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Service d’Endocrinologie, Département de Médecine (Z.K., N.G.-P.), and Département de Physiologie et Biophysique (N.B., P.K., M.D.P.), Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service d’Endocrinologie, Département de Médecine, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001, 12e avenue Nord, Sherbrooke, Québec, Canada J1H 5N4. E-mail: nicole.gallo-payet{at}usherbrooke.ca.
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Introduction |
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Compared with other MCRs, MC2R is unique in that it binds only ACTH and does not possess affinities for other melanocortins, whereas other MCRs, in contrast, can be activated by both ACTH and MSH (9, 10). MC2R is predominantly expressed in the adrenal cortex, with highest expression levels in the zonae glomerulosa and fasciculata and weaker expression in the zona reticularis (1, 2, 3, 9). MC2R signaling is inherently complex. In 1970, cAMP was identified as the main second messenger (8), cooperating with Ca2+ influx for maximal ACTH-induced steroid secretion (11 ; for review, see Ref.12).
The majority of GPCRs undergo a desensitization process within seconds to minutes of ligand binding, characterized by an uncoupling from the cognate effector system, followed by internalization. Desensitization is initiated by receptor phosphorylation by second messenger-regulated kinases such as PKA or protein kinase C (PKC), with subsequent binding of proteins referred to as arrestins (13, 14). After several minutes, desensitization is generally followed by receptor sequestration from the cell surface, typically leading to down-regulation (15). The regulation of MC2R expression is atypical, because, in contrast to the aforementioned general properties, MC2R is known for its capacity for self-controlled up-regulation. Indeed, in bovine primary cultures of fasciculata cells, ACTH pretreatment for 48 h causes an increase in cAMP production, mediated by an increase in the number of ACTH-binding sites (16) and in the number of MC2R mRNA transcripts (17, 18).
All of the currently available data regarding specific receptor regulation properties have used a strategy of heterologous receptor expression in cell lines. However, one difficulty with the MC2R was to find the appropriate model for cell expression (10). Indeed, the only successful transfection studies were carried out with cell lines already expressing one of the MCRs such as Cloudman melanoma (M3) cells (19), COS-7 cells (20), and adrenocortical cell lines Y6 (21) and OS3 (22). This is most likely due to cell type-restricted expression of a recently identified protein necessary for MC2R cell membrane targeting termed melanocortin-2 receptor accessory protein (23). Using M3 cells, Penhoat et al. (24) demonstrated that the properties of transfected MC2R are very similar to those of endogenous MC2R observed in adrenal fasciculata cells in primary cultures, with high binding affinity and efficient coupling to cAMP production. Other studies have also successfully expressed MC2R in the Y1-related cell line, Y6, which is devoid of endogenous MC2R (21, 25). However, the EC50 value for ACTH-induced cAMP production was 2 orders of magnitude higher than the Kd value for MC2R binding, making this model less physiologically relevant than M3 cells. Thus, compared with other GPCRs, research in the field of MC2R regulation has indubitably been hampered by such difficulties.
The aim of the present study was to investigate the short-term regulation of the human (h) MC2R receptor by generating a functional expression system in M3 cells allowing us to monitor and track a c-Myc-tagged hMC2R with a specific antibody. The expression, subcellular localization, and pharmacology of hMC2R in response to ACTH stimulation were thoroughly examined. Furthermore, hMC2R responsiveness to repeated ACTH exposure as well as cellular redistribution of the receptor and involvement of ß-arrestin-2 were also studied. Lastly, the roles of two different second messenger-dependent kinases, PKA and PKC, in hMC2R loss of responsiveness and sequestration were assessed using specific pharmacological inhibitors. This study brings new detailed insight into the mechanisms involved in short-term regulation of hMC2R activity while revealing some unique properties of the ACTH receptor hMC2R.
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Materials and Methods |
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Construction of c-Myc-tagged human MC2R
The c-Myc epitope was added by PCR amplification of the hMC2R cDNA with Taq polymerase. The primers were designed to amplify the c-Myc-MC2R and to create an XhoI restriction site (underlined) at the N terminus, followed by the human c-Myc epitope sequence (italics) in-frame with the MC2R coding sequence (5'-agt ccg ctc gag ATG GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG ATG AAG CAC ATT ATC AAC TCG TAT G –3') and an XbaI restriction site (underlined) at the C-terminus (5'-agt ccg tct aga CTA CCA GTA CCT GCT GCA GAA GAT CAT C-3'). The purified PCR product was digested with XhoI and XbaI, then ligated into XhoI-XbaI-opened pcDNA3 expression vector. The construct was confirmed by sequencing.
Cell culture and transfections
M3 cells were purchased from American Type Culture Collection and cultured in Kaighn’s modification of Ham’s F-12 medium (F-12K) supplemented with 15% horse serum, 2.5% fetal bovine serum, and 1% GlutaMAX. The cells were kept in a humidified atmosphere of 95% air and 5% CO2 at 37 C according to the manufacturer’s instructions. One day before transfection, 300,000 cells were plated in 35-mm culture dishes and used at 70% confluence. Transfections were carried out with 1.5 µg cDNA using Lipofectamine Plus reagent according to the manufacturer’s instructions. Transiently transfected cells were harvested 72 h after transfection. G418 was used as the selection agent of stably transfected clones.
RT-PCR
M3 cell total RNA was prepared using the RNAqueous-4PCR kit (Ambion, Inc., Austin, TX) according to the manufacturer’s instructions. The product was treated with deoxyribonuclease I to eliminate contamination of genomic DNA. Total RNA was reverse transcribed with 200 U Moloney murine leukemia virus reverse transcriptase using oligo(deoxythymidine) primers in the presence of 25 U RNasin ribonuclease inhibitor. Two micrograms of cDNA were then used for PCRs for 30 cycles each, with specific primers for mouse glyceraldehyde-3-phosphate dehydrogenase and hMC2R. The primers used for hMC2R were 5'-TCTTCAGCCTGTCTGTGATTG-3' and 5'-GGCACAGGATGAAGACCAG-3', with an expected fragment size of 222 bp. The primers used for mouse glyceraldehyde-3-phosphate dehydrogenase were 5'-ACTCCTTGGAGGCCATGTAGG-3' and 5'-CAACTCCCACTCTTCCACCTTC-3', with an expected fragment size 143 bp.
Membrane preparation
Confluent native or hMC2R-transfected M3 cells cultured on 100-mm culture dishes were washed twice with ice-cold PBS; resuspended in 5 ml ice-cold homogenization buffer containing 50 mM Tris-HCl, 0.1 mM EDTA, 1 mM dithiothreitol, and complete EDTA-free protease inhibitor cocktail (Roche, Laval, Canada); sonicated; and centrifuged at 150 x g for 10 min at 4 C. The membrane-containing supernatant was then recentrifuged at 20,000 x g for 30 min at 4 C. The crude membrane preparation was washed twice with the same buffer, recentrifuged, snap-frozen in liquid nitrogen, and transferred to –80 C. On the day of the experiment, the membranes were thawed, centrifuged, and resuspended in buffer containing 50 mM Tris-HCl (pH 7.6), 2 mM EGTA, and 1 mM dithiothreitol.
Western blotting
Western blot studies were carried out on membrane preparations according to the method described previously (26). Proteins were resolved on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. After blocking in 5% nonfat milk, hMC2R was detected using anti-Myc monoclonal antibody 9E10 and a horseradish peroxidase-coupled secondary antibody (1:1000) and visualized with enhanced chemiluminescence (ECL Plus).
cAMP production assay
Intracellular cAMP production was determined by measuring the conversion of [3H]ATP to [3H]cAMP, as previously described (27), in 1 x 106 cells plated in 35-mm culture dishes. Briefly, cells were incubated for 1 h at 37 C with complete culture medium containing 2 µCi/ ml [3H]adenine. Cells were then washed and incubated for 15 min in 1 mM IBMX in Hanks’ balanced solution (HBS) containing 130 mM NaCl, 3.5 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 2.5 mM NaHCO3, and 5 mM HEPES, supplemented with 1 g/liter glucose. Hormones and drugs were then added to the incubation medium for 15 min at 37 C. Separation of [3H]cAMP from [3H]ATP was obtained by chromatography on Dowex and alumina columns according to the method of Salomon et al. (28). cAMP formation was calculated as follows: % conversion = [3H]cAMP/([3H]cAMP + [3H]ATP) x 100/15 min. In some figures, values are expressed as the fold increase above basal cAMP accumulation.
Desensitization studies
In dose-dependent hMC2R desensitization protocols, native or stably hMC2R-transfected M3 cells were preincubated with the indicated ACTH concentrations from 1 pM to 10 nM for 15 min without the phosphodiesterase inhibitor IBMX. After two washings with HBS, cells were incubated for an additional 15 min with incubation medium containing various concentrations of ACTH in the presence of IBMX to generate a dose-response curve. For desensitization kinetics experiments, cells were preincubated with 10 nM ACTH without IBMX for 1–60 min, then washed and incubated for an additional 15 min with 10 nM ACTH in the presence of IBMX. cAMP accumulation was measured as described above. Preliminary results showed that use of an acid wash step [130 mM NaCl, 3.5 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 2.5 mM NaHCO3, and 5 mM HEPES (pH 4.0) adjusted with glacial acetic acid] between exposures to ACTH had no effect on the desensitization profile.
For studies using second messenger kinase inhibitors, an incubation step with the inhibitor diluted in incubation medium was performed before ACTH pretreatment. The PKA inhibitor H89 was added at 10 µM for 15 min, and the PKC inhibitor GF109203X was used at 1 µM for a 30-min incubation period. Each respective inhibitor was also present during the ACTH treatment period. Control cells treated with 0.1% and 0.01% dimethylsulfoxide, corresponding to H89 and GF109203X dilutions, respectively, did not show any difference in c-Myc labeling either in control conditions or after 10 nM ACTH stimulation (data not shown).
To investigate the relationship between internalization and cAMP production (desensitization), cells were incubated with or without 450 mM sucrose in HBS for 30 min at 37 C. This treatment inhibits internalization through a clathrin-dependent pathway (29). The cells were then pretreated with 10 nM ACTH, washed twice, and restimulated with 10 nM ACTH in the presence of IBMX. Sucrose was maintained throughout the experiment. Cells were then harvested, and cAMP was measured as described above.
Immunofluorescence microscopy
Cells were seeded onto 35-mm dishes, transfected in accordance with the standard protocol, and transferred onto poly-L-lysine-coated coverslips 1 d before the experiment. To confirm the localization and correct targeting of c-Myc-MC2R, cells were fixed with 2% paraformaldehyde/PBS for 15 min at room temperature without permeabilization. Cells were then washed twice with PBS, and nonspecific binding was blocked with 5% nonfat milk/PBS for 30 min at room temperature. Cells were incubated with the primary anti-c-Myc antibody clone 9E10 in blocking solution at a concentration of 4 µg/ml for 1 h at room temperature. After three washes with PBS, goat antimouse Alexa Fluor488-coupled secondary antibody was applied for 1 h at room temperature (dilution, 1:500). After one brief PBS wash, nuclei were stained with 300 mM DAPI, diluted in blocking solution for 3 min at room temperature. Cells were then washed three times with PBS at room temperature and mounted with coverslips in Vectashield mounting medium.
For internalization studies, after two brief PBS washes, cell surface receptors were first labeled by incubation with anti-c-Myc antibody (1:500 dilution) for 1 h at 4 C in F12-K medium supplemented with 1% BSA as described by Parent et al. (30). Cells were then washed twice with PBS and treated without or with 10 nM ACTH in complete F12-K medium for 30 min at 37 C. After two washes with PBS, cells were fixed in –20 C methanol for 15 min. Nonspecific binding was blocked with 5% nonfat milk/PBS for 30 min at room temperature. Goat antimouse Alexa Fluor488-coupled secondary antibody was added at a 1:500 dilution in blocking solution for 1 h at room temperature. After one PBS wash, nuclei were stained with DAPI as described above. After three final PBS washes, coverslips were mounted in Vectashield mounting medium. For studies using pharmacological kinase inhibitors, an incubation step with inhibitor diluted in complete medium was performed before ACTH treatment. Kinase inhibitor was also added to the ACTH incubation solution.
For hMC2R and ß-arrestin-GFP colocalization studies, a cotransfection ratio of 1 µg hMC2R-GFP and 0.25 µg ß-arrestin-2-GFP cDNAs was used. In these experiments, hMC2R was visualized using goat antimouse Alexa Fluor597-coupled secondary antibody. For studies investigating internalization by clathrin-coated pits, the membrane-bound hMC2Rs were first labeled with anti-Myc antibody as described for internalization experiments. An additional sucrose incubation step was added before ACTH stimulation. Sucrose (450 mM) in complete medium was also present throughout ACTH stimulation.
Cells were examined on an Eclipse 2000 microscope (Nikon, Mississauga, Canada) equipped for epifluorescence, and images were acquired using a Hamamatsu Orca digital camera (Nikon) connected to a personal computer running SimplePCI software (Nikon). The microscope was also equipped with a computer-controlled motorized focus drive, allowing acquisition of serial z-scans. Cells were thus scanned from top to bottom in incremental 0.5-µm steps. The figures represent images taken at midplane across the cell body, unless specified otherwise. All images were deconvolved using AutoDeblur software (AutoQuant Imaging, Inc., Troy, NY). Cells displaying intermediate fluorescence intensity were chosen for imaging and analysis. Typically, 150–200 cells were examined for each experiment.
Data analysis
Dose-response curves were constructed using PRISM 3 software (GraphPad, Inc., San Diego, CA). Data from each experiment were fitted using nonlinear fitting of sigmoidal dose-response curves using the equation: y = bottom + (top-bottom)/(1 + 10exp(logEC50 – x)), where x is the logarithm of concentration, and y is the response. EC50 and maximal stimulation (plateau) values were calculated as the mean ± SE individual EC50 and maximal stimulation (plateau) values for the number of separate experiments indicated, each performed in triplicate. The dose-response curves in Figs. 3
and 4 were fitted on data representing the mean ± SE of all respective experiments using the equation given above. Statistical analysis was performed by two-way ANOVA (for Figs. 5 and 10) and one-way ANOVA, followed by Tukey’s or Dunnett’s post hoc statistical test for Fig. 4.
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(upper panel), native M3 cells and pcDNA3 mock-transfected cells did not express hMC2R, whereas M3 cells transfected transiently with the c-Myc-tagged hMC2R as well as the stably transfected clone expressed hMC2R mRNA. Using specific primers (see Materials and Methods), a single band of 222 bp was detected, corresponding to the expected size (GenBank accession no. X65633). Possible DNA contamination of RNA preparations was excluded by amplification on respective RNA samples that were not reverse transcribed from all samples assessed (lane B for each cell type). Moreover, as assessed by RT-PCR, M3 cells do not express endogenous mouse MC2R (data not shown).
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Cellular localization of hMC2R in M3 cells
To investigate subcellular localization of the expressed protein, indirect immunofluorescence studies were performed in unpermeabilized cells transiently overexpressing c-Myc-tagged hMC2R. Figure 2 shows images of the same cell taken at different focal planes: at midcell (A and C) and at the cell top (D and F). Visualization of hMC2R was performed using monoclonal anti-Myc antibody (Fig. 2, A and D) with DAPI staining of the nuclei (Fig. 2, B and E). As clearly shown in colocalization panels (Fig. 2, C and F), the cell to the left (arrowhead) had an intense green staining corresponding to a positive hMC2R signal, in contrast to the untransfected cell (arrow), which was considered a negative control. The results illustrated in Fig. 2 also indicate that the transfected hMC2R was correctly inserted into the plasma membrane, as revealed by an intense fluorescence signal of the N-terminal c-Myc epitope (Fig. 2, A and D). Of note, the hMC2R protein appears to be localized in discrete submembrane domains, evidenced by the punctate fluorescence pattern. Together, these experiments demonstrate that hMC2R transfected in M3 cells is expressed at both mRNA and protein levels and is correctly targeted and inserted into the plasma membrane.
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, all hMC2R-transfected cell types exhibited comparable ACTH responsiveness. The EC50 values varied from 7.6 ± 1.2 to 11.9 ± 1.4 nM, as reported in Table 1. EC50 values of all cell types assessed were significantly different from that obtained for native untransfected cells. All hMC2R-transfected cell types responded to ACTH challenge with approximately the same efficacy, with the maximal response ranging from a 20.9 ± 0.7- to 24.7 ± 0.7-fold increase over basal cAMP levels (Table 1). cAMP accumulation reached a plateau at a concentration of 100 nM ACTH. The maximal ACTH response in native M3 cells was only a 6.5 ± 0.2-fold increase over basal values and appeared solely at ACTH concentrations greater than 50 nM, corresponding to the contaminating response from MC1R (data not shown), which exhibits a lower affinity for ACTH (6, 33). These results confirm the functionality of hMC2R expression in both transiently and stably transfected M3 cells while exhibiting equivalent properties regarding ACTH-induced cAMP production.
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, the hMC2R response demonstrated a time-dependent attenuation of cAMP formation upon stimulation by a second exposure to ACTH. Maximal loss of responsiveness was reached after 15 min of ACTH pretreatment (30% responsiveness of unpretreated control), followed by a steady plateau up to 60 min (maximal time tested).
To characterize this ACTH-induced loss of hMC2R responsiveness, stable transfectants were preincubated with increasing ACTH concentrations (from 1 pM to 10 nM) without IBMX for 15 min, then washed and additionally stimulated with various ACTH concentrations in the presence of IBMX to generate respective dose-response curves of cAMP accumulation. As shown in Fig. 4B, increasing concentrations of ACTH pretreatment induced a significant dose-dependent rightward shift of the dose-response curves, with an increase in EC50 from 9.6 ± 3.2 nM in cells preincubated with medium alone to 46.4 ± 3.1 nM in cells preincubated with 10 nM ACTH (P < 0.05; n = 4; Table 2). These values represent a dose-dependent decrease in ACTH potency, which is an indication of desensitization of the hMC2R.
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). ANOVAs revealed a statistically significant difference between inhibitors and ACTH treatment (F = 4.79; df = 3; P < 0.004). Under control conditions, stimulation with ACTH induced cAMP accumulation from 0.072 ± 0.006 to 1.918 ± 0.198 (26.6-fold increase; P < 0.001; n = 4; –/–; 
). Preincubation with ACTH increased the basal level of cAMP (from 0.072 ± 0.006- to 0.660 ± 0.142, 9.2-fold increase; P < 0.001; n = 3; –/– vs. +/–; 
) and decreased the subsequent ACTH-stimulated hMC2R response in terms of cAMP accumulation by 30.6% (from 1.918 ± 0.198 to 1.332 ± 0.274; P < 0.05; n = 3; –/– vs. +/–; ). Preincubation with 10 µM H-89 alone increased cAMP accumulation after single ACTH stimulation (from 1.918 ± 0.198 to 2.973 ± 0.075; 1.6-fold increase; P < 0.001; n = 3; –/– vs. –/+; ). Moreover, when pretreated with both 10 nM ACTH and H-89, the PKA inhibitor restored ACTH-induced loss of hMC2R responsiveness to the level observed in control cells (+/+ vs. –/– compared with +/–; ; Fig. 5A).
Pretreatment with 1 µM GF109203X alone did not alter the basal level of cAMP accumulation in response to a single ACTH challenge. However, PKC inhibition restored hMC2R activity reduced by ACTH-induced desensitization (Fig. 5B). Moreover, inhibition of both PKA and PKC induced a synergistic effect on ACTH-desensitized hMC2R activity and raised receptor responsiveness above the hMC2R activity observed under control conditions [2.834 ± 0.309 vs. 1.918 ± 0.198; 2.5-fold increase; P < 0.05; n = 4; Fig. 5C; compare +/+ () in Fig. 5, A–C]. Of note, PKA and PKC inhibition was associated with an enhancement of basal hMC2R activity [0.660 ± 0.142 to 1.447 ± 0.138; 2.2-fold increase; P < 0.001; n = 3; compare +/+ () in Fig. 5, A–C]. These results suggest that hMC2R is desensitized in a PKA- and PKC-dependent manner.
ACTH induced hMC2R internalization
Because GPCR desensitization is generally accompanied by receptor internalization, immunofluorescence studies were performed to determine whether hMC2R was also internalized in response to ACTH. M3 cells were transiently transfected with c-Myc-tagged hMC2R and incubated with specific anti-Myc antibody at 4 C before any treatment to label only receptors present at the cell surface. Cells were then incubated in the absence or presence of 10 nM ACTH and processed for immunofluorescence detection as described in Materials and Methods. As shown in Fig. 6A, in unstimulated cells, the population of hMC2R was clearly visible at the cell surface (arrowhead). A 15-min exposure to 10 nM ACTH resulted in the appearance of intracellular vesicles, distributed throughout the cell cytoplasm (Fig. 6B, arrowhead), with MC2R also present at the membrane (Fig. 6B, arrow). After 30 min of ACTH stimulation, hMC2R labeling had practically disappeared from the cell membrane (Fig. 6C, arrow) and displayed a striking redistribution to intracellular, endocytic compartments, clustered near the nucleus (Fig. 6C, arrowhead). After 60 min of stimulation with 10 nM ACTH, the majority of hMC2R remained intracellular, with a diffuse distribution throughout the cytoplasm (Fig. 6D, arrowhead) as well as the reappearance of hMC24 labeling at the cell membrane, an indication of receptor recycling (Fig. 6D, arrow).
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, arrowhead), whereas ß-arrestin-2-GFP fluorescence exhibited diffuse and homogenous distribution throughout the cytoplasm (Fig. 7B), with no observable colocalization (Fig. 7C). However, after a 30-min treatment with ACTH, receptor immunofluorescence (Fig. 7D) and ß-arrestin-2-GFP fluorescence (Fig. 7E) showed extensive colocalization (yellow) in endocytic compartments, clustered near the nucleus (Fig. 7F).
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, G and H, ß-arrestin-2-GFP exhibited a diffuse and homogenous distribution throughout the cytoplasm in both control and stimulated cells, thus excluding participation of MC1R in redistribution of ß-arrestin-2-GFP in hMC2R-transfected cells. These results indicate that ß-arrestin-2-GFP specifically colocalizes with internalized hMC2R in endocytic vesicles.
Certain receptors may also be associated with caveolin-1 to either promote internalization or act as a molecular chaperone; caveolin-1 is involved in membrane receptor trafficking or as a scaffold protein, directly implicated in interactions with signaling molecules (34, 35). As shown in Fig. 8, under control conditions, hMC2R was present at the plasma membrane (Fig. 8A, arrowhead), and caveolin-1 was distributed as small dots, in the intracellular vesicles and plasma membranes. After a 30-min incubation with ACTH, hMC2R was sequestered in endocytic compartments, clustered near the nucleus (Fig. 8B, arrow), whereas caveolin-1 distribution remained unchanged, indicating that hMC2R did not exhibit colocalization with caveolin-1 either at the membrane or during the sequestration process.
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, red, arrowhead) internalized in endocytic compartments where it colocalized with ß-arrestin-2-GFP (Fig. 9 B, yellow, arrow), whereas sucrose pretreatment completely blocked hMC2R internalization (Fig. 9D, arrow, vs. Fig. 9C, arrowhead). In contrast, this treatment prevented the decrease in cAMP production, thus preventing desensitization (Fig. 10). To determine whether hMC2R desensitization and internalization are interrelated processes, hMC2R stably transfected cells were pretreated with 450 mM sucrose, after which receptor loss of responsiveness was assessed in terms of cAMP accumulation. Under control conditions, ACTH stimulated cAMP accumulation (P < 0.001; n = 3). This hMC2R responsiveness was desensitized after pretreatment with 10 nM ACTH. ACTH responsiveness decreased by 67.3% compared with control nonpreincubated cells (P < 0.001; n = 3; –/–; ; difference between ACTH stimulation and basal level under control conditions compared with difference between ACTH stimulation and basal level after ACTH pretreatment. Sucrose pretreatment completely reversed hMC2R loss of responsiveness and restored receptor responsiveness to control levels. These results indicate that hMC2R internalization occurs via clathrin-coated pits, and that hMC2R desensitization and internalization are indeed interrelated.
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) internalized into intracellular vesicles after 30 min of 10 nM ACTH stimulation (Fig. 11B). In cells pretreated with 10 µM H-89 (Fig. 11C) and subsequently stimulated with 10 nM ACTH, the entire hMC2R population clearly remained localized at the cell membrane, indicating that H-89 completely abolished agonist-induced receptor sequestration (Fig. 11D). In contrast, pretreatment with the PKC inhibitor (1 µM GF109203X) did not modify the process of internalization (Fig. 11, F vs. E). However, intracellular labeling displayed a different pattern compared with that under basal conditions; the puncta were visible mostly in the perinuclear zone and appeared as large vesicles compared with the small and diffuse vesicles observed under control conditions (Fig. 11, F vs. B).
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We were also able to confirm the presence and localization of hMC2R protein by indirect immunofluorescence studies. Moreover, the extracellular c-Myc epitope located at the N terminus of hMC2R in conjunction with a permeabilization-free immunofluorescence approach allowed us to determine not only the presence of the hMC2R, but also its correct orientation in the cell membrane. Fusing a small tag, such as Flag, hemagglutinin, or c-Myc, to GPCRs is a common strategy used to bypass the lack of specific antibody (26, 30). Interestingly, the hMC2R protein appears to be localized in discrete submembrane domains, as previously described for endothelin receptor type A (36). Other GPCRs, such as ß2AR (37), angiotensin II type 1A receptor (38), and cholecystokinin receptor type A (39), have been shown to be distributed evenly throughout the plasma membrane.
Heterologous hMC2R was also found to be functional in terms of stimulation of cAMP accumulation, with EC50 values ranging from 7.6–11.9 nM, comparable to previously reported data (19, 40, 41, 42). Moreover, hMC2R heterologously expressed in M3 cells demonstrated a biological activity comparable to that in primary cultures of rat glomerulosa and fasciculata cells (27). M3 cells did not express endogenous mouse MC2R, as confirmed by RT-PCR (data not shown), but did express endogenous MC1R, which has a weak affinity for ACTH (EC50, 100 nM). The threshold ACTH concentration necessary to induce a cAMP response in native M3 cells was 30 nM compared with 0.3 nM in hMC2R-transfected cells. Moreover, maximal cAMP stimulation in native M3 cells was 6-fold compared with 25-fold stimulation in hMC2R-transfected cells. Thus, cAMP levels in response to ACTH stimulation in this study cannot be attributed to background ACTH response due to endogenous MC1R. Together, these data indicate that the biological properties of M3 hMC2R-transfected cells are similar to those of the MC2R expressed in adrenocortical cells (12), thereby enhancing the value of this model for the study of hMC2R regulation.
The second portion of this study focused on hMC2R short-term regulation and involvement of second messenger-dependent kinases in receptor functionality. Cells were tested for their ability to respond to a second ACTH challenge after a 15-min preexposure to various concentrations of ACTH or for various durations using one elevated concentration of ACTH. ACTH pretreatment induced a significant dose- and time-dependent decrease in sensitivity to subsequent ACTH exposure, indicating a dose- and time-dependent desensitization of hMC2R. Maximal hMC2R desensitization was reached with 10 nM ACTH pretreatment, consistent with a previous study of murine MC2R (43).
The roles of PKA and PKC in GPCR desensitization are well known. PKA contribution to GPCR desensitization has been largely described for ß2AR containing two canonic PKA phosphorylation sites (44, 45). Involvement of PKC phosphorylation in desensitization has also been reported for many Gi- and Gs-linked GPCRs, such as the ß2AR (46), and, more recently, for the D2 dopamine receptor (47) and the muscarinic acetylcholine receptor (48). As shown previously, PKA is necessary for inducing sustained steroid secretion (49), but has also been described in desensitization of murine MC2R (43). Furthermore, in keeping with the effect of ACTH on adrenal steroidogenesis, one study reported PKC involvement in ACTH-induced attenuation of aldosterone secretion after a 4-h treatment of bovine glomerulosa cells (50).
In light of the above observations, we verified whether PKA and/or PKC could participate in the process of ACTH-induced cAMP desensitization using well-known pharmacological agents. Firstly, pretreatment with H-89, but not GF109203X, increased cAMP accumulation after a single exposure to ACTH. This indicates an involvement of PKA in the regulation of basal hMC2R responsiveness and suggests a very early onset of PKA-induced loss of hMC2R responsiveness, which may be present even in the basal state. A similar observation was reported by Swords et al. (51); the researchers proposed a model in which only a small population of receptors was in an active state capable of generating signals leading to a desensitization process. Our results support such a hypothesis, because hMC2R did not display any detectable constitutive activity (data not shown), also consistent with previous studies (51, 52). However, this minimal fraction of active receptors would be sufficient to induce PKA activation.
Secondly, preincubation with the PKC inhibitor GF109203X reestablished hMC2R responsiveness to ACTH. In addition, coincubation with both PKA and PKC inhibitors had a synergistic effect on the reversal of hMC2R desensitization and enhanced ACTH responsiveness even above that of control, nonpreincubated cells. These results indicate that both kinases play an important role in hMC2R desensitization and act in synergy, supporting the hypothesis of a constitutive desensitization effect of both kinases on hMC2R activity in the basal state.
The next step involved determining whether hMC2R undergoes agonist-induced internalization in addition to receptor desensitization, because both phenomena are interconnected in prototypical GPCR (for review, see Refs.13 and 14). Time-course studies indicated maximal sequestration of hMC2R occurring after a 30-min exposure to ACTH. This process was relatively slow compared with ß2AR, which exhibits maximal sequestration after only 15 min of stimulation (53). Similar delayed internalization kinetics were observed for hMC4R (54). hMC4R internalization involves both PKA and G protein-coupled receptor kinase (GRK) phosphorylation of residues in the C-terminal tail, whereas hMC2R does not possess potential site for GRK phosphorylation in the C-terminal tail. Indeed, the only serine residue (Ser294) is not localized in a serine/threonine cluster or in proximity of an acidic amino acid such as aspartate or glutamate, conditions typically associated with serine-threonine residue phosphorylation by GRKs (55, 56). Although we did observe partial recycling of the receptor after 60 min of agonist treatment, this recycling to the membrane was not accompanied by receptor resensitization. One could speculate that the recycled receptor requires resensitization by a specific protein. However, the exact mechanism involved in this process remains to be investigated.
Internalization of hMC2R was shown in this study to be clathrin dependent, as demonstrated through the use of sucrose, a specific inhibitor of clathrin-coated pit formation (57, 58, 59, 60). Moreover, the internalized receptor colocalized with ß-arrestin-2-GFP in endocytic compartments, suggesting the involvement of ß-arrestin-2 in this internalization process. Another member of the MCR family, hMC4R, is also internalized in a ß-arrestin-1-dependent manner (59). In contrast, studies with murine MC2R in Y1 cells have described an internalization process that is clathrin dependent, but ß-arrestin independent (61). Collectively, the present data nevertheless suggest that hMC2R displays a desensitization and internalization pattern typical of the majority of GPCRs, such as the ß2AR (62).
The relationship between desensitization and internalization of GPCRs remains controversial. In the case of ß2AR, there is a general consensus that these two processes are independent of one another (44). In contrast, our results indicate that hMC2R desensitization and internalization are strongly interrelated processes, because inhibition of clathrin-dependent internalization by sucrose treatment completely reversed receptor desensitization. We thus addressed the potential involvement of second messenger-dependent kinases (which control the loss of receptor responsiveness) in hMC2R internalization. In contrast to receptor desensitization, sequestration appears to be differentially controlled by these two kinases. Although the role of second messenger-activated kinases has typically been studied in the context of GPCR desensitization, their involvement in receptor sequestration has only recently begun to emerge. In the case of the secretin receptor, PKA induces receptor phosphorylation and internalization, but not desensitization (63, 64). In contrast, PKA is involved in both the desensitization and sequestration of the ß1AR (65). Our findings indicate that agonist-induced internalization of the hMC2R appears to be primarily dependent on PKA, in contrast to murine MC2R, where PKA was shown to have no influence on internalization (61). These results suggest that GPCR sequestration is most likely species specific. Moreover, according to the present data, PKA is not only necessary for hMC2R desensitization, but also plays a critical role in the internalization of hMC2R.
Conversely, our results suggest that PKC is not required for hMC2R internalization. Such differential involvement of second messenger kinases has been shown for ß2AR, where PKA-mediated phosphorylation induces receptor desensitization, but is not required for its sequestration (53). Thus, the role of PKC in hMC2R regulation is restricted to the desensitization process, as opposed to the D2-dopamine receptor (47), for which both PKC-induced desensitization and sequestration were reported. It should be emphasized that despite preservation of hMC2R sequestration in the presence of the PKC inhibitor, there was a marked difference in the morphology and intracellular distribution of vesicles compared with those with agonist incubation alone. The larger size and perinuclear localization of the puncta observed during inhibition of PKC are typical of lysosomes. This would indicate that proper intracellular trafficking of the hMC2R, once agonist-induced sequestration has begun, requires intact PKC activity.
The level of hMC2R expression at the cell membrane represents dynamic steady-state equilibrium. To ensure this equilibrium, the population of functional hMC2R on the cell surface represents the sum of proteins residing in the membrane and those recycled into intracellular compartments. According to this study, this process is tightly regulated by PKA, which plays the essential role of early receptor desensitization as well as its sequestration. The other second messenger-regulated kinase, PKC, enhances hMC2R desensitization and guides intracellular trafficking, but is not involved in the onset of receptor internalization. Thus, the amount of functional membrane receptors is differentially regulated by both kinases. The establishment of an equilibrium among mobilization of available receptors from intracellular pools, desensitization by PKA and PKC, and internalization by PKA could represent essential mechanisms ensuring adequate ACTH responses in various situations where glucocorticoid secretion requires a high level of regulation, allowing, in turn, the adequate control of glycolytic homeostasis, metabolism, and responses to stress.
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Z.K., N.B., P.K., M.D.P., and N.G.P. have nothing to declare.
First Published Online February 23, 2006
Abbreviations: ß2AR, ß2-Adrenergic receptor; DAPI, 4',6-diamido-2-phenylindole hydrochloride; GFP, green fluorescence protein; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; h, human; HBS, Hanks’ balanced solution; IBMX, isobutylmethylxanthine; MCR, melanocortin receptor; PKA, protein kinase A; PKC, protein kinase C.
Received August 3, 2005.
Accepted for publication February 3, 2006.
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