here wedemonstrate that MC2 and MC5 receptorsare also differentially regulated by MRAP,which exerts opposite effects on surfaceexpression and dimerization of the tworeceptors.

EXPERIMENTAL PROCEDURES

Materials- hMC2, MC5 and β2-adrenergic receptors with three N-terminalHA tags and RAMP3 were obtained fromMissouri S&T cDNA Resource Center,RAMP constructs from Dr. Ian Dickerson(University of Rochester, Rochester, NY),and YFP-F1 and YFP-F2 constructs fromDr. Catherine Berlot (Weis Center forResearch, Geisinger Clinic, Danville, PA)(13). Construction of mouse MRAP andMRAP2 plasmids has been described (4).ER-tracker blue-white DPX was purchasedfrom Invitrogen (Carlsbad, CA). Antibodieswere from AbDSerotec (Kidlington, UK)(monoclonal anti-V5), Covance (Princeton,NJ) (monoclonal HA11 anti-HA), Biorad(Hercules, CA) (HRP-coupled anti-mouse),or Molecular Probes (Carlsbad, CA) (Alexa546-coupled anti-mouse). MC2 and MC5receptors with N-terminal V5 tags weremade by PCR; primer sequences areavailable on request.

Cell Culture and Transfection- CHOcells were grown in DMEM/F-12supplemented with 5% fetal bovine serum.Plasmids were transiently transfected 24hbefore experiments using Fugene HD(Roche).

Construction of Split-YFP Vectors-To make 3HA-MC5R-YFP-F1 and 3HA-MC5R-YFP-F2, 3HA-MC5R with exclusionof the stop codon was amplified from the3HA-MC5R in pcDNA3.1+ plasmid usingthe following primers: Forward:TATATATATAGCTAGCGTTTAAACTTAAGCTTGGTACC and Reverse:TATATATATAACGCGTATCCCTTCTGGGAAAGCTGCAGGCG. 3HA-MC2R-YFP-F1 and 3HA-MC2R-YFP-F2 weremade by the same strategy using thef o l l o w i n g p r i m e r s : F o r w a r d :TATATATATAGCTAGCGTTTAAACTTAAGCTTGGTACC and Reverse:TATATATATAACGCGTCCAGTACCTGCTGCAGAAGATCATC. Products anddestination vector (YFP-F1 or YFP-F2 inpciNeo) were digested with NheI and MluIand the amplified receptor product wasinserted 5’ of the coding sequence of YFP-F1 or YFP-F2. To make V5-MRAP-YFP-F2, YFP-F2 was amplified from the YFP-

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F2stop in pcDNA1 using the followingp r i m e r s : F o r w a r d :TATACGCGTAAGAACGGCATCAAGGTG a n d R e v e r s e :ATAGAATTCTTACTTGTACAGCTCGTCCAT. The product and destination vector(V5-MRAP-3Flag in pciNeo) were thendigested with MluI and NotI (removing the3 Flag epitopes and the stop codon fromMRAP). YFP-F2 was inserted after theMRAP coding region, producing V5-MRAP-YFP-F2 in pciNeo.

RT-PCR- mRNA extraction frommouse adrenal glands and Y1 cells wasperformed using the RNeasy kit and RT-PCR using the SuperScript III One-Step RT-PCR system from Invitrogen followingmanufacturer’s instructions and appropriateprimers (primer sequences available onrequest).

Surface and Total Epitope Detectionby Fixed Cell ELISA- To measure epitopeson the extracellular side of the plasmamembrane, cells in 12- or 24-well plateswere washed with PBS, fixed for 10 minwith 2% paraformaldehyde, washed,blocked in 5% nonfat dry milk in PBS,incubated with 1:5000 mouse monoclonalanti-HA or anti-V5 antibody in blockingbuffer for 2 h at room temperature, washed 3times for 5 min in PBS, and incubated withsecondary antibody and processed forELISA as described (3). The same protocolwas performed in permeabilized cells tomeasure total expression of proteins ofinterest. In this case the blocking buffer usedwas 5% milk in RIPA buffer (150 mMNaCl, 50 mM Tris, 1 mM EDTA, 10 mMNaF, 1% Triton X-100, 0.1% SDS, pH 8.0).

Live Cell Imaging. Cells on glasscoverslips were rinsed and incubated withanti-V5 antibody at 1:100 in DMEM-F12media supplemented with 5% FBS for 1 h at37°C, washed and incubated with 1:100anti-mouse antibody coupled to Alexa546for 5 min at room temperature. Whereindicated, 1 µM of ER tracker blue-whiteDPX from Invitrogen was added with thesecondary antibody. A Nikon Diaphotinverted microscope with 100x/1.3 N.A oilobjective, Photometrics CoolSNAP ES

camera and appropriate filter sets fromChroma were used. Images were capturedwith Metamorph software from UniversalImaging and transferred to Powerpoint forlabeling. Fluorescence intensitymeasurements were made using Metamorphsoftware. Micrographs displayed in a groupwere exposed and processed identically.

Immunoprecipitation and WesternBlotting. Cells were lysed with 0.1% n-dodecyl-β -maltoside, centrifuged, andsupernatants incubated with anti-HA or anti-V5 antibody at 1:5000 overnight at 4°C andimmunoprecipitates were collected onprotein A/G beads. Where noted,deglycosylation was carried out usingPNGase from NE Biolabs as recommended.Beads were washed three times, suspendedin loading buffer with 100 mMdithiothreitol, boiled 5 min and centrifuged.Proteins were resolved by SDS-PAGE on10% gels from Lonza. Routine Westernblotting was performed as previouslydescribed (3) using chemiluninescentdetection methods. For quantitation, blotswere probed with IRDye 800CW goat anti-mouse IgG from LiCor, scanned on a LiCorImaging System and analyzed usingOdyssey software.

RESULTS

Effect of MRAP on MC5 ReceptorExpression-Unlike the MC2 receptor, MC5receptor traffics readily to the plasmamembrane when expressed in CHO cells inthe absence of MRAP (3,6,14). Since MC2and MC5 receptors are expressed together inseveral systems including adipocytes (11),we investigated the effect of MRAP on theMC5 receptor using receptors epitope-tagged at the extracellular N-terminus.

To determine whether MRAPmodulates the surface expression of MC5receptor, we transfected CHO cells with aconstant concentration of HA-MC2 receptoror HA-MC5 receptor along with increasingconcentrations of MRAP-V5. Surfacereceptor was quantified by ELISA on non-permeabilized cells and MRAP expressionby ELISA in permeabilized cells in parallel

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dishes. As expected, MC2 receptorexpression at the cell surface increased withincreasing concentrations of MRAP(Fig.1A). Surprisingly, we found that MC5receptor surface expression was greatlydecreased by MRAP in a concentration-dependent manner (Fig.1B). MRAP had nosignificant effect on the surface expressionof the β2-adrenergic receptor (Fig.1C),establishing that the decrease of MC5receptor density at the plasma membrane inthe presence of MRAP is not due to celltoxicity and that this effect is specific.

To determine if this effect of MRAPon the MC5 receptor is of possiblephysiological relevance, we tested theexpression of MC2 receptor, MC5 receptor,MRAP and MRAP2 in mouse adrenalglands and in the Y1 adrenal tumor cell lineby RT-PCR. mRNAs coding for all fourproteins were detected in mouse adrenalglands and in Y1 cells (Fig.1D). When thesame experiment was performed omittingthe reverse transcriptase in order to verifythat the bands observed were due toamplification of cDNA and notcontaminating genomic DNA, noamplification was detectable (Fig.1D).

We speculated that MRAPdecreased the amount of MC5 receptor onthe plasma membrane by causing it to beretained within intracellular compartments,likely the endoplasmic reticulum (ER)and/or the Golgi apparatus. To test thishypothesis, we expressed MC2 or MC5receptor in CHO cells with or withoutMRAP, lysed the cells, immunoprecipitatedreceptor and resolved the proteins by SDS-PAGE. MC2 receptor was detected in themature glycosylated form only in thepresence of MRAP (Fig.1E), in agreementwith published findings (3). In contrast,MC5 receptor was glycosylated in thepresence or absence of MRAP (Fig.1F),showing that MRAP does not prevent post-translational glycosylation of the MC5receptor. Interestingly, in the presence ofMRAP the MC5 receptor ran slightly fasterthan it did when MRAP was not expressed.To verify that the bands described earlierwere indeed representative of glycosylated

forms of the MC5 receptor, we expressedMC5 receptor with or without MRAP,immunoprecipitated the receptor and treatedthe samples with PNGase F. Thisexperiment confirmed that MC5 receptorwas glycosylated even in the presence ofMRAP since the bands hypothesized to beglycosylated forms of MC5 receptor, in bothconditions, collapsed to a lower molecularweight band after enzymatic deglycosylation(data not shown). There are four potentialglycosylation sites on the MC5 receptor, andfaster migration of receptor could be due tothe use of fewer glycosylation sites orincomplete maturation of the carbohydratein the presence of MRAP. Alternatively,MRAP could inhibit formation of homo- orhetero-multimers, or alter an unidentifiedpost-translational modification.

Localization of Receptors in LiveCells-To localize MC2 and MC5 receptorsin live cells, we expressed the receptorsfused to a red fluorescent protein, tandem(td)-tomato, with or without MRAP.Because the td-tomato reporter contains twocopies of the fluorescent protein, it is notprone to form intermolecular dimers (15). Inthe absence of MRAP, the MC2 receptorwas retained in the cell and the fluorescencelargely coincided with a marker for the ER.When MRAP was co-expressed, MC2receptor was localized at the plasmamembrane (Fig.2A). MC5 receptordemonstrated exactly the opposite behavior.Without MRAP, the receptor was at theplasma membrane, but when MRAP was co-expressed, MC5 receptor was retainedwithin the cell (Fig.2B) and partially co-localized with the ER marker. It isinteresting that much of the MC5 receptorwas found in the ER, even though mostreceptor had undergone N-linkedglycosylation, a process that is completed inthe Golgi apparatus. These results suggestthat MRAP may lead to MC5 receptorretrieval by the ER.

The findings described abovesuggest that MRAP can, directly orindirectly, differentially regulate MC2 andMC5 receptor surface expression. Todetermine if this regulation is due to a direct

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or close interaction between receptors andMRAP, we tested whether each receptor co-immunoprecipitated with MRAP. MC2receptor co-precipitated with MRAP (Fig.3A), as reported previously (3). Moreimportantly, MC5 receptor also co-precipitated with MRAP (Fig.3A). We alsoshowed that the unrelated β2-adrenergicreceptor does not co-immunoprecipitate withMRAP to establish that the interactions ofMRAP with MC2 and MC5 receptors arespecific (Fig.3B).

To visualize receptor-MRAPcomplexes in live cells and compare theoverall localization of MC2 and MC5receptors with localization of receptor-MRAP complexes, we used bimolecularfluorescence complementation (16). Wefused an N-terminal fragment of the yellowfluorescent protein YFP (YFP-F1) to the C-terminus of MC2 and MC5 receptors and theC-terminal fragment of YFP (YFP-F2) to theC-terminus of MRAP. Since YFPfluorescence can be achieved only if the twofragments are close enough to reconstitutethe full YFP, fluorescence reports solely thelocalization of either MC2 receptor-MRAPor MC5 receptor-MRAP complexes,depending on the constructs transfected. TheMC2 receptor-MRAP complex was visiblemainly at the plasma membrane (Fig.3C),supporting the idea that MRAP isresponsible for the trafficking of the MC2receptor from the ER to the surface. On theother hand, the MC5 receptor-MRAPcomplex was localized in intracellularcompartments (Fig.3D), further suggestingthat a direct interaction of MRAP with MC5receptor is responsible for preventing thereceptor from trafficking to the plasmamembrane. We previously describedextensive control experiments to validate thebimolecular fluorescence complementationapproach in this model system (4). Weobtained negligible fluorescence when bothYFP fragments were fused to the C-terminusof MRAP (i.e. parallel dimers were notseen), or when YFP fragments were fused toeither end of MRAP and expressed with thecomplementary YFP fragments fused toRAMP3, G protein β or G protein γ

subunits. These results show that the splitYFPs do not artificially induce significantdimerization.

Effect of MRAP on ReceptorDimerization- Several of the melanocortinreceptors have been reported to formhomodimers and heterodimers (17-19). Todetermine if MC2 or MC5 receptors formhomodimers or oligomers and if suchcomplexes are affected by MRAP, we co-expressed MC2 receptor C-terminally fusedto YFP-F1 with MC2 receptor fused to YFP-F2 in the presence or absence of MRAP. Inthe absence of MRAP, the YFPfluorescence, reporting two MC2 receptorsin close proximity, was readily detectable(Fig.4A). When MRAP was expressed,fluorescence was still detectable and moreconcentrated on the plasma membrane(Fig.4B). To quantify bimolecularfluorescence complementation, we measuredthe average of YFP fluorescence intensity inall successfully transfected cells, which wereidentified by staining with antibody to theV5 epitope that was present on both RAMPand MRAP. MRAP did not alter the YFPfluorescence intensity of cells expressingMC2 receptors fused to YFP fragments(Fig.4C), nor did MRAP change the overallexpression of MC2 receptor-YFP fragmentsmeasured by ELISA in permeabilized cells(Fig.4D). These results suggest that MC2receptors form homodimers or highermultimers and that MRAP does not alter theformation of such complexes.

When the same experiment wasperformed for the MC5 receptor, YFPfluorescence was detected in the absence ofMRAP (Fig.5A) but was greatly diminishedwhen MRAP was present (Fig.5B).Quantitative analysis revealed that theaverage YFP fluorescence was significantlydecreased by MRAP (Fig.5C). Thesefindings indicate that MC5 receptors formhomodimers or oligomers in cells, and thatMRAP inhibits the formation of multimers.The overall concentration of MC5-YFPfusion proteins, again measured by ELISAin permeabilized cells, was not affected byMRAP, so the loss of fluorescence was notdue to an MRAP-mediated decrease in

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MC5-YFP fusion protein expression(Fig.5D). Furthermore, as shown above inFig.3D, the same MC5 receptor-YFP fusionprotein yielded readily measurablefluorescence when it was co-expressed withthe complementary MRAP-YFP fragment.

To confirm that MC5 receptorsdimerize and that the stability of the receptordimer is altered by MRAP, we expressedHA-MC5 receptor and V5-MC5 receptorwith or without MRAP, immunoprecipitatedthe V5-tagged receptor and measured thefraction of HA-tagged receptor co-precipitated with the V5-MC5 receptor byWestern blot. A significant fraction of theHA-MC5 receptor was immunoprecipitatedalong with V5-MC5 receptor in the absenceof MRAP (Fig.6A). In the presence ofMRAP, almost no HA-MC5 receptor co-precipitated with V5-MC5 receptor(Fig.6B). Quantitative analyses confirmedthat MRAP caused a very significantdecrease in co-immunoprecipitation of HA-MC5 receptor with V5-MC5 receptor(Fig.6C). The results of both the co-precipitation and bimolecular fluorescencecomplementation studies indicate that theMC5 receptor homodimer is destabilized byMRAP. We were able to detect dimers ofMC2 and MC5 receptors by bimolecularfluorescence complementation but wereunable to assess the effect of MRAP becausethe concentration of heterodimers was lowresulting in a very dim signal (data notshown).

Regions of MRAP Important forEffects on MC5 Receptors- To determine theregions of MRAP that are necessary toretain MC5 receptor in intracellularcompartments, we generated severaltruncation mutants and chimeras of MRAPand RAMP3 and expressed these with MC5receptor. Surface receptor concentration wasmeasured by ELISA on non-permeabilizedcells. MC5 receptor surface expression wasgreatly reduced when the receptor wasexpressed with a C-terminally truncatedform of MRAP (MRAPΔc). Surface MC5receptor was also decreased by an MRAPchimera where the transmembrane domainof MRAP was replaced by the

transmembrane region of RAMP3, anaccessory protein that does not regulatemelanocortin receptor trafficking.Importantly, deletion of the amino acids 31to 37, which are responsible for the dualtopology of MRAP (4), did not prevent thenegative effect of MRAP on surfacelocalization of the MC5 receptor. Finally,deletion of the first 30 amino acids ofMRAP (MRAPΔ1-30) completely abolishedthe effect of MRAP on MC5 receptortrafficking (Fig.7A). The relative amounts ofMRAP and RAMP3 proteins were measuredon Western blots (Fig.7B). The lack ofeffect of MRAPΔ1-30 on MC5 receptordimerization was not due to poor expression,because this mutant was expressed atcomparable or higher levels than wild typeMRAP (Fig.7B). In fact, the mutant that wasexpressed at the lowest concentration wasMRAPΔc, which still significantly inhibitedsurface expression of the MC5 receptor.

These results demonstrate that theconserved N-terminus of MRAP is requiredfor MRAP to inhibit MC5 receptorlocalization on the plasma membrane.Surprisingly, the unusual dual topology ofMRAP, which appears to be essential for itsinteractions with the MC2 receptor, does notseem to be necessary for MRAP to restrictMC5 receptor movement to the cell surface.The newly identified mouse MRAP2protein, which can promote surfaceexpression of MC2 receptor but not efficientACTH signaling, acts like MRAP with theMC5 receptor, preventing its surfaceexpression (4).

We used the bimolecularfluorescence complementation approach totest if there was a correlation between theability of MRAP, MRAP mutants andMRAP2 to prevent MC5 receptor traffickingand prevent receptor dimerization. Toquantify MC5 receptor dimer formation, wemeasured YFP fluorescence intensity incells expressing MC5R-YFP-F1 and MC5R-YFP-F2 together with V5-tagged RAMP3,MRAP, MRAP mutant or MRAP2. Asdescribed earlier, the average YFPfluorescence intensity of transfected cells, asdetermined by surface staining with an anti-

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V5 antibody, was determined (Fig.7C). Inthe presence of RAMP3, YFP fluorescencewas readily detectable, confirming thatRAMP3 does not interfere with MC5receptor homodimerization. When MRAP,M R A PΔ31-37, MRAP(RAMP3TM),MRAPΔc or MRAP2 was present, YFPfluorescence was significantly decreased,consistent with a decrease in MC5 receptorhomodimer formation. Again, MRAPΔ1-30did not inhibi t MC5 receptorhomodimerization. Comparable results wereobtained when the intensity of YFPfluorescence in individual cells wasanalyzed by a fluorescence-activated cellsorter (data not shown). These results showthat there is a direct correlation between theability of MC5 receptor to homodimerizeand its presence at the plasma membrane,suggesting that MC5 receptor monomersmay be trapped intracellularly.

DISCUSSION

The observations described hereexpand the known functions of accessoryproteins in melanocortin receptor biology.We have shown that MRAP hasdiametrically opposed effects on thestructurally similar MC2 and MC5receptors. The impact of MRAP issubstantial. The MC2 receptor requiresMRAP in order to traffic to the plasmamembrane, while MRAP prevents the MC5receptor from reaching the cell surface.When MC2 and MC5 receptors wereexpressed together in the same cells, MRAPstimulated surface expression of MC2receptors and trapped MC5 receptorsintracellularly, so one action does notpredominate (data not shown). The absenceof receptor on the cell surface with orwithout MRAP (MC5 and MC2 receptors,respectively) precludes analysis of theinfluence of MRAP on the structure-activityprofile of either receptor. The concentrationsof MRAP required for these opposite actionsare the same.

MRAP did not modify the surfaceexpression of the β2-adrenergic receptor(Fig.1C), another Gs-coupled class AGPCR, suggesting that the action of MRAPon MC2 and MC5 receptors is specific.Individuals lacking MRAP protein havesevere glucocorticoid deficiency resultingfrom resistance to ACTH (2) but noadditional problems that might indicate arole for MRAP in other receptor systems.The lack of any noticeable MC5 receptor-related phenotype in patients lacking MRAPcould be explained by the presence ofMRAP2 since the latter is, like MRAP, ableto regulate surface expression of the MC5receptor. The potential redundancy ofMRAP and MRAP2 will make it difficult todetermine which GPCRs are regulated bythese accessory proteins until knockoutanimals lacking MRAP, MRAP2 and bothare available.

It is not known what characteristicsmake the MC2 receptor dependent onMRAP for trafficking when the other fourmelanocortin receptors can function withoutit. The transmembrane regions of themelanocortin receptors are quitehomologous and in several cases have beeninterchanged with little effect on receptorexpression, ligand affinity or cAMPresponses (20,21). The effects of MRAP onboth MC2 and MC5 receptors require theconserved MRAP aminoterminus, butbecause MRAP has dual topology it isunclear whether this domain interacts withMC2 receptor from the exoplasmic orcytoplasmic side of the membrane. For theMC5 receptor, however, our results suggestthat the N-terminal region of MRAPinteracts with one of the extracellulardomains of the receptor since theexclusively Nexo-Ccyto mutant MRAPΔ31-37strongly inhibits MC5 receptor surfaceexpression.

MRAP co-precipitates with bothMC2 and MC5 receptors, and formscomplexes in which YFP fragments are ableto interact, implying direct or at least veryclose interactions. MRAP interactions withthe MC2 receptor have at least twoconsequences: more efficient receptor

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trafficking to the plasma membrane, andincreased ligand binding and signaling.Receptor trafficking may be secondary toproper receptor folding facilitated by MRAPserving as a chaperone, and high affinityligand binding may be secondary tointeraction of the MRAP-MC2 receptorcomplex with G proteins, which increasesaffinity of receptor agonists. With the MC2receptor, the trafficking function of MRAPrequires dual topology and the MRAPtransmembrane domain, but is retained inMRAP mutants missing much of theaminoterminus. The ability of MRAP toenhance ACTH binding to the MC2 receptorrequires four amino acids from residues 18-21, LDYI (4). In contrast, MRAP inhibitionof MC5 receptor trafficking does not dependon the transmembrane domain of MRAP,dual topology, the LDYI motif (missing inMRAP2) (4) or the C-terminal region, but itdoes require the N-terminal region.

Several lines of evidence show thatMRAP powerfully inhibits formation ofMC5 receptor dimers. MRAP reducesbimolecular fluorescence complementationbetween MC5 receptor-YFP fragment fusionproteins. In addition, MRAP reduces co-precipitation of MC5 receptors tagged withdifferent epitopes. The MC2 receptorappears to dimerize equally well with orwithout MRAP. There is no direct evidencethat melanocortin receptors must dimerize totraffic to the cell surface, but homo- andhetero-dimerization of class A GPCRs iswell documented and MC1 and MC3receptors have been shown to form homo-and heterodimers (17-19). In general,dimerization of GPCRs occurs in the ER atearly stages in receptor biosynthesis, anddimerization explains dominant negativeeffects of some naturally occurring receptormutations (22,23), including several in themelanocortin receptor family (24). The datadescribed here can be explained if MRAPdisrupts a critical dimerization step in MC5

receptor maturation and trafficking. There isa perfect correlation between the ability ofdifferent MRAP mutants and MRAP2 todisrupt MC5 receptor dimerization and toprevent receptor trafficking to the plasmamembrane. Interestingly, heterodimerizationof µ and δ opioid receptors is promoted byRTP4 (25), a member of the RTP family ofaccessory proteins that facilitate odorant andtaste receptor expression (26,27).

Because suitable antibodies are notavailable, there is little information aboutwhere MRAP, MRAP2, MC2 and MC5receptor proteins are expressed in vivo.Analysis of mRNA levels by Northern blot,RT-PCR and in situ hybridization suggeststhat both receptors are expressed in certainadipose tissues, immune system cells, andadrenal cortex, although it is not clear if theyreside in the same cells. We showed that themRNA coding for all four proteins is presentin normal mouse adrenal glands as well as inthe tumor-derived Y1 adrenal cell line,demonstrating that MRAP, MRAP2, MC2and MC5 receptor may be expressedtogether under physiological conditions.Endogenous agonists and antagonistsregula te melanocor t in receptorsphysiologically. Such dual regulation is veryrare, even in the large and diverse GPCRfamily. Another unusual feature of themelanocortin system is the ability ofdifferent peptides derived from a commonprecursor, pro-opiomelanocortin, to activatethe five receptors with varying degrees ofselectivity. The work described here showsthat an increase in MRAP expression willfavor responsiveness to ACTH and decreaseresponsiveness to melanocyte stimulatinghormone in any tissue expressing MC2 andMC5 receptors, while a fall in MRAP willshift responses in the opposite direction. Inthis way the accessory proteins MRAP andMRAP2 add a new dimension to themelanocortin signaling systems.

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FOOTNOTES

*To whom correspondence should be addressed: Dept. of Pharmacology and Physiology,University of Rochester Medical Center, Box 711, Rochester, NY 14642. Tel.: 585-275-4933;

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Fax: 585-273-2652; E-mail: Patricia_Hinkle@urmc.rochetser.edu. This work was supported byNational Institutes of Health (NIH) Grant DK19974 (to P.M.H.).

1The abbreviations used are: ACTH, adrenocorticotropic hormone; ER, endoplasmicreticulum; GPCR, G protein-coupled receptor; MC2, melanocortin-2; MC5, melanocortin-5;MRAP, MC2-receptor accessory protein; RAMP, receptor activity modifying protein; YFP,yellow fluorescent protein.

FIGURE LEGENDS

Figure 1: Regulation of surface MC2 and MC5 receptors by MRAP. Surface expression of A.HA-MC2, B. HA-MC5 or C. HA-β2-adrenergic receptor (β2ADR) measured by ELISA in non-permeabilized CHO cells using anti-HA antibody, as a function of the total expression of MRAP-V5 measured in parallel dishes by ELISA in permeabilized cells using anti-V5 antibodies. D.Detection of MC2R, MC5R, MRAP and MRAP2 mRNA in mouse adrenal glands or mouse Y1adrenal cell line mRNA extracts by RT-PCR. Cells expressing E. HA-MC2 or F. HA-MC5receptor in the presence or absence of MRAP-V5 were lysed, and receptors wereimmunoprecipitated with anti-HA antibody and resolved on SDS-PAGE. Blots were probed withanti-HA antibody. * IgG heavy chain.

Figure 2: MRAP-dependent MC2 and MC5 receptor localization. A. MC2 and B. MC5receptors fused to the red fluorescent protein td-Tomato were expressed with or without MRAPand imaged in live CHO cells expressing either receptor in the presence or absence of MRAP. ERwas stained with ER-tracker.

Figure 3: MRAP can interact and co-localize with both MC2 and MC5 receptors. A and B.CHO cells expressing MRAP-V5 together with A. HA-MC2 or HA-MC5 receptor or B. HA-β2-adrenergic receptor were lysed and receptors were immunoprecipitated with anti-HA antibodyand MRAP with anti-V5 antibody. Proteins were resolved on SDS-PAGE and receptors wereprobed with anti-HA antibody. C. Live CHO cells transfected with MC2 receptor-YFP-F1 andMRAP-YFP-F2. D. Live CHO cells transfected with MC5 receptor-YFP-F1 and MRAP-YFP-F2.* IgG heavy chain.

Figure 4: Effect of MRAP on MC2 receptor homodimerization. Live CHO cells expressingHA-MC2 receptor-YFP-F1 and HA-MC2 receptor-YFP-F2 A. without or B. with MRAP-V5.YFP fluorescence is shown in green (left panel). Surface MRAP in the same cells detected withanti-V5 antibody and secondary anti-mouse Alexa546 is shown in red (right panel). C. AverageYFP fluorescence intensity was measured in five or more randomly selected fields. Allsuccessfully transfected cells, identified by staining with anti-V5 antibody, were analyzed (25-88cells/condition). YFP fluorescence reports the formation of receptor homodimers. Shown aremean ± SE in relative fluorescence units (RFU). D. Total expression of HA-MC5 receptor-YFP-F1 and HA-MC5 receptor-YFP-F2 without or with MRAP-V5 measured by ELISA inpermeabilized cells using anti-HA antibody.

Figure 5: Effect of MRAP on MC5 receptor homodimerization measured by bimolecularfluorescence complementation. A and B. Live CHO cells expressing HA-MC5 receptor-YFP-F1and HA-MC5 receptor-YFP-F2 A. without or B. with MRAP-V5. YFP fluorescence is shown ingreen (left panel). Surface MRAP in the same cells detected with anti-V5 antibody and secondaryanti-mouse Alexa546 is shown in red (right panel). C. Average YFP fluorescence intensity inrelative fluorescence units (RFU). D. Total expression of HA-MC5 receptor-YFP-F1 and HA-

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MC5 receptor-YFP-F2 without or with MRAP-V5. YFP fluorescence and total expression ofreceptors was quantified as described in the legend to Fig.4.

Figure 6: Effect of MRAP on MC5 receptor homodimerization measured by co-immunoprecipitation. A and B. Cells expressing HA-MC5 receptor and V5-MC5 receptor A.without or B. with MRAP were lysed and MC5 receptor was immunoprecipitated with anti-HA oranti-V5 antibody. Proteins were resolved by SDS-PAGE and HA-MC5 receptor was detectedwith anti-HA antibody. * IgG heavy chain. C. Quantitative analysis of HA-MC5 receptor and V5-MC5 receptor co-immunoprecipitation in the presence or absence of MRAP was performed asdescribed in Methods. It is expected that only a fraction of MC5 receptor dimers will have HA/V5tags and be visualized, while others will have HA/HA or V5/V5 tags. The mean ± SE from threeseparate experiments are shown. * p<0.01 compared to control.

Figure 7: Regions of MRAP required for regulation of MC5 receptor homodimerizationand trafficking. A. Surface HA-MC5 receptor was measured by ELISA on non-permeabilizedCHO cells transfected with HA-MC5 receptor alone or with RAMP3 or the noted MRAPs. Bargraph represents the mean ± SE of three separate experiments, each performed in duplicate ortriplicate, for surface expression of MC5 receptor normalized to control expression in thepresence of RAMP3. B. Western blot analysis of RAMP3, MRAP, MRAP mutants or MRAP2expression in cell lysates from parallel dishes in the experiment depicted in A. All of theaccessory proteins were tagged with an N-terminal V5 epitope and made in the same pCI-Neovector. C. YFP fluorescence intensity in live CHO cells expressing HA-MC5 receptor-YFP-F1and HA-MC5 receptor-YFP-F2 with V5-tagged RAMP3 or the noted MRAPs, analyzed asdescribed in the legend to Fig.4. Sequences of MRAP mutants have been reported previously(3,4). * p<0.05 compared to control.

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Figure 1

D

MC

2R

MC

5R

MR

AP

MR

AP

2

No RT

Y1Adrenal

Cells

MouseAdrenalGlands No RT

0 0.2 0.4 0.6 0.8 10

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Total MRAP (V5 A450)

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Total MRAP (V5 A450)

Sur

face

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Total MRAP (V5 A450)

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β2 A

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ImmatureMC2R

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MC5R

ImmatureMC5R

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+-

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Figure 2

MC2R

MC5R

Con

trol

MR

AP

Con

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MR

AP

ER Overlay

ER Overlay

A

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Figure 3

C

MRAP-YFP-F2

MC2R-YFP-F1

YFP-F1YFP-F2

YFP-F2

MC2R-MRAP Dimers

MRAP-YFP-F2

MC5R-YFP-F1

YFP-F1YFP-F2

YFP-F2D MC5R-MRAP Dimers

β2ADR

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HA-β2ADRMRAP-V5 M

ock

*

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k HA-MC2RMRAP-V5

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HA

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Figure 4

C

MC2R

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P3

MC2R

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RAP0

100

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+

0.0

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MC2R Homodimers(YFP)

MRAP(V5)

MC2R-YFP-F1

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MRAP-V5

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YFP-F1YFP-F2

MC2R-YFP-F2B

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Figure 5

C

MC5R

/ RAM

P3

MC5R

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RAP0

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)

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Figure 6

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Figure 7

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Julien A. Sebag and Patricia M. HinkleMC5 receptor dimerization and trafficking

Opposite effects of the melanocortin-2 receptor accessory protein MRAP on MC2 and

published online June 17, 2009J. Biol. Chem. 

  10.1074/jbc.M109.022400Access the most updated version of this article at doi:

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