Melanocortin 2 receptor accessory protein plasmids that have not been described previously (4, 8, 11) were constructed using QuikChange from Stratagene with standard molecular biological techniques and verified by sequencing. MRAP–V5–3xFlag contained mMRAP followed by GKPIPNPLLGLDSTGRDYKDHDGDYKDHDIDYKDDDDK at the C-terminus. AP–MRAP contained the minimal AP sequence GLNDIFEAQKIEWHE immediately after the initiating Met followed by mMRAP or mMRAP–V5–3xFlag. MRAP–AP contained the same AP sequence at the C-terminus of mMRAP or mMRAP–V5–3xFlag followed by a stop codon. MRAPs containing the AP sequence but no epitope tags were constructed similarly. On figures, these constructs are shown as AP–MRAP or MRAP–AP and epitope tags are described in the legends. V5–MART1–AP and AP–MART1–V5 were constructed by adding V5 epitope (GKPIPNPLLGLDST) and AP sequence (GLNDIFEAQKIEWHE) to opposite ends of mMART1. (4K to R)MRAP refers to V5–mMRAP–3xFlag with the four Lys in native mMRAP mutated to Arg. 3xHA-hMC2 receptor, referred to as MC2 receptor, was from Missouri cDNA Resource Center and plasmid encoding eGFP from Clontech. BirA in pcDNA3 and BirA_ER in pDisplay vectors were generously donated by Dr. Alice Ting (Massachusetts Institute of Technology) and CRE-luciferase by Dr. George Holz (SUNY Upstate Medical University). Plasmid encoding HA-ubiquitin (HA-Ub) has been described (17). Monoclonal antibodies were from the following: Sigma (M2 anti-Flag and immobilized M2 anti-Flag), Covance (HA-11 anti-HA), and AbDSerotec (anti-V5). Streptavidin–HRP and streptavidin–agarose were from Thermo Scientific, immobilized Protein A/G beads from Santa Cruz Biotechnology, and HRP-labeled antibody against mouse heavy and light chains from BioRad.

CHO cells were from American Type Culture Collection and OS3 adrenal cells (18) from Dr. Bernard Schimmer (University of Toronto). CHO and OS3 cells were maintained in DMEM/F12 medium containing 5% fetal bovine serum at 37°C in 5% CO₂-95% air and passaged with trypsin. Transfection was performed using FugeneHD or Lipofectamine 3000 according to manufacturers’ instructions. Stable cell lines were generated by transfecting with plasmids encoding an MRAP, BirA, or BirA_ER and selecting with 1 mg/ml G418. Pooled cells were used for experiments. Biotin-labeling experiments were performed by adding 2.5 μM biotin to complete medium at the time of transfection unless otherwise noted.

In brief, cells were grown in white 96-well plates and transfected with 40–50 ng total DNA/well, usually with equal parts of MC2 receptor, MRAP construct and CRE-luciferase, a reporter containing multiple cAMP response elements from the rat insulin promoter (19). After 24 h cells were challenged for 4–5 h with hACTH(1–24) or 20 μM forskolin in DMEM with 0.1% BSA. Medium was replaced with Firefly Reagent from Nanolight and luminescence read in a BioTek platereader. Unless noted, results are expressed as percent of the forskolin response measured in the same experiment.

Cells were washed with PBS and lysed on ice in Tris/Mg/EGTA buffer (150 mM NaCl, 50 mM Tris-Cl, 1 mM EDTA, and 1% Triton X-100, pH 8.0) containing protease inhibitors. Iodoacetamide (10 mM) was included in lysis buffers when ubiquitin labeling was being assessed. Samples were centrifuged at 10,000 × g for 20 min to remove nuclei and supernatants used for analysis. In some cases, HA- or Flag-labeled proteins were immunoprecipitated by incubating overnight at 4°C with 1:1000 dilutions of antibody and collected on Protein A/G beads. Biotin-labeled proteins were pelleted following overnight incubation with streptavidin–agarose beads. Samples were taken up in NuPage LDS sample buffer with a final concentration of 50 mM dithiothreitol. For analysis by SDS-PAGE samples were run on Lonza PagR or BioRad TGX gels. Proteins were transferred to nitrocellulose, blocked in TBST (Tris-buffered saline with 0.05% Tween 20) with 5% non-fat dry milk, and incubated overnight at 4°C in either monoclonal anti-V5, anti-HA, or anti-Flag antibody at 1:5000 in TBST/milk. After washing, antibody blots were incubated in 1:5000 HRP-anti-mouse heavy and light chain, washed and visualized using Western Lightning chemiluminescent reagent. To visualize biotin-labeled proteins, blots were incubated in 1:10,000 HRP–streptavidin in TBST with 0.5–1% BSA, because milk contains biotin, washed and developed. In some cases, blots were subsequently incubated in 0.1% sodium azide to inactivate HRP and blotted with antibody as described above.

Expression of proteins on the plasma membrane was quantified as described previously (4, 11). Cells grown in 12- or 24-well plates were fixed with 2 or 3% paraformaldehyde for 10–20 min at room temperature, washed with PBS, and incubated for ~1 h with 1:5000 monoclonal anti-HA, anti-V5, or anti-Flag antibodies in PBS containing 5% non-fat dry milk. Cells were then washed extensively and incubated for ~1 h with 1:5000 HRP-labeled anti-mouse IgG, and washed prior to the addition of tetramethylbenzidine substrate (Sigma) and measurement of absorbance at 450 nm.

For microscopy, CHO cells stably expressing BirA_cyt or BirA_ER were grown on glass coverslips and transfected with AP-tagged MRAP constructs. After overnight incubation, live cells were incubated with anti-Flag antibody (1:250 in serum-containing medium) for 30 min at 37°C, washed, and then incubated with 1:500 Alexa488-labeled anti-mouse M2 and 1:500 Alexa555-labeled streptavidin and incubated for 30 min at room temperature. Cells were washed, fixed in paraformaldehyde, washed again and mounted in Prolong Gold (InVitrogen), and viewed in a Nikon epifluorescence microscope with a 40× oil objective. Images showing Flag and streptavidin were obtained using 500 and 2500 ms exposures, respectively. All Flag and all streptavidin micrographs were processed identically.

To deglycosylate proteins, cell lysates were denatured and incubated with PNGaseF from New England Biolabs following manufacturer’s instructions; control lysates were incubated identically in reactions lacking enzyme. All experiments were repeated a minimum of two times. Error bars indicate mean and range or SE of duplicate or triplicate determinations in representative experiments. Significance of differences between two values was determined by Student’s t-test.

The strategy used to determine whether MRAP is first synthesized in dual orientations is outlined schematically in Figure 1A. Biotin labeling of a protein modified to contain a decarboxylase biotin acceptor domain was used previously to characterize membrane insertion of bacterial lactose permease (20). The biotin-labeling approach described here uses a small acceptor sequence and exogenous BirAs to provide a new means to analyze MRAP architecture. The bacterial enzyme biotin ligase (BirA) attaches a biotin residue to the single Lys residue in a specific acceptor peptide sequence (AP). When expressed in mammalian cells, bacterial biotin ligase is localized in the cytoplasm; addition of a carboxyterminal KDEL sequence targets the enzyme to the ER (21, 22). Because there are relatively low concentrations of biotin in standard serum-supplemented tissue culture media, it is straightforward to initiate biotin labeling of a protein tagged with an AP sequence by adding exogenous biotin. Mouse MRAP contains a single N-linked glycosylation site at Asn3 of the aminoterminus and N-glycosylation occurs exclusively in the inside of the ER and Golgi apparatus, which is topologically equivalent to the exoplasmic face of the plasma membrane. Glycosylated MRAP, which migrates more slowly than non-glycosylated MRAP on SDS-PAGE, must, therefore, be in the N_exo/C_cyt orientation unless it has changed orientations during trafficking. We engineered MRAPs with an AP sequence at either the aminoterminal or carboxyterminal end of the protein; each of these MRAP constructs also contained a Flag epitope. As shown in Figure 1B, control MRAP, AP–MRAP (N-terminal AP sequence), and MRAP–AP (C-terminal AP sequence) all supported comparable ACTH-stimulated increases in a cAMP reporter.

MRAP, AP–MRAP, and MRAP–AP were expressed in CHO cells stably expressing either cytoplasmic BirA or ER-targeted BirA_ER. The upper left panel of Figure 2 shows a Flag immunoblot identifying total MRAP. In every case, monomeric glycosylated and non-glycosylated MRAP species ran as a doublet at ~20 and 25 kDa. Enzymatic degradation with the glycosidase PNGaseF confirmed that the two major bands represented MRAP with and without N-linked glycosylation (4) and Figure 11 below. Higher MW bands are likely MRAP multimers and their mobility shifts following PNGaseF treatment (not shown). The upper right panel of Figure 2A shows the same blot probed with streptavidin to detect only biotin-labeled proteins. Despite having identical molecular weights, AP–MRAP and MRAP–AP consistently displayed different mobilities on SDS-PAGE, as do AP–MART1 and MART1–AP (Figure 3 below). It is possible that the proteins undergo different post-translational modifications but the behavior of membrane proteins on SDS-PAGE is not always predictable.

Endogenous biotinylated carboxylases are visible in all lanes at high molecular weight (75 kDa and above) in the blot on the upper right. These metabolically labeled proteins do not depend on transfected BirA. The three control lanes shown on the left of each blot in Figure 2 demonstrate that MRAP lacking the biotin acceptor peptide was expressed well but not labeled with biotin. Cytoplasmic BirA catalyzed the addition of biotin to the faster-migrating, non-glycosylated form of AP–MRAP (situation a in Figure 2B), whereas ER-targeted BirA_ER added biotin to the higher molecular weight glycosylated form of AP–MRAP (situation b). The reverse was found when cytoplasmic BirA and ER-targeted BirA_ER were expressed with MRAP–AP. In this case, BirA_cyt preferentially labeled glycosylated MRAP–AP and BirA_ER preferentially labeled non-glycosylated MRAP–AP (situations c and d, respectively). These results support the conclusion that the great majority of MRAP molecules achieve an antiparallel orientation when first synthesized. The finding of some biotin-labeled but non-glycosylated AP–MRAP with BirA_ER could result from (1) biotin labeling of AP–MRAP that is incompletely glycosylated, (2) incomplete import of overexpressed BirA_ER into the ER leaving some active enzyme in the cytoplasm, or (3) a change in orientation of a small portion of MRAP after synthesis. The finding of a biotin-labeled and glycosylated MRAP–AP band with BirA_ER is consistent with incomplete BirA_ER import. After overnight incubation in biotin-supplemented media, essentially all AP-tagged MRAP could be collected on streptavidin–agarose beads, implying nearly quantitative biotinylation.

As an additional control for this approach, we studied MART1 (also called Melan-A), a melanosome protein with a single transmembrane domain, strongly predicted N_exo/C_cyt orientation, and size close to that of mouse MRAP (18 vs. 14 kDa without epitopes or carbohydrate). We engineered plasmids encoding MART1 with AP tags at one end or the other and V5 tags at the opposite ends. We used a previously described fixed cell ELISA protocol (4) in which antibodies were added to intact cells to detect epitopes localized on the outside of the plasma membrane (Figure 3A). MART1 was found in the predicted N_exo/C_cyt orientation, whereas MRAPs with the AP sequence at either end were found in dual N_exo/C_cyt and N_cyt/C_exo orientations. MRAP but not MART1 increased surface expression of co-expressed MC2 receptor (Figure 3B). MART1 was then tested in an experiment identical to that shown in Figure 2 for MRAP. AP–MART1 was expressed strongly but labeled only by ER-localized BirA_ER and labeling was rather weak (Figure 3C). By contrast, MART1–AP was expressed weakly but labeled strongly by cytoplasmic BirA and slightly by BirA_ER. Labeling by BirA_ER probably occurred because of incomplete import of BirA_ER into the ER, which left some biotin ligase in the cytoplasm.

To isolate MRAP that had trafficked to the plasma membrane, we localized cell surface biotin-labeled MRAP by fluorescence microscopy. Cells stably expressing cytoplasmic or ER-targeted BirA were transiently transfected to express either AP–MRAP–Flag or MRAP–Flag–AP. After incubation with biotin, live cells were stained with monoclonal anti-Flag antibody or fluorescent streptavidin. Because MRAP forms an antiparallel structure, both AP–MRAP–Flag and MRAP–Flag–AP were detectable on the cell surface with anti-Flag antibody, as expected (Figure 4A). On the other hand, if AP–MRAP–Flag and MRAP–Flag–AP did not change orientations following biosynthesis, only ER-localized BirA_ER would be able to label acceptor peptide in the ER lumen and, after trafficking, the extracellular side of the plasma membrane (Figure 4B). This result was obtained, with cell surface MRAP clearly labeled in all configurations but cell surface biotin-labeled MRAP visible only in cells expressing BirA_ER. The results of this experiment are not confounded by incomplete import of BirA_ER into the ER.

A kinetic approach was also undertaken to determine whether MRAP changes orientation after biosynthesis (Figure 5). To avoid the ambiguity present in experiments using BirA_ER, cells were transfected with cytoplasmic BirA and either AP–MRAP or MRAP–AP and incubated overnight in the absence of added biotin. During this period MRAP synthesis took place with very little biotin labeling (zero time point). Cells were then treated with added biotin for times up to 24 h. Immunoblotting for total AP–MRAP and MRAP–AP with anti-Flag antibodies is shown in the left panels. As seen previously (Figure 2A), cytoplasmic BirA labeled the faster-moving non-glycosylated AP–MRAP band. Increased biotin labeling could be detected as early as 6 min after biotin supplementation and labeling continued over time; after 24 h, there was only a trace amount of a higher molecular weight biotin-labeled AP–MRAP (Figure 5A). This result does not support the idea that MRAP flips orientations after synthesis. In the case of MRAP–AP, a minor fraction of non-glycosylated biotin-labeled MRAP was visible but the proportion did not increase with time, indicating that MRAP does not gradually flip but rather that a small amount of N_exo/C_cyt MRAP does not become fully glycosylated (Figure 5B).

To characterize MRAP topology in its natural setting, we took advantage of the OS3 line of mouse adrenal cells that does not express MC2 receptors. ACTH responses were measured in parallel in OS3 and CHO cells transfected with the cAMP reporter CRE-luciferase with or without MC2 receptor and MRAP (Figures 6A,B). ACTH stimulated a 26-fold increase in the cAMP response in CHO cells expressing MRAP and MC2 receptor. Mock-transfected OS3 cells did not respond to ACTH, consistent with the reported absence of MC2 receptors (18). When the adrenal cells were transfected with MRAP and MC2 receptor, ACTH produced a striking 273-fold increase in cAMP reporter activity. OS3 cells must express MRAP, because MC2 receptors are functional when transfected alone. Interestingly, responses to forskolin were also exceptionally strong in OS3 cells. Forskolin acts directly on adenylyl cyclase and amplifies both basal and Gs-stimulated enzyme activity. Together, these findings suggest that adrenal OS3 cells have unusually efficient signal transduction at the G protein/adenylyl cyclase level.

We also explored MRAP orientation and the effects of MC2 receptor and ACTH (Figures 6C,D). OS3 cells and parallel cultures of CHO cells were transfected with BirA and AP-tagged MRAPs with or without MC2 receptors and incubated overnight with or without 10 nM ACTH. For reasons that are not clear, expression of AP–MRAP was higher when MC2 receptor was co-expressed and that of MRAP–AP was lower. ACTH had little effect. Biotin labeled the non-glycosylated AP–MRAP band, as predicted if topology does not change. In OS3 cells, there was a non-specific biotin-labeled protein at ~37 kDa that did not correspond to any MRAP band. MRAP–AP was weakly expressed in these cells and biotin-labeled species were partially obscured by the non-specific band.

Having established that MRAP topology is stable, we examined the overall stability of the accessory protein. The stability of MRAP was estimated in CHO cells stably expressing Flag-tagged MRAP together with GFP or MC2 receptor. Cultures were treated for 4 h with the protein synthesis inhibitor cycloheximide (CHX), the proteasome inhibitor MG132 or both (Figure 7A). MRAP was quite labile, with little protein remaining 4 h after protein synthesis blockade. Surprisingly, MG132 by itself caused an enormous increase in the concentration of MRAP. On the other hand, MG132 did little to protect MRAP from degradation when CHX was present. A similar pattern was seen with the MC2 receptor, although the rate of degradation was slower (half-life between 3 and more than 8 h in different experiments) and effect of MG132 alone was much less marked. Mature MC2 receptor, which contains multiple sites for N-linked glycosylation (23, 24), ran as a broad band at high molecular weight, whereas immature core glycosylated and non-glycosylated receptor ran in more distinct bands close to the predicted molecular weight of 3xHA-hMC2 receptor, 37.3 kDa. Figure 7B shows a similar experiment demonstrating the rapidity of CHX and MG132 effects. The half-life of MRAP was estimated in similar experiments in which CHO cells were incubated with CHX from 0 to 5 h and MRAP levels were quantified densitometrically in immunoblots assuming first order kinetics. In most experiments, glycosylated MRAP was lost less rapidly than the non-glycosylated form, but this was not entirely consistent. The half-life of total MRAP averaged 1.7 ± 0.5 h (n = 7). The half-life of MRAP was short when it was transiently expressed with or without MC2 receptors in CHO cells but longer in adrenal OS3 cells, which grow less rapidly than CHO cells (Figure 7C). To study turnover of MRAP on the plasma membrane, CHO cells were incubated for 1 h with protein synthesis or proteasome inhibitors and then either lysed or incubated with antibody to label cell surface MRAP selectively (Figure 7D). The effects of CHX and MG132 were not as extreme but clearly present when plasma membrane MRAP was isolated.

The use of a global protein synthesis inhibitor to study degradation of a particular protein is complicated by potential effects on proteins other than the one under study. To avoid this problem, we studied cells stably expressing cytoplasmic BirA and then measured the amount of biotinylated-MRAP–AP at intervals after the removal of extracellular biotin with or without addition of CHX (Figure 8). Since biotin cannot be removed from a protein until it has been thoroughly degraded, this approach is conceptually equivalent to a radiolabeling pulse-chase experiment. For a low abundance protein, the method has the advantage of labeling only the protein of interest that can be concentrated on streptavidin beads prior to analysis. A limitation is the delay caused by the time required for washout of intracellular biotin. Following biotin withdrawal, total MRAP pools were quite stable over 20 h while normal synthesis and degradation were ongoing. The half-life of biotin-labeled MRAP was only 2.8 h, however, indicating that MRAP has a short half-life. In the presence of CHX, the corresponding values were 2.8 h for the total MRAP pool and 2.2 h for biotin-labeled MRAP. Because the half-lives measured by metabolic labeling and protein synthesis inhibition were strikingly similar, rapid turnover of MRAP was not an indirect effect of CHX.

In light of the effect of MG132, we investigated the possibility that MRAP itself undergoes ubiquitin-mediated degradation. In the canonical pathway ubiquitin is added to Lys residues and mouse MRAP has only four lysines, all located on the N-terminal side of the transmembrane helix in the juxtamembrane region. To test the importance of Lys to overall MRAP stability, we mutated these four residues to Arg (4K to R), retaining positive charge but removing lysine ɛ-amino groups that can be ubiquitylated. Arg-substituted MRAP was tagged with a V5 epitope on the N-terminus and a Flag epitope on the C-terminus, enabling analysis of its topology. Although (4K to R)MRAP was not expressed as highly as wild-type MRAP, it still assumed dual topology with both V5 and Flag epitopes on the plasma membrane (Figure 9A). Despite a lower level of MC2 receptor on the plasma membrane, cells expressing (4K to R)MRAP responded to ACTH with a cAMP increase equivalent to that supported by wild-type MRAP (Figure 9B). MG132 by itself caused an enormous increase in (4K to R)MRAP concentration just as it did with wild-type. Replacing all of the MRAP Lys residues with Arg delayed MRAP degradation by at most threefold (Figure 9C).

We tested whether ubiquitin is directly added to MRAP lysines by co-expressing HA-ubiquitin with either wild-type or (4K to R)MRAP in CHO cells and incubating for 3 h with MG132 to allow accumulation of any ubiquitylated species. Co-immunoprecipitation and immunoblotting revealed that Flag-tagged MRAP undergoes ubiquitin modification (Figure 10A). Multiple ubiquitylated MRAP bands were observed and only one of them was lost in the Arg-substituted mutant, suggesting that ubiquitin is added to Lys and also to one of the amino acids less frequently ubiquitylated: the aminoterminal amino group, the hydroxyl group of Ser or Thr, or the sulfhydryl group of Cys (25). On average, ubiquitylated MRAP species totaled ~5% of total MRAP after incubation for 3 h with a proteasome inhibitor.

To rule out the possibility that MRAP ubiquitylation was occurring on one of the Lys residues in the Flag sequence, we tested AP–MRAP and AP-(4K to R)MRAP that contained the biotin acceptor peptide sequence but no epitope tag in cells expressing cytoplasmic BirA (Figure 10B). Once labeled with biotin, the AP tag has no lysine amino group available for ubiquitin addition. Once again, multiple ubiquitylated MRAP bands were observed and only one was lost in the Arg-substituted mutant, suggesting that ubiquitin was added to MRAP at an atypical site.

The glycosylation status of various MRAP bands was analyzed in lysates of cells expressing HA-Ub and MRAP before and after enzymatic deglycosylation with PNGaseF. None of the ubiquitylated MRAP bands was lost following PNGaseF treatment, whereas the slower-migrating MRAP bands collapsed to the mobility of the faster-migrating non-glycosylated species (Figure 11).