Molecular and Cellular Endocrinology 361 (2012) 69–79

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Molecular and Cellular Endocrinology

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Melanocortin 5 receptor signaling and internalization: Role of MAPK/ERKpathway and b-arrestins 1/2

Adriana R. Rodrigues a,b, Henrique Almeida a,b, Alexandra M. Gouveia a,b,c,⇑a Department of Experimental Biology, Faculty of Medicine, Universidade do Porto, Portugalb IBMC- Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugalc Faculty of Nutrition and Food Sciences, Universidade do Porto, Portugal

a r t i c l e i n f o

Article history:Received 17 October 2011Received in revised form 19 March 2012Accepted 19 March 2012Available online 28 March 2012

Keywords:Melanocortin receptorsERK1/2G-protein coupled receptor (GPCR)b-arrestins

0303-7207/$ - see front matter � 2012 Elsevier Irelanhttp://dx.doi.org/10.1016/j.mce.2012.03.011

Abbreviations: CREB, cAMP-responsive element bilular-regulated protein kinase; GAPDH, glyceraldehnase; GFP, green fluorescent protein; GPCR, G-prohorseradish peroxidase; MC5R, melanocortin 5 recreceptors; MSH, melanocyte-stimulating hormone;activated protein kinase 1; p90RSK, 90-kDa ribosomkinase A; PKC, protein kinase C.⇑ Corresponding author at: Department of Exper

Medicine, Universidade do Porto, 4200-319 Porto, Portfax: +351 22 551 36 55.

E-mail address: agouveia@med.up.pt (A.M. Gouvei

a b s t r a c t

The Melanocortin 5 receptor (MC5R) is a G-protein coupled receptor (GPCR) that exhibits high affinity fora-MSH. Here we present evidence for MC5R-GFP internalization and subsequent recycling to cell surface,in a-MSH-stimulated HeLa cells. This melanocortin induces a biphasic activation of ERK1/2 with an earlypeak at 15 min, a Gi-protein driven, b-arrestins 1/2 independent process, and a late sustained activationthat is regulated by b-arrestins 1/2. ERK1/2 lead to downstream phosphorylation of 90-kDa ribosomal S6kinases (p90RSK) and mitogen- and stress-activated protein kinase 1 (MSK1). Only a small fraction (10%)of phosphorylated p90RSK and ERK1/2 translocates to the nucleus inducing c-Fos expression. a-MSH alsoactivates CREB through cAMP/PKA pathway. In 3T3-L1 adipocytes, where MC5R is endogenouslyexpressed, a-MSH also induces phosphorylation and cytosolic retention of the same signaling molecules.These findings provide new evidence on the signaling mechanisms underlying MC5R biological responseto a-MSH.

� 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction induce lipolysis (Cho et al., 2005; Moller et al., 2011). Since there is

Melanocortins are neuropeptides derived from pro-opiomelano-cortin (POMC), a polypeptide that upon processing originates theadrenocorticotropic hormone (ACTH), a, b and c-melanocyte stim-ulating hormone (MSH) (Mountjoy, 2010), which are then recog-nized by a family of five melanocortin receptors (MCRs) termedMC1R to MC5R (Cooray and Clark, 2011). MC5R was the last mela-nocortin receptor to be cloned and although the precise functionalrole of this receptor is still unclear it seems to be related with theperipheral regulation of lipid metabolism. MC5R activation wasdirectly implicated in the stimulation of fatty acid oxidation inskeletal muscle (An et al., 2007) and MC5R null mice show adown-regulation of sebaceous lipid secretion, leading to a defectin water repulsion and thermoregulation (Chen et al., 1997).Furthermore, in 3T3-L1 adipocytes, a-MSH and other melanocortins

d Ltd. All rights reserved.

nding protein; ERK, extracel-yde-3-phosphate dehydroge-tein coupled receptor; HRP,eptor; MCRs, melanocortin

MSK1, mitogen- and stress-al S6 kinases; PKA, protein

imental Biology, Faculty ofugal. Tel.: +351 22 551 36 54;

a).

evidence that this cell line expresses only MC2R and MC5R (Normanet al., 2003; Boston and Cone, 1996), and MC2R has specificity forACTH binding (Cooray and Clark, 2011), a-MSH effect on lipolysisshould be mediated by MC5R.

MC5R belongs to the large family of GPCRs, plasma membranereceptors with seven transmembrane domains. After beingactivated by specific agonists, GPCRs trigger G proteins-dependentand independent signaling pathways (Woehler and Ponimaskin,2009) and, subsequently, most of them are desensitized byinternalization, primarily via clathrin-coated pits (Jean-Alphonseand Hanyaloglu, 2011). Usually this process involves thephosphorylation of the receptors by members of the GPCR kinasefamily and also the recruitment of cytoplasmic b-arrestins 1 and2 (Jean-Alphonse and Hanyaloglu, 2011). Although the mechanismof GPCRs internalization is commonly linked to desensitization,some receptors continue to signal when integrated in endosomesmainly in complex with b-arrestins, which activate MAPK signalingmolecules (Calebiro et al., 2010). Once internalized, GPCRs can berecycled back to the plasma membrane, targeted to lysosomes tobe degraded or retained into endosomes. Thus, the final destinationof GPCRs has also important functions in signal extinction orpropagation.

The MCR trafficking mechanisms have been intensively studiedin last years. It was shown that the cell surface targeting of MC5R isnegatively regulated by the MC2 receptor accessory protein(MRAP), which is required for MC2R expression at cell membrane

70 A.R. Rodrigues et al. / Molecular and Cellular Endocrinology 361 (2012) 69–79

(Chan et al., 2009; Sebag and Hinkle, 2009). MC5R localizes at theplasma membrane in the absence of MRAP, but is trapped intracel-lularly when co-expressed with this accessory protein (Chan et al.,2009; Sebag and Hinkle, 2009). Regarding MC5R internalizationmechanisms, there is only one report referring that the receptoractivation leads to b-arrestins recruitment to plasma membraneand receptor internalization occurs by a clathrin-dependent endo-cytosis process (Cai et al., 2006).

The signaling mechanisms following MC5R activation are notcompletely understood but in skeletal muscle, PKA and AMP-acti-vated protein kinase were implicated in the regulation of fatty acidoxidation (An et al., 2007). In lymphocytes, a-MSH binding to MC5Ractivates the Jak/Stat pathway (Buggy, 1998) and in HEK293 stablyexpressing the receptor it increases the production of cAMP and cal-cium (Hoogduijn et al., 2002; Mountjoy et al., 2001). We also haveshown that in HEK293 cells stably expressing MC5R-GFP, the recep-tor uses a specific transduction pathway to activate ERK1/2 inde-pendently from adenylyl cyclase, PKA, PKC and Akt/PKB pathwaysbut involving PI3K (Rodrigues et al., 2009). MC3R also induces aPI3K-dependent ERK1/2 phosphorylation independently from PKAand PKC but exhibits alterations on Akt phosphorylation (Nyanet al., 2008); MC4R promotes a PI3K-dependent ERK1/2 phosphory-lation but requires PKC activation (Cai et al., 2007). MC1R triggersthe ERK pathway by receptor tyrosine kinase transactivation,independently of cAMP and PKC (Herraiz et al., 2011). DifferentMCRs share the same agonist (a-MSH), so a distinct intracellularnetwork seems to define the specificity and function of each MCR,in which ERK1/2 seems to play a pivotal role.

The present study is aiming to comprehensively investigate thesignaling pathways promoted by a-MSH-activated MC5R and ad-dress the regulation of the receptor internalization/desensitizationmechanisms.

2. Materials and methods

2.1. Materials

DMEM/Ham’s F-12 and DMEM media, glutamine, insulin, fetalbovine and fetal calf serum (FBS and FCS, respectively) werepurchased from Biochrom AG. 3-isobutyl-methyl-xantine (IBMX),a-MSH, dexamethasone, monensin sodium, pertussis toxin (PTX),cholera toxin (CTX), forskolin, phosphatase and protease inhibitorcocktails and siRNA oligonucleotides were obtained from Sigma–Aldrich. MEK1/2 inhibitors PD98059 and U0126 were purchasedfrom Calbiochem and Cell Signaling, respectively. Rp-adenosine-3’,5’-cyclic mono-phosphorothioate triethylamine salt (Rp-cAMP)was from Santa Cruz. PKC inhibitor 2-[1-(3-dimethylaminopro-pyl)-1H-indol-3-yl]-3-(1H-indol-3-yl) maleimide (GF109203X)was obtained from Calbiochem. Lipofectamine 2000 was fromInvitrogen.

2.2. HeLa cells culture and transfection

The pMC5R–EGFP vector was obtained as previously described(Rodrigues et al., 2009). HeLa cells were cultured in DMEM/Ham’sF-12 medium supplemented with 10% FBS and 2 mM glutamineuntil 70–80% confluence was reached. Then, cells were transfectedwith pMC5R–EGFP or pEGFP using Lipofectamine 2000, as recom-mended by the manufacturer, and selected for outgrowth of iso-lated colonies using 0.6 lg/ll Geneticin (GIBCO). Two coloniesderived from independent transfection experiments were picked,after screening by inverted fluorescence microscopy. Oneclone was used in all the experiments presented in the paper. Thecharacterization of the second clone involved the study of ERK1/2

activation by a-MSH in order to guarantee that observations werenot clone specific.

2.3. 3T3-L1 differentiation

3T3-L1 pre-adipocytes were grown to confluence in DMEM sup-plemented with 10% FCS and 2 mM glutamine. Two days later, adi-pogenesis was induced by incubation with DMEM containing 10%FBS, 10 lg/ml insulin, 250 nM dexamethasone and 0.5 mM IBMX.After 3 days, the medium was replaced to DMEM, FBS and insulinfor further 2 days. Cells were then allowed to differentiate for 7more days in medium supplemented only with FBS.

2.4. Decrease of b-arrestins 1/2 function

siRNA sequences targeting human b-arrestin 1 and 2 and GAP-DH were already described (Shenoy et al., 2006; Gong et al., 2008).HeLa cells stably expressing MC5R-GFP (2.5 � 105 cells) were pla-ted in 6-well dishes and 24 h later were transfected with 60 nMb-arrestin 2 and GAPDH siRNAs and 6 nM b-arrestin 1 siRNAs usingLipofectamine 2000, according to the manufacturer’s protocol.Forty-eight hours later, cells were split into 24-well dishes andserum-deprived during 16 h. All assays were performed 72 h aftersiRNA transfection. Silencing levels were confirmed by b-arrestin1/2 immunoblotting detection using a mouse anti-b-arrestin 1antibody (BD Biosciences) that cross-reacts with b-arrestin 2 (Luoet al., 2008; Fernandez et al., 2008).

The interference on b-arrestin 1 and 2 function was also ob-tained by transfecting HeLa cells stably expressing MC5R-GFP withdominant negative b-arrestin 1 and 2 mutants (delta-LIELD-F391Aand pcDNA3-b-arrestin 2 290–409, respectively) and with bothmutants. Cells transfected with the pcDNA3.1 vector were usedas control. All assays were performed 48 h after transfection. Thedominant negative b-arrestin 1 and 2 mutants were kindly pro-vided by Dr. Jeffrey Benovic (Department of Biochemistry andMolecular Biology, Thomas Jefferson University) and Dr. Jean-LucParent (Rheumatology Division of the Faculty of Medicine at Uni-versity of Sherbrooke), respectively.

2.5. Cell treatment and preparation of cellular extracts

HeLa cells expressing MC5R-GFP and 3T3-L1 differentiated adi-pocytes were serum starved 16 h prior to stimulation with 1 lM a-MSH for different periods of time. The concentration of a-MSHused was selected after ERK1/2 activation analysis by western-blotting of a dose–response experiment (Supplementary Fig. 1).Thirty minutes before a-MSH stimuli, cells were treated withPD98059 (100 lM) and UO126 (10 lM) for ERK1/2 signaling inhi-bition, with PKC inhibitor GF109203X (5 lM) and with the PKAblocker Rp-cAMP (10–100 lM). Cells incubated with PdBU(1 lM) and Forskolin (10 lM), during 15 min, were used asGF109203X and Rp-cAMP positive controls, respectively. The spe-cific inhibition of G protein isoforms Gas and Gai was carried outby incubation with CTX (100 ng/ml) and PTX (100 ng/ml), respec-tively, overnight before a-MSH stimulus. Although CTX is acAMP-elevating agent when administered for a short period oftime, CTX sustained treatment results in a Gs down-regulationand, consequently, in a decline of cAMP levels (Lin et al., 1993;Mizuno et al., 2002). The selection of the proper inhibitors concen-trations was based on the product datasheets and on the literature(Nishihara et al., 2004; Fricke et al., 2004). Cells were then washedwith ice-cold PBS and solubilized in lysis buffer (50 mM Tris–HClpH 7.6, 10 mM NaCl, 5 mM EDTA, 1 mM b-glycerophosphate, phos-phatase and protease inhibitor cocktails) containing 0.25% TritonX-100. Lysates were sonicated and protein concentrations weredetermined using the Bradford protein assay (BioRad).

A.R. Rodrigues et al. / Molecular and Cellular Endocrinology 361 (2012) 69–79 71

2.6. Western-blotting analysis

Twenty micrograms of cell lysates were used for immunoblot-ting as described previously (Rodrigues et al., 2009). Specific pri-mary antibodies were used for detection of phosphorylated formsof c-Raf, MEK1/2, ERK1/2, p90RSK, MSK1 (1:1000, Cell Signaling)and CREB (1:1000, Millipore) and also expression of c-Fos(1:10,000, Abcam), lamins A/C (1:600, Santa Cruz) and a-tubulin(1:10,000, Sigma). Chemiluminescent detection was performedusing the SuperSignal West Pico reagent (Pierce) after incubationwith a secondary HRP-conjugated antibody (1:5000, JacksonImmunoResearch). After detection of phosphorylated ERK1/2,membranes were stripped with 10% SDS for 30 min at room tem-perature and then incubated with anti-ERK1/2 antibody (1:1000,Cell Signaling).

2.7. Subcellular fractionation

After a-MSH treatment, cells were washed with ice-cold PBS,gently scrapped in lysis buffer containing 0.1% Triton X-100 and dis-rupted in a glass-Teflon homogenizer. Half volume of total-cell ex-tracts was centrifuged at 1300g for 10 min. Equivalent amounts oftotal, cytoplasmic (supernatant) and nuclear (pellet) fractions andmultiple equivalent amounts of nuclear extracts (5 and 10-fold)were subjected to western-blotting analysis as described above.

2.8. cAMP assay

After serum starvation, HeLa cells stably expressing MC5R-GFPwere treated for 15 min with 1 lM a-MSH in the presence of IBMX(phosphodiesterase inhibitor), after overnight incubation with CTX(100 ng/ml), PTX (100 ng/ml) or vehicle. The medium was re-moved, cells were immediately lysed and intracellular cAMP wasquantified following the manufacturer instructions using thecAMP-Screen� System (Applied Biosystems).

2.9. Biotinylation of cell surface proteins and quantification of MC5R-GFP levels at the plasma membrane

HeLa cells stably expressing MC5R-GFP or GFP alone, with orwithout b-arrestins 1/2 or GAPDH siRNA silencing, were grownin 24 well plates until 80–90% confluence. After serum-deprivation,cells were stimulated with 1 lM a-MSH for several time-points.Cells were then washed with ice-cold PBS and labeled with0.4 mM EZ-Link Sulfo-NHS-SS-Biotin (Pierce), a membrane-imper-meable biotinylation reagent, for 30 min at 4 �C. The cross-linkerexcess was quenched with TBS. Cells were subsequently scrapedin lysis buffer containing 0.25% Triton X-100 and total protein con-centration was determined by Bradford assay (BioRad). Five hun-dred nanograms of total protein, supplemented with 0.5% sodiumdeoxycholate plus 1% Triton X-100, were incubated in neutravidincoated plates (Pierce). After binding of biotin-labeled proteins,MC5R-GFP was detected using an anti-GFP primary antibody(1:2000, Abcam) and a HRP-conjugated secondary antibody(1:15,000, Jackson ImmunoResearch). The signal was revealed bychemiluminescence and captured using a microplate reader (Te-can). The signal obtained from HeLa cells stably transfected withGFP alone was considered as background. MC5R levels at cell sur-face, after a-MSH stimuli, were determined as a ratio related to anon-stimulated condition, after subtraction of background values.

2.10. Fluorescence microscopy

HeLa cells stably transfected with MC5R-GFP were cultured onglass coverslips. Thirty minutes before stimulation with 1 lMa-MSH, cells were treated with vehicle, 10 lM monensin,

300 mM sucrose or subjected to potassium depletion as describedelsewhere (Vercauteren et al., 2010). For the recycling experi-ments, monensin-treated cells were stimulated with a-MSH for30 min and, thereafter, the agonist was removed by washing andcells were further incubated in agonist-free medium until 120and 360 min. Then cells were fixed with 4% paraformaldehydeand permeabilized for 5 min with 1% Triton X-100. Immunodetec-tion of c-Fos was carried out by incubating cells with the anti-c-Fosantibody (1:1500, Abcam) overnight at 4 �C and a secondary anti-body Alexa�568 (Molecular Probes), after blocking with 5% bovineserum albumin. Nuclei were counterstained with 40,6-diamidino-2-phenylindole (DAPI) and the images were captured with anApoTome fluorescence microscope (Zeiss).

2.11. Data analysis

Digital images of Western-blots were analyzed by densitometryusing Scion Image. Each result represents the mean ± standarddeviation of at least three independent experiments. All data wereanalyzed using the two-tailed Student’s t-test.

3. Results

3.1. MC5R-GFP internalization and recycling in HeLa cells

HeLa cells stably expressing MC5R fused to GFP at the C-termi-nus were obtained. These cells have no endogenous expression ofMCRs and therefore the cross-reactions between a-MSH and thediverse MCRs are avoided in this system. Moreover, a GFP-taggedversion of the receptor allows the study of MC5R trafficking, an is-sue that has been compromised by the lack of specific antibodies.

As demonstrated in Fig. 1A, MC5R-GFP efficiently targets to cellsurface and internalizes after sustained exposure to a-MSH (Mock,0 and 120 min) resulting in a noteworthy accumulation of thereceptor in intracellular vesicles after 2 h of stimulus. MC5R inter-nalization is thought to occur via clathrin-dependent endocytosis(Cai et al., 2006) and, in fact, when HeLa cells over-expressingMC5R-GFP are subjected to hyperosmotic sucrose or potassiumdepletion conditions, prior to a-MSH stimulus, the receptor is effi-ciently retained at cell surface (Fig. 1A, Sucrose and K+ depletion).Hyperosmotic sucrose and potassium depletion are said to inhibitclathrin-dependent endocytosis by preventing clathrin and adapt-ors from interacting or by dissociating the clathrin lattices at theinner leaflet of the plasma membrane, respectively (Hansen et al.,1993; Heuser and Anderson, 1989; Larkin et al., 1986).

Direct evidence of MC5R-GFP recycling was achieved by fluo-rescence analysis of GFP-tagged receptor localization upon re-moval of the agonist after 30 min of stimulation. As shown inFig. 1B (Mock), the amount of MC5R-GFP at cell surface is clearlyincreased at 120 and 360 min after removal of the agonist, indicat-ing that the internalized receptor shuttles back to the plasmamembrane. As shown in Fig. 1B (Monensin) the recycling ofMC5R-GFP is completely abolished by monensin, an acidotropicionophore that raises pH within endosomes and inhibits recyclingof internalized proteins to the plasma membrane (Basu et al., 1981;Mollenhauer et al., 1990). Monensin also disrupts the function oftrans-Golgi apparatus cisternae and probably for this reason, innon-stimulated monensin-treated cells (Fig. 1A, Monensin,0 min), MC5R-GFP was already detected in intracellular vesicles.In addition this observation could be justified by monensin impair-ment of the constitutive receptor recycling that occurs in basalconditions.

The quantification of MC5R-GFP levels at cell surface, which is aresult of both internalization and recycling mechanisms, was as-sessed by cell surface ELISA experiments. In brief, after a-MSH

Fig. 1. MC5R-GFP internalization and recycling. A, Fluorescence microscopy of HeLa cells stably expressing MC5R-GFP before (0 min) or after 1 lM a-MSH stimulation for 15and 120 min. Prior to a-MSH addition, cells were incubated with 10 lM monensin (monensin), 300 mM sucrose (sucrose), potassium depletion buffer (K+ depletion) orvehicle (Mock) during 30 min (scale bar – 10 lM). B, Mock and monensin-treated cells were incubated with a-MSH during 30 min and, thereafter, cells were washed in orderto remove the agonist and incubated with agonist-free medium for a further 90 and 330 min (120 and 360 min, respectively) (scale bar – 10 lM). C, MC5R-GFP levels at cellsurface were determined after a-MSH treatment during 15, 30, 60, 90 min and 2, 3, 4, 5, 6 h by ELISA quantification of biotinylated receptor at plasma membrane. Data werenormalized to MC5R-GFP levels at cell surface on control cells (non-stimulated cells; 0 min), which was considered 100%. The results represent the mean ± standard deviationof at least three independent experiments. All groups statistically differ from non-stimulated group (P < 0.001) with exception of the 15 min time point (ns, P = 0.17).

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treatment of HeLa cells expressing MC5R-GFP for several timepoints, extracellular domains of total plasma membrane proteinswere biotinylated and, after cell lysis, were bound to a neutravi-din-coated plate. MC5R-GFP levels at cell surface were detectedby chemiluminescence quantification of the anti-GFP antibodybinding capacity. As shown in Fig. 1C, MC5R-GFP internalizes grad-ually with increasing time of a-MSH exposure, similarly to fluores-cence microscopy observations. Two hours of a-MSH contactinduces an endocytosis of around 50% of the initial amount ofcell-surface MC5R-GFP. In order to sustain the recycling idea inopposition to degradation of the receptor at later time points thatcould justify the decrease of the cell surface levels, we analyzed theMC5R-GFP expression by In-Cell Western experiments. InSupplementary Fig. 2 it is possible to observe that the levels ofMC5R-GFP are the same in all time points tested, supporting therecycling mechanism.

3.2. MC5R signaling downstream of ERK1/2 pathway

We have previously reported that MC5R activation induces asustained ERK1/2 phosphorylation in HEK293 cells stably express-ing the receptor (Rodrigues et al., 2009). Similarly, in HeLa cells sta-bly expressing MC5R-GFP we also observe the activation of ERK1/2pathway in consequence of a-MSH treatment (Fig. 2A). The kineticanalysis of MAPK cascade activation reveals that a-MSH treatmentrapidly induces a phosphorylation of c-Raf, MEK and ERK1/2 (peak-ing at 15 min) (Fig. 2A, B). These kinases also present a statisticallysignificant second wave of activation at 120 min (Fig. 2A, B).

A major downstream target of ERK1/2 is the family of 90-kDaribosomal S6 kinases (p90RSKs), which consists of RSK1 to RSK4

(Hauge and Frodin, 2006). As shown in Fig. 2A, a-MSH treatmentcauses a similar p90RSK phosphorylation pattern. Both ERK1/2and p90RSK are effectors for mitogen- and stress-activated proteinkinase 1 (MSK1) phosphorylation, and in fact the kinetics of MSK1phosphorylation obtained after a-MSH treatment also resemblesERK1/2 and p90RSK activation (Fig. 2A).

The transcription factor cAMP response element binding pro-tein (CREB) and the immediate-early gene c-Fos are also found tobe activated in a-MSH-stimulated MC5R-GFP expressing cells(Fig. 2A). The receptor induces a sustained CREB phosphorylationwith a peak at 15 min of agonist treatment. c-Fos expression is onlydetected after 60 min of a-MSH stimulation (the maximum activa-tion occurs at 180–360 min). Western-blotting analysis also dem-onstrates that c-Fos shifts from a lower molecular weight to ahigher molecular weight band between 60 and 180 min (Fig. 2A).ERK1/2 and p90RSK phosphorylate c-Fos on multiple residuesresulting in increased molecular weight and protein stability(Murphy et al., 2002; Pellegrino and Stork, 2006). In fact, it is thehigher molecular weight of c-Fos form that remains visible after12 h of a-MSH treatment. c-Fos expression was also detected byimmunofluorescence (Fig. 2C). After 120 min of a-MSH treatment,c-Fos strongly accumulates in the nuclei (red) and, as demon-strated previously in Fig. 1, MC5R-GFP (green) is efficientlyinternalized.

To further ascertain the contribution of ERK1/2 signaling top90RSK, MSK1, CREB and c-Fos activation, the cell line over-expressing MC5R-GFP was incubated with the MEK1/2 inhibitorsPD98059 or U0126 for 30 min and subsequently treated with a-MSH for 15 or 120 min. ERK1/2 inhibition strongly reducesp90RSK and MSK1 activation and c-Fos expression but has no

Fig. 2. Characterization of MC5R-GFP signaling pathways. A, HeLa cells stably expressing MC5R-GFP were mock-treated (0 min) or incubated with 1 lM a-MSH for theindicated periods of time (5 min to 24 h). Thereafter, 20 lg of cell lysates were analyzed by western-blotting for detection of phosphorylated forms of c-RAF, MEK1/2, ERK1/2,p90RSK, MSK1, CREB and expression levels of c-Fos and a-tubulin. B, Densitometry analysis of ERK1/2 activation from at least three independent experiments, as described inA, was performed. Data are expressed as fold increase of phospho/total-ERK1/2 levels over mock-treated samples. All a-MSH-stimulated groups are statistically different fromnon-stimulated cells using two-tailed student’s t-test (P < 0.001); statistical significance was also achieved for the second wave of ERK1/2 phosphorylation at 2 h whencompared to adjacent time points 1 and 3 h (#P < 0.05). C, Immunofluorescence detection of c-Fos (red) was performed in MC5R-GFP (green) expressing cells with or withouta-MSH incubation during 2 h. Nuclei (blue) were counterstained with DAPI. Scale bar- 10 lm. (For interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)

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effect on CREB phosphorylation (Fig. 3A), suggesting that CREB nu-clear signaling may be conveyed by other pathway than ERK1/2. Infact, the classical pathway implicated in CREB activation involvessynthesis of cAMP and activation of PKA. To test this hypothesis,MC5R-GFP expressing cells were incubated with the PKA inhibitorRp-cAMP prior to a-MSH treatment. In this experiment it is ob-served a decrease in CREB phosphorylation levels, which are com-pletely abolished with the highest concentration of the inhibitor(Fig. 3B). Thus, we can conclude that CREB activation occurs inde-pendently of ERK1/2 pathway but is PKA-driven. It is noteworthythat ERK1/2 is activated independently of PKA in this system(Fig. 3B), in agreement with the results obtained in HEK293 cellsover-expressing MC5R-GFP (Rodrigues et al., 2009).

Since the activation of MAPK cascade seems to occur in twotemporal distinct periods, a rapid peaking at 15 min and a sus-tained at 2 h, we tried to understand which ERK1/2 wave of phos-phorylation is responsible for the increase of c-Fos expression. Forthis, cells were incubated with a-MSH during 120 min and theMEK1/2 inhibitors were added only 60 min after starting the mel-anocortin stimulus, when the ERK1/2 phosphorylated levels areminimum (Fig. 3C). On those conditions the time persistentERK1/2 activation was inhibited and the levels of c-Fos expressionremain unaltered (Fig. 3C). The results make possible to concludethat the first wave of ERK1/2 activation is the only requisite forc-Fos induction.

3.3. Subcellular location of phosphorylated forms of ERK1/2 andp90RSK

Spatiotemporal regulation of ERK1/2 activation plays an impor-tant role in determining the functions of these kinases. To establishthe subcellular distribution of phosphorylated forms of ERK1/2 andp90RSK, cell fractionation studies were performed in HeLa cellsover-expressing MC5R-GFP, after a-MSH stimulation. Proteinequivalents of total (T), nuclear (N) and cytoplasmic (C) fractionswere analyzed by western-blotting. It is possible to conclude that

the majority of activated forms of ERK1/2 and p90RSK is found atthe cytosol in all different time points analyzed, including 15 and120 min of a-MSH treatment (Fig. 4A). The purity of the nuclearand cytoplasmic fractions was confirmed by lamins A/C anda-tubulin detection, nuclear and cytosol markers, respectively.

In order to quantitatively determine the levels of these kinasesin cytoplasm and nuclear fractions, multiple amounts of the nucle-ar fraction (plus 5 and 10 times, 5 N and 10 N respectively) werealso analyzed by western-blotting (Fig. 4B). Blots quantification re-veals that almost 90% of pERK1/2 and pp90RSK are retained at thecytoplasm, whereas only about 10% of these activated proteinstranslocate to the nucleus after 5 and 15 min of a-MSH treatment(Fig. 4B and C).

3.4. MC5R signaling: the role of b-arrestins and G-proteins

It is well documented that activation of ERK1/2 by severalGPCRs can occur through b-arrestins, which are scaffold proteinslinking receptors to downstream signaling cascades, includingthe MAPK pathway and promoting cytoplasmatic retention ofphosphorylated ERK1/2 (DeFea, 2011). Since MC5R activationwas reported to promote binding of b-arrestin 2 (Cai et al., 2006),we tested whether b-arrestins 1/2 are involved in a-MSH-inducedERK1/2 activation. We started to deplete by siRNA the expressionof these proteins in MC5R-GFP-overexpressing HeLa cells. The b-arrestin 1 and 2 siRNA duplexes used were already tested (Shenoyet al., 2006) and, as expected, western-blotting experimentsrevealed that they specifically decrease protein expression levels(Fig. 5A). We observed that the silencing of b-arrestins 1/2 hasno significant effect on the early phase (15 min) of ERK1/2 activa-tion, but it considerably decreases the long-lasting ERK1/2 activa-tion (2 h) (Fig. 5B). To corroborate the b-arrestin siRNA data, thefunctional performance of these proteins was decreased by trans-fection of Hela cells over-expressing MC5R-GFP with dominantnegative b-arrestin 1 and 2 mutants. As expected, the over-expres-sion of these non-functional proteins has no effect on ERK1/2

Fig. 3. Contribution of ERK1/2 signaling to p90RSK, MSK1, CREB and c-Fos activation. A, MC5R-GFP over-expressing cells were incubated with MEK1/2 inhibitors PD98059(100 lM) and U0126 (10 lM) during 30 min and then were stimulated with 1 lM a-MSH for subsequent 15 or 120 min. The effect on p90RSK, MSK1 and CREBphosphorylation and also on c-Fos expression was evaluated by western-blotting. B, To test the PKA involvement on CREB phosphorylation, the same cells were incubatedwith several concentrations of Rp-cAMP (10–100 lM) and the result was evaluated by western-blotting using an antibody directed to CREB phosphorylated forms. C, Cellswere stimulated with 1 lM a-MSH for 120 min and MEK1/2 inhibitors PD98059 and U0126 were added 60 min after starting the melanocortin treatment. The activation levelof all proteins was analyzed as described above in A. Blots are representative of three independent experiments.

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activation at 15 min of a-MSH treatment but interferes on the lateERK phosphorylation (Fig. 6). In conclusion, the two b-arrestin fam-ily members have crucial functions on the second and sustainedwave of ERK1/2 signaling. However, a different mechanism regu-lating the activation of these kinases at 15 min must exist.

Several data report that G proteins, like Gas or Gaq, have a con-tribution to rapid and transient MAPK activation (Lefkowitz andShenoy, 2005; Goldsmith and Dhanasekaran, 2007). However, thespecific inhibition of Gas activity with a long-term administrationof the cholera toxin (CTX) has no influence on ERK1/2 phosphory-lation at 15 min and also at 2 h (Fig. 7B). It is of note that CTX activ-ity was controlled by quantification of cAMP levels and, as shownin Fig 7A, CTX markedly decreases the high cAMP levels achievedafter alpha-MSH stimulus. Since Gas signals mainly through PKA,the use of Rp-cAMP, a PKA inhibitor, supports the Gas-independentERK phosphorylation (Figs. 7C and 3B). In fact, this inhibitor doesnot interfere with MC5R-induced ERK1/2 activation, although for-skolin effect was abolished. To test if ERK1/2 phosphorylation at15 min is mainly activated by Gaq, we analyzed the activity ofPKC on ERK1/2 activation (Fig 7C). Once again, the inhibition ofPKC with GF109203X (GF) does not interfere with a-MSH medi-ated ERK1/2 activation, in opposition to PdBU effect. The role ofGai was accomplished by the use of pertuxis toxin (PTX), a specificinhibitor of this particular isoform of G proteins. Since PTX effi-ciently decreases Gai activity, we observed an increase in cellularcAMP in a-MSH-stimulated and PTX pre-treated cells (Fig. 7A).Regarding ERK1/2 phosphorylation levels, it is possible to observean inhibition at 15 and also 120 min (approximately 20% and 30%,

respectively) by treatment of MC5R-GFP expressing cells with PTXprior to a-MSH stimulus (Fig. 7D). In conclusion, Gai (or the re-leased subunits Gcb) have an active role on the regulation of bothtransient and sustained MC5R-mediated ERK1/2 activation.

3.5. The role of MC5R internalization and recycling on ERK1/2signaling

The involvement of b-arrestins 1/2 on sustained ERK1/2 activity(Figs. 5B and 6) suggests that MC5R internalization could be med-iated by b-arrestins binding. In fact, in most cases, the signalingfunctions of b-arrestins are linked to the removal of GPCRs fromthe cell membrane by linking them to clathrin-coated pits (DeFea,2011). To analyze the b-arrestins 1/2 role on MC5R-GFP internali-zation, the expression of these proteins was inhibited by siRNA andthe receptor levels at plasma membrane were quantified by cellsurface ELISA experiments. As shown in Fig. 5C, the percentage ofMC5R-GFP integrated in the cell membrane at 15, 90 and120 min of a-MSH treatment is not influenced by the silencing ofb-arrestins 1/2, suggesting that MC5R internalization (and recy-cling) does not depend on b-arrestins binding.

Besides internalization, recycling mechanisms of GPCRs are alsokey events for spatiotemporal regulation of ERK1/2 signaling. Tofurther elucidate the role of MC5R recycling on ERK1/2 sustainedactivation, HeLa cells over-expressing MC5R-GFP were treatedwith monensin prior to a-MSH stimulus during 15 and 120 minand the effect on ERK1/2 activation was tested by western-blotting.

Fig. 4. MC5R-mediated ERK1/2 and p90RSK phosphorylation are retained in the cytosol. A, HeLa cells over-expressing MC5R-GFP were stimulated with 1 lM a-MSH forseveral periods of time. Then the cell membrane was disrupted and nuclei were separated from the cytoplasmic fraction by centrifugation. Equivalent amounts of the total (T),cytoplasmic (C) and nuclear (N) fractions were subjected to western-blotting analysis, using primary antibodies directed to phosphorylated ERK1/2 and p90RSK. The analysisof lamins A/C and a-tubulin expression were used to evaluate the purity of nuclear and cytosolic fractions, respectively. B, Multiple (five – 5 N and ten – 10 N) equivalentamounts of nuclear fractions were also analyzed as described above. C, Quantification of the relative amount of these kinases on the nucleus and cytosol was performed bydensitometric analysis of three independent western-blotting results. The phosphorylation levels of ERK1/2 and p90RSK in the cytosol or nucleus were normalized toa-tubulin and lamins A/C, respectively.

A.R. Rodrigues et al. / Molecular and Cellular Endocrinology 361 (2012) 69–79 75

As illustrated in Fig. 8, monensin treatment decreases ERK1/2 sig-naling only after 120 min of a-MSH stimulus.

All these data suggests that sustained ERK1/2 phosphorylationis a dual process regulated by b-arrestins expression and receptorrecycling.

3.6. MC5R signaling pathway in 3T3-L1 adipocytes

Data presented so far characterized a model of MC5R signalingand internalization in a heterologous expression system. To verifyif this model is still valid under MC5R endogenous expression con-ditions, we took advantage of the murine adipocyte cell line 3T3-L1where, consensually, only MC2R and MC5R are present (Normanet al., 2003; Boston and Cone, 1996; Boston, 1999). This cell lineis also the best characterized system to study the energy balanceregulation at the adipocyte. As shown in Fig. 9A, in differentiated3T3-L1 adipocytes, a-MSH induces phosphorylation of ERK1/2,p90RSK, MSK1 and CREB, similarly to MC5R-GFP signaling in HeLacells. Also in 3T3-L1 adipocytes, phosphorylated forms of ERK1/2are mostly retained at the cytoplasm (Fig. 9B).

4. Discussion

The expression of MC5R coupled to GFP was accomplished inHeLa cells and made possible the unraveling of signaling and inter-nalization mechanisms activated by the receptor after a-MSHstimulus. In agreement with published data (Rodrigues et al.,2009), we observed that a-MSH activated-MC5R induces at leasttwo signaling pathways: PKA and ERK1/2 (MAPK). Temporal and

spatial differences in ERK1/2 activation modify downstream sig-naling and dictate cell outcomes. In this work we conclude thata-MSH activated-MC5R induces a biphasic phosphorylation ofERK1/2, with one peak at 15 min and another one more sustainedin time at 2 h. It is generally accepted that, upon phosphorylation,ERK1/2 dissociate from their anchoring proteins, such as MEK1/2,being able to phosphorylate several cytoplasmatic targets (suchas cytoskeleton and downstream kinases) (Kondoh et al., 2005)or, more commonly, translocate into the nucleus, where they acti-vate various transcription factors (Goldsmith and Dhanasekaran,2007). Here we provide evidence that phosphorylated ERK1/2 pool,obtained after MC5R activation, remains almost totally in the cyto-sol and is responsible for p90RSK phosphorylation, also with majorcytoplasmic location. The small fraction of pERK1/2 that translo-cates to the nucleus (only 10%) is responsible for the increase ofc-Fos expression. The biological relevance of this observation isstill unknown.

The classical role of b-arrestins on GPCRs function is related todesensitization mechanisms, which include the uncoupling ofGPCRs from G proteins or GPCRs internalization, and also to signaltransduction by acting as scaffold molecules (DeFea, 2011). Our re-sults show that b-arrestins 1/2 are not involved in MC5R internal-ization, but are essential to ERK1/2 sustained activation. ManyGPCRs exhibit a distinct temporal pattern of ERK1/2 activation,which initially includes a robust, transient phase that is followedby an intermediate, sustained phase. For instance, the pituitaryadenylate cyclase-activating receptor (Broca et al., 2009), parathy-roid hormone (Gesty-Palmer et al., 2006), b-2 adrenergic receptor(Shenoy et al., 2006) and angiotensin II receptors (Ahn et al.,2004), when properly stimulated with their ligands, activate

Fig. 5. Silencing of b-arrestins 1/2 expression by siRNA. A, HeLa cells stablyexpressing MC5R-GFP were transfected with siRNAs directed to b-arrestin1, 2, bothor GAPDH (as negative control). After 72 h, cells were lysed and analyzed forb-arrestins 1/2 expression by western-blotting using a mouse monoclonal antibodyagainst b-arrestins 1 that cross-reacts with b-arrestins 2. Protein levels weredetermined by densitometric analysis of the blots and then normalized witha-tubulin levels. Data are expressed as a percentage of b-arrestins levels in GAPDHdepleted cells. B, Cells subjected to siRNA were stimulated with 1 lM a-MSH for 15or 120 min. The effect on ERK1/2 phosphorylation was evaluated by western-blotting. Phospho-ERK1/2 levels were normalized with total ERK1/2 and data areexpressed as a percentage of phospho/total ERK1/2 levels from GAPDH depletedcells. C, After b-arrestins and GAPDH siRNA silencing, HeLa cells stably transfectedwith MC5R-GFP were mock-treated (0 min) or stimulated with 1 lM a-MSH for 15,90 and 120 min. Cell surface MC5R-GFP levels in GAPDH and b-arrestins depletedcells were quantified by ELISA as described in Fig. 1B. Data are expressed as apercentage of the MC5R-GFP levels in the plasma membrane obtained in mock-treated cells. All experiments were repeated at least three times and asterisksindicate groups that are statistically different from controls (GAPDH depleted cells)using two-tailed Student’s t-test (⁄P < 0.05; ⁄⁄P < 0.001).

Fig. 6. Decrease of b-arrestins 1/2 function by over-expression of dominantnegative mutants. Hela cells stably expressing MC5R-GFP were transfected withthe dominant negative b-arrestin1 (Db1) and 2 (Db2) mutants, with both mutants(Db1 + 2) or with the pcDNA3.1 plasmid as control (pcDNA). Forty-eight hours after,cells were stimulated with 1 lM a-MSH for 15 min or 2 h and the effect on ERK1/2phosphorylation was evaluated by western-blotting, as described in legend to Fig 5.All experiments were repeated at least six times and asterisks indicate groups thatare statistically different from controls using two-tailed Student’s t-test (⁄⁄P < 0.01).

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ERK1/2 also by a biphasic mechanism and b-arrestins were foundto regulate only one of these phases of ERK1/2 phosphorylation.As already proposed for the angiotensin II receptor (Ahn et al.,2004), the prolonged ERK1/2 activation by MC5R could be a conse-quence of b-arrestins shielding from MAPK phosphatases. Thebinding of b-arrestins and their role as scaffold, could be likelyimplicated in the retention of ERK1/2 at the cytosol (or associatedat the plasma membrane) controlling their trafficking to the nu-cleus. Besides b-arrestins, Sef (similar expression to fgf genes)and PEA-15 are two other proteins that play a pivotal role in regu-lation of spatial control of ERK1/2 signaling, promoting the cyto-solic location of these kinases (Kondoh et al., 2005).

In HeLa cells over expressing MC5R-GFP, both transient andsustained ERK1/2 activation occur by a mechanism that dependspartially on Gai activity. Other MCRs, like MC3R and MC4R, werealso found to respond to melanocortins activating the pertussistoxin-sensitive Gi pathway (Chai et al., 2007; Buch et al., 2009).Gai can modulate ERK1/2 phosphorylation mainly by two mecha-nisms: inhibiting cAMP synthesis and PKA activity or recruitmentof Rap1 GTPase activating protein in a Ras dependent process(Woehler and Ponimaskin, 2009; Goldsmith and Dhanasekaran,2007). The first mechanism involving PKA may be excluded sincewe verified an independence of PKA on ERK1/2 activation. The sec-ond one is a hypothesis that we cannot rule out. Nevertheless, aregulatory mechanism of MAPKs cascade involving the bc-subunitof Gi proteins was also reported for some GPCRs (Woehler andPonimaskin, 2009; Goldsmith and Dhanasekaran, 2007). Briefly,Gbc dimer dissociates from activated Gai subunit to directly impli-cate effector proteins like PI3K that modulate MAPK activity. Thisseems the most likely mechanism since we have previously shownthe implication of PI3K in MC5R-mediated ERK1/2 phosphorylation(Rodrigues et al., 2009). However, Gai inhibition only reduces 20–30% of ERK1/2 activity suggesting that other pathways should alsobe responsible for the ERK1/2 signaling. For instance, our resultsdemonstrate that the late activation of ERK1/2 by a-MSH is also

Fig. 7. The role of G proteins on MC5R-stimulated ERK1/2 phosphorylation. MC5R-GFP expressing cells were stimulated with 1 lM a-MSH for 15 min in the presence ofvehicle (mock-treated cells), 100 ng/ml CTX or 100 ng/ml PTX and cAMP levels were quantified (A). Statistical differences were obtained for PTX and CTX treated groups whencompared with mock-treated cells (⁄P < 0.05). MC5R-GFP expressing cells were mock-treated (0 min) or stimulated with 1 lM a-MSH for 15 or 120 min, after overnightincubation with 100 ng/ml CTX (B) or 100 ng/ml PTX (D). PKC inhibitor GF109203X (GF; 5 lM) and PKA inhibitor Rp-cAMP (10 lM) were added 30 min before a-MSHtreatment. PdBU (1 lM) and Forskolin (10 lM) were used as positive controls for GF109203X and Rp-cAMP activity (C). ERK1/2 phosphorylation levels were evaluated bywestern-blotting and all results are representative of three independent experiments. ERK1/2 phosphorylation levels were determined by densitometric analysis of the blotsand then normalized with total ERK1/2 levels (C). Statistical differences were obtained for PTX-treated groups when compared with mock-treated cells, at both 15 and120 min of a-MSH stimulation (⁄P < 0.05).

Fig. 8. The role of MC5R-GFP recycling in ERK1/2 activation. HeLa cells over-expressing MC5R-GFP were incubated with monensin (0.1–10 lM) to block the receptorrecycling. After 30 min, cells were mock-treated (0 min) or a-MSH stimulated for 15 and 120 min. ERK1/2 phosphorylation was detected by western-blotting analysis. Blotsare representative of three independent experiments.

A.R. Rodrigues et al. / Molecular and Cellular Endocrinology 361 (2012) 69–79 77

b-arrestins dependent. This complexity of MC5R-activated ERK1/2signaling that results from both G-protein dependent and indepen-dent pathways bring new insights for future MCRs signalingstudies.

MC5R is a GPCR whose function is far from being completelyelucidated, but there is evidence in the literature that a-MSH in-creases fatty-acid oxidation in skeletal muscle, in which MC5Rplays a major role (An et al., 2007). The murine 3T3-L1 adipocytes,that endogenously express MC2R and MC5R and respond to a-MSHincreasing the lipolysis rate, is another evidence of the involve-ment of the melanocortin system on lipid metabolism (Molleret al., 2011). Since MC2R binds only ACTH, the effect of a-MSH

on lipolysis should be mediated by MC5R, although direct evidenceon that is lacking. We have used the 3T3-L1 adipocytes to verify ifthe signaling mechanisms described in a heterologous system withover-expression of MC5R also occur in a native system. We ob-served that in adipocytes the same signaling molecules and path-ways are activated by a-MSH, although the kinetics slightlydiffers. In fact, in contrast to HeLa cells, we did not observe the sec-ond wave of ERK1/2 activation on adipocytes. Although we did notdetermine the density of a-MSH binding sites on HeLa cells over-expressing MC5R and 3T3-L1 adipocytes we are convinced that itis lower on adipocytes, resulting in weaker activation of ERK1/2and preventing the detection of the second wave of ERK1/2

Fig. 9. MC5R signaling in 3T3-L1 adipocytes. Fully differentiated adipocytes were serum starved during the night and subsequently stimulated with 1 lM a-MSH for severaltime points (0–24 h). A 20 lg of cell lysate were analyzed by western-blotting for detection of ERK1/2, p90RSK, MSK1 and CREB phosphorylation. B, 3T3-L1 adipocytes werecell fractionated by centrifugation and equivalent amounts of total (T), cytosolic (C) and nuclear (N) fractions were also subjected to western-blotting analysis. Blots arerepresentative of at least three independent experiments.

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phosphorylation. This phenomenon is not a particularity of HeLacells since it is also observed on HEK293 cells over-expressingMC5R-GFP (Rodrigues et al., 2009).

Cho et al. (2005) show that the incubation of adipocytes withthe MEK inhibitor PD98059, prior to a-MSH stimulus, leads to a de-crease on lipolysis levels, supporting the involvement of ERK1/2signaling pathway on lipolysis regulation. In contrast, Mooleret al. (2011) conclude that ERK1/2 is not essential for NDP-a-MSH and PG-901 (a-MSH analogues) lipolysis mediation. How-ever, these authors do not evaluate the involvement of ERK1/2on a-MSH-induced lipolysis, an issue that needs to be clarified.Furthermore, future experiments involving MC5R silencing wouldhelp to accurately clarify its role on adipocytes.

Acknowledgments

Rodrigues A.R. was supported by POCI 2010, FSE and ‘‘Fundaçãopara a Ciência e Tecnologia’’ (SFRH/BD/41024/2007).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.mce.2012.03.011.

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