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Molecular Endocrinology, doi:10.1210/me.2007-0041
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Molecular Endocrinology 21 (7): 1656-1669
Copyright © 2007 by The Endocrine Society

Differential Regulation of the Human Adrenocorticotropin Receptor [Melanocortin-2 Receptor (MC2R)] by Human MC2R Accessory Protein Isoforms {alpha} and ß in Isogenic Human Embryonic Kidney 293 Cells

Simon Roy, Mohamed Rached and Nicole Gallo-Payet

Service d’Endocrinologie, Département de Médecine, 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 Ave Nord, Sherbrooke, Québec, Canada J1H 5N4. E-mail: Nicole.Gallo-Payet{at}USherbrooke.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ACTH receptor [melanocortin-2 receptor (MC2R)] is the smallest known G protein-coupled receptor (GPCR). Herein, human MC2R accessory protein (MRAP) isoforms {alpha} and ß, cloned from a human fetal adrenal gland, were expressed with c-Myc-tagged MC2R (Myc-MC2R) in 293/Flp recombinase target site cells by homologous recombination. Although insertion of Myc-MC2R at the plasma membrane occurred without MRAP assistance, ACTH stimulation of cAMP production was only detected in cells coexpressing MC2R with either MRAP isoform. On the other hand, a MC2R-green fluorescent protein fusion transfected with either MRAP{alpha} or MRAPß was impaired both in cell membrane localization and signaling. MRAP isoforms were also tagged with either Flag or 6xHis epitopes. In cell populations coexpressing transiently and/or stably Myc-MC2R with MRAP{alpha} or MRAPß, stimulation with ACTH induced production of cAMP with EC50 values lower in MRAP{alpha}- than in MRAPß-expressing cells. ACTH only bound Myc-MC2R in the presence of MRAP. Higher Myc-MC2R cell surface density was observed in the presence of MRAPß comparatively to MRAP{alpha}, possibly contributing to higher ACTH binding capacity and higher maximal cAMP responses observed in MRAPß-expressing cells. Immunofluorescence studies indicated that MRAP isoforms were localized near the plasma membrane and in the vicinity, but not colocalized, with Myc-MC2R. In summary, through the generation of a new all-human experimental model devoid of endogenous MCRs, we present evidence that human MRAP isoforms, although not essential for MC2R localization at the plasma membrane, are essential for ACTH binding and ACTH-induced cAMP production and that they differentially regulate, although modestly, cell membrane density and functional properties of MC2R.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ACTH, THROUGH THE melanocortin-2 receptor (MC2R), is the most potent trophic stimulus of the adrenal cortex, stimulating both steroidogenesis and protein synthesis (1, 2). By stimulating aldosterone and glucocorticoid synthesis and secretion (corticosterone in rodents and cortisol in humans and bovine), ACTH binding to MC2R plays a pivotal role in homeostasis, metabolism, and stress response (3). MC2R is the second member of the five known melanocortin receptors (MCRs) belonging to the Rhodopsin-like family of G protein-coupled receptors (GPCRs) (4, 5). These five MCRs constitute a distinct family of GPCRs, characterized by their unusually short coding sequence and the absence of highly conserved amino acid residues or motifs common to most GPCRs (6, 7, 8). MC2R is both the smallest MCR and the smallest known GPCR (297 amino acids). In comparison with other MCRs, MC2R is unique in that it binds only to ACTH and does not possess affinities for other melanocortins ({alpha}-, ß-, and {gamma}-MSH). On the other hand, other MCRs are activated by both ACTH and MSHs (mainly {alpha}-MSH) (for review see Refs. 5 , 7 , and 8).

All known MCRs are coupled to Gs proteins, thus stimulating adenylyl cyclase, resulting in cAMP production and protein kinase A activation (8). Although numerous studies have been conducted on ACTH action in the adrenal gland, several aspects in the various steps from its initial binding up to ensuing cell responses remain poorly understood (9). In contrast to most GPCRs, difficulties linked to the specific functional expression of MC2R have made it impossible to express MC2R in a system that is free of any endogenous MCR expression (10). Indeed, successful transfection studies are limited to those cell lines that are known to express one of the MCRs such as in Cloudman melanoma 591 (M3) cells (11, 12, 13), African green monkey kidney cell line COS-7 cells (14), or the mutant adrenocortical cell lines Y6 (15) and OS3 (16). However, as reported for other GPCRs (17, 18), MCRs can form constitutive homo- and heterodimers (19, 20). Because MC3R and MC4R are also expressed in the adrenal gland (21, 22) and mouse Mc1r is expressed in M3 cells (13), such interactions could impede efforts to better understand the exact nature of MC2R’s structure-function relationship.

Thanks to the recent discovery of the MC2R accessory protein, MRAP (23), MC2R heterologous expression difficulties can now be overcome. Mouse Mrap, a transmembrane protein, was first identified in adipocytes after differentiation of mouse 3T3-L1 fibroblasts (24) and initially named fat tissue low-molecular weight protein. Fat tissue low-molecular weight protein has since been renamed "melanocortin-2 receptor accessory protein" (designated henceforth as human MRAP and mouse Mrap). Interaction between Mc2r-GFP (green fluorescent protein) (GFP fused to the C terminus of Mc2r) and Mrap-Flag (Mrap tagged at its C terminus with Flag) has been described in the Chinese hamster ovarian cell line (CHO-K1) and in the human Caucasian bone marrow neuroblastoma cell line (SK-N-SH) (23). Mrap is known to increase ACTH responsiveness in some cell lines (23, 25). In humans, there are two distinct forms of MRAP proteins, isoform {alpha} (exons 3–4-5, 172 amino acids) and isoform ß (exons 3–4-6, 102 amino acids) (23), both of which share the same N terminus and transmembrane domain (approximately from residue 38–58), but are otherwise highly divergent in their C termini. However, nothing is known regarding their functional relationship with MC2R.

The human embryonic kidney 293 (HEK293) cell line is known to be MCR free and to be nonresponsive to ACTH and NDP-MSH (26, 27). It has been reported by immunofluorescence studies that MC2R-GFP (GFP fused to MC2R C-terminus) does not reach the cell surface in these cells and thus is retained in intracellular compartments (10, 27). In the present study, the combination of our recently developed N-terminal-c-Myc-tagged human MC2R construction (Myc-MC2R) (13) and the Flp recombinase-mediated homologous recombination system in HEK293 cells [Flp-In system; 293/Flp recombinase target site (FRT) cell line] (28, 29) was used to generate isogenic populations expressing both Myc-MC2R with either of the appropriately tagged MRAP{alpha} or MRAPß isoforms. These various constructions were also used to investigate Myc-MC2R and MC2R-GFP expression at the cell membrane and the capacity of these cells to produce cAMP upon ACTH stimulation. Through a multifaceted approach consisting of cell-surface ELISAs, immunofluorescence studies, and cAMP and ligand-binding assays, our results provide evidence that the human MC2R alone is inserted in the plasma membrane, but fails to bind ACTH and to produce cAMP in the absence of MRAP isoforms. In addition, MRAP{alpha} and MRAPß confer specific and slightly different functional properties to MC2R.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Myc-MC2R Alone, But Not MC2R-GFP, Is Inserted at the Plasma Membrane of 293/FRT Cells
Localization of Myc-MC2R was assessed by indirect immunofluorescence and cell-surface ELISA in unpermeabilized 293/FRT cells expressing Myc-MC2R either transiently or stably. Immunofluorescence images were acquired at the plane of the nuclei (Fig. 1Go, A and C) or at the plane of cell adhesion to the coverslip (Fig. 1Go, B and D) to visualize only the area in close proximity to the cell membrane. Results reveal an intense fluorescence signal corresponding to the detection of the N-terminal c-Myc epitope of Myc-MC2R in both transient (Fig. 1Go, A and B) and stable (Fig. 1Go, C and D) conditions. Myc-MC2R was localized in discrete plasma membrane subdomains present at the cell surface, as evidenced by the punctuated fluorescence pattern (Fig. 1CGo, inset). As shown by cell surface ELISA measurements, when Myc-MC2R was transiently transfected at 61% efficiency, the level of cell surface expression was 3.6-fold higher than in stable isogenic cells, where more than 95% of the cells stably expressed Myc-MC2R at lower levels (P < 0.001 and P < 0.01, n = 3, respectively compared with control 293/FRT cells) (Fig. 1EGo). In contrast to Myc-MC2R, observations at both the nuclear and cell adhesion to coverslip planes in Fig. 1FGo and 1GGo, respectively, reveal that transient expression of MC2R-GFP in 293/FRT cells yielded fluorescence localized in the cytoplasm in a punctuate pattern. Hence, in 293/FRT cells, Myc-MC2R was inserted into the plasma membrane, but not MC2R-GFP.


Figure 1
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  		Fig. 1. Expression of Myc-Tagged Human MC2R in 293/FRT Cells

Myc-MC2R was transiently (A and B) or stably (C and D) expressed in 293/FRT cells, as described in Materials and Methods. Cells were fixed but not permeabilized before incubation with anti-c-Myc antibody and were detected using the antimouse Alexa-Fluor488 secondary antibody. Images were acquired at the nuclear plane (A and C) or at the cell adhesion to coverslip plane (B and D) and are representative illustrations of more than 100 cells from at least six experiments. Inset is a magnification (2.8-fold) of a portion of the cell membrane. Because large variations of signal intensity were observed from cell to cell in transiently transfected cells, low-expressing cells were selected for panels A and B and used in a qualitative approach. E, Transient and stable cell surface expression of Myc-MC2R as assessed by ELISA on unpermealized cells. Results represent the mean ± SEM of three experiments, each performed in triplicate. Statistical significance: **, P < 0.01; ***, P < 0.001, when compared with control conditions. F and G, MC2R-GFP was transiently overexpressed in 293/FRT cells and analyzed using the GFP reporter at the nuclear plane (F) and at the cell adhesion to the coverslip level (G). Scale bar: 10 µm for all panels and 3.6 µm for the insets.

 
MRAP{alpha} and ß Expression Is Required for cAMP Signaling of MC2R
We first verified whether human mRNA transcripts encoding MRAP{alpha} and MRAPß were present in the human 293/FRT cell line and in human fetal and adult adrenal glands. In 293/FRT cells, RT-PCR analysis was negative for the amplification of either transcript, whereas both MRAP isoform transcripts were present in human fetal adrenal gland as well as in zona glomerulosa and zona fasciculata of adult human adrenal glands (Fig. 2AGo).


Figure 2
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  		Fig. 2. Involvement of MRAP in ACTH-Induced cAMP Production

A, Reversed-transcribed RNA from indicated human specimens was used to amplify the entire coding sequences for MRAP{alpha} and MRAPß by RT-PCR (30 cycles, 3 µl load each), as described in Materials and Methods using cloning primers pairs listed in Table 1Go. Legend titles above lanes represent 293/FRT cells (293), human fetal adrenal gland of 18 wk-old (F), human zona fasciculata (ZF), and glomerulosa cells (ZG) of a 46-yr-old male. B, cAMP production in 293/FRT cells transiently cotransfected with pcDNA3/Myc-MC2R, pcDNA3/MRAP{alpha}, and pcDNA3/MRAPß combinations as indicated (0.4 µg /plasmid, total DNA fixed at 1.2 µg using pEGFP). Cells were stimulated with or without 100 nM ACTH for 15 min in the presence of 1 µM IBMX. Results represent the mean ± SEM of three experiments, each performed in triplicate. C, 293/FRT/MycMC2R cells were transiently transfected with pcDNA3/MRAP{alpha} or pcDNA3/MRAPß and assessed for cAMP production in the presence of saline (basal), 100 nM of NDP-MSH, ACTH 1–24, or ACTH 1–39. This experiment represents the mean ± SE of one experiment in triplicate. Statistical significance: ***, P < 0.001, when compared with control conditions.

 
Second, to assess ACTH responsiveness in terms of cAMP production, Myc-MC2R was transiently cotransfected in 293/FRT cells with either MRAP{alpha} or MRAPß. As shown in Fig. 2BGo, cells transfected with MRAP{alpha}, MRAPß, or Myc-MC2R alone did not respond to ACTH stimulation. ACTH responsiveness was detected only in cells coexpressing MC2R with either MRAP{alpha} or MRAPß, with 100 nM ACTH stimulation inducing 187 ± 5- and 192 ± 5-fold increases over their respective basal values (P < 0.001). Moreover, in stable 293/FRT/MycMC2R cells, transiently transfected with either MRAP isoform, ACTH 1–24 and 1–39 were equipotent in terms of cAMP production whereas NDP-MSH was unable to stimulate cAMP production in these cells (Fig. 2CGo). In addition, cells transfected with MRAPß had a tendency to generate greater cAMP accumulation after 15 min stimulation comparatively to cells transfected with MRAP{alpha}. Together, these results highlight that 293/FRT cells are devoid of endogenous MCR and MRAP expression and that ACTH only stimulates cAMP production when MC2R and MRAP are coexpressed, even if Myc-MC2R is readily present at the cell surface without MRAP assistance.

ACTH Responsiveness Remains Intact with Myc-MC2R, But Not with MC2R-GFP
The following experiments were designed to compare the ability of 293/FRT/MRAP{alpha} and 293/FRT/MRAPß cell lines (stably expressing MRAP{alpha} and MRAPß respectively), transfected with untagged-MC2R, Myc-MC2R, or MC2R-GFP, to respond to increasing concentrations of ACTH. As shown in Fig. 3AGo, ACTH induced similar dose-responses curves with untagged- and Myc-MC2R in both cell lines. As previously observed, maximal cAMP stimulation was lower in 293/FRT/MRAP{alpha} cells, reaching 154 ± 4- and 158 ± 4-fold increases over control for untagged- and Myc-MC2R, compared with 197 ± 1 and 188 ± 9 -fold increase, respectively, over basal values in the 293/FRT/MRAPß cells. The threshold concentration was 1 pM and 10 pM in MRAP{alpha} and MRAPß isogenic cell lines, respectively. In MC2R-GFP transfected cells, responses were lower, exhibiting 25 ± 1- and 19 ± 1-fold increases over basal values in MRAP{alpha} and MRAPß cell lines, respectively, with a threshold concentration established at 3 nM ACTH in both cell lines. When illustrated as normalized data (Fig. 3BGo), EC50 values for MC2R-GFP expressed in MRAP{alpha} and MRAPß isogenic cell lines [2680 (2240, 3210) and 3030 (2930, 3140) pM, respectively] were clearly right shifted compared with untagged- [79 (44, 143) and 248 (242, 254) pM, respectively] and Myc-MC2R [58 (34, 100) and 189 (68, 519) pM] cell lines. There was no statistical difference between untagged- and Myc-MC2R-transfected cells. Maximal responses and EC50 values from MC2R-GFP- transfected cells were different from those of untagged-MC2R (P < 0.001, n = 3, for each cell line). Thus, as previously described in M3 cells (27), the presence of GFP at the C terminus of MC2R alters the potency and sensitivity of the ACTH response.


Figure 3
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  		Fig. 3. Tagged and Untagged Comparison of MC2R Functionality with MRAP Isoforms

Stable isogenic cells expressing each MRAP isoform separately (293/FRT/MRAP{alpha} and 293/FRT/MRAPß) were transiently transfected with pcDNA5/FRT/untagged-MC2R, pcDNA5/FRT/Myc-MC2R, or pCEP4/MC2R-GFP and assessed for cAMP production in the presence of 1 µM IBMX for 15 min using indicated ACTH concentrations. A, The data are expressed as fold increases over basal cAMP levels to illustrate maximal effects. B, The data from panel A were normalized from 0–100% to visualize shifts in dose-response curves. Results are the mean ± SEM or three experiments performed in duplicate.

 
MRAP{alpha} and MRAPß Isoforms Differentially Affect ACTH-Induced cAMP Production and ACTH Binding
A more in-depth analysis of the differences in cAMP production by MRAP{alpha} and MRAPß was assessed through the construction of bicistronic vectors, containing one open reading frame for Myc-MC2R and another open reading frame for either MRAP{alpha}-Flag or MRAPß-Flag, inserted in a Flp-In-compatible vector and each driven by independent cytomegalovirus promoters. This strategy was used to gain similar expression levels for MC2R as well as both MRAP isoforms and to render all transfected cells responsive to ACTH. The 293/FRT cell line, transiently transfected with pcDNA5/FRT/MycMC2R/MRAPß-Flag or pcDNA5/FRT/MycMC2R/MRAP{alpha}-Flag bicistronic vectors, responded to increasing ACTH concentrations with maximal responses of 170 ± 6-fold and 231 ± 8-fold increase over basal values, respectively (P < 0.01, n = 3) (Fig. 4AGo). As illustrated in the normalized data (Fig. 4BGo), the EC50 values were 49 [31, 78] and 193 [127, 293] pM, respectively, for MC2R/MRAP{alpha}- and MC2R/MRAPß-transfected cells (P < 0.001). With the two double stable isogenic cell lines, 293/FRT/MycMC2R/MRAP{alpha}-Flag and 293/FRT/MycMC2R/MRAPß-Flag, obtained by stable integration of the bicistronic vectors in the 293/FRT genome, maximal stimulations were 673 ± 21- and 891 ± 14-fold increase over basal values (P < 0.001), and EC50 values were 60 [41, 88] and 252 [211, 301] pM, respectively (P < 0.001, n = 3) (Fig. 4Go, C and D). Thus, in both models (transient or double stable isogenic cell lines), MC2R coexpressed with MRAP{alpha} was less efficient than MC2R coexpressed with MRAPß under maximal ACTH-induced cAMP production, whereas in MRAP{alpha}-transfected cells, the cells were more sensitive to low ACTH concentrations.


Figure 4
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  		Fig. 4. Modulation of ACTH-Induced cAMP Production by MRAP{alpha} and MRAPß

293/FRT cells transiently transfected with either bicistronic vectors, pcDNA5/FRT/MycMC2R/MRAP{alpha}-Flag or pcDNA5/FRT/MycMC2R/MRAP{alpha}-Flag (0.5 µg) (A and B), or double stable isogenic cell lines, 293/FRT/MycMC2R/MRAP{alpha}-Flag or 293/FRT/MycMC2R/MRAPß-Flag (C and D), were stimulated with increasing concentrations of ACTH for 15 min at 37 C in the presence of 1 µM IBMX. cAMP was measured as described in Materials and Methods. Results represent the mean ± SEM of three experiments, each performed in triplicate. Data are expressed as fold increases over basal value (denoted as B in the x-axis) (A and C) or normalized from 0–100% (B and D). E, 293/FRT cells transiently transfected with pcDNA5/FRT/MycMC2R/MRAP{alpha}-Flag or pcDNA5/FRT/MycMC2R/MRAPß-Flag were assessed for binding experiments using competition of [125I]iodotyrosyl22 ACTH(1–39) with unlabeled ACTH(1–24), as described in Materials and Methods. F, Bound/free vs. bound representations (Scatchard plots) of the results presented in E. Straight lines have a slope of –1/Kd. Results were normalized to 270,000 cells. Results represent the mean ± SEM of three experiments, each performed in duplicate. Bo, Specific binding in the presence of radiolabeled peptide only; Bmax, maximal binding capacity.

 
Binding studies were thus conducted to verify whether the observed differences in ACTH-induced cAMP production were due to ACTH binding properties. None of the native 293/FRT cells transfected with either MRAP{alpha}, MRAPß, or Myc-MC2R alone were able to bind specifically to ACTH (data not shown). However, in transient Myc-MC2R/MRAP{alpha}-Flag-transfected cells (bicistronic vector), unlabeled ACTH reduced [125I]iodotyrosyl23-ACTH binding to MC2R in a dose-dependent manner, with 50% inhibition (IC50) obtained at 2.9 [2.5, 3.5] and 4.8 [3.8, 6.2] nM for MC2R with MRAP{alpha} and MRAPß, respectively (P < 0.01, n = 3) (Fig. 4EGo). Scatchard analyses revealed a Kd of 1.1 ± 0.6 nM in MRAP{alpha}-expressing cells and 2.1 ± 0.2 nM in MRAPß-expressing cells (P < 0.05), with a binding capacity of 68 ± 4 fmol/liter x 106 cells (41,000 ± 2,300 sites per cell) and 140 ± 9 fmol/1iter x 106 cells (75,200 ± 4,800 sites per cell), respectively, for MC2R/MRAP{alpha}- and MC2R/MRAPß-expressing cells (P < 0.05) (Fig. 4FGo).

MRAP{alpha} and -ß Enhance MC2R Cell Surface Density
In stable 293/FRT/MycMC2R cells transiently transfected with native untagged MRAP{alpha} or MRAPß, ELISA measurements indicated that cell surface MC2R expression increased by 1.9 ± 0.2-fold in MRAP{alpha}-transfected cells (P < 0.01) compared with 2.8 ± 0.2-fold in MRAPß-transfected cells (P < 0.001) over basal Myc-MC2R density in control cells (P < 0.01; difference between MRAP transfected cells, n = 4) (Fig. 5AGo).


Figure 5
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  		Fig. 5. Modulation of MC2R and MRAP Isoform Expression in Transient and Stable Conditions

Cell surface detection of Myc-MC2R using anti-c-Myc antibodies (A and C) and MRAP constructions using anti-Flag antibodies (B and D) was measured by cell surface ELISA as described in Materials and Methods. A, 293/FRT transfected with pEGFP (substracted background OD) or stable 293/FRT/Myc-MC2R cells transiently transfected with pEGFP (control), pcDNA3/MRAP{alpha}, or pcDNA3/MRAPß were assayed for c-Myc detection. B, Epitope-tagged MRAP constructions were expressed transiently from pcDNA5/FRT/GOI vectors in 293/FRT cells (shown) or stable 293/FRT/Myc-MC2R cells (data not shown; similar results) and tested for Flag epitope detection. C, Stable 293/FRT (control), 293/FRT/MRAP{alpha}, and 293/FRT/MRAPß cells were transiently transfected with pEGFP (substracted background OD) or pcDNA3/Myc-MC2R and assayed for c-Myc epitope detection. D, Stable 293/FRT (control), 293/FRT/Flag-MRAP{alpha}, 293/FRT/MRAP{alpha}-Flag, 293/FRT/Flag-MRAPß, and 293/FRT/MRAP{alpha}-Flag cells were assayed for cell surface Flag epitope detection. Results represent the mean ± SEM of three experiments, each performed in triplicate. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, compared with control or indicated conditions.

 
To further investigate the functional properties of MRAP{alpha} and MRAPß, 10 MRAP constructions were created, consisting of Flag and 6xHis epitope tags fused to N- and/or C termini of MRAP{alpha} and MRAPß (Table 2Go, and supplemental data 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). As illustrated in Fig. 5BGo, in transient overexpression conditions, Flag-MRAP{alpha} in 293/FRT cells was poorly detected at the cell surface in comparison with MRAP{alpha}-Flag, with MRAP{alpha}-Flag being significantly detected over control cells and over Flag-MRAP{alpha} (P < 0.001, n = 3). On the other hand, Flag-MRAPß and MRAPß-Flag were both significantly detected at the cell surface compared with control cells and compared with Flag-MRAP{alpha} control (P < 0.05 each). Results were similar when 6xHis-tagged MRAP isoforms were assayed (data not shown).


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  		Table 2. Schematic Representation, Full Names, and Abbreviations of the Epitope-Tagged MRAP Constructions

 
On the other hand, cell membrane expression of transient Myc-MC2R was verified in 293/FRT/MRAP{alpha} and 293/FRT/MRAPß stable isogenic cells. A significant increase in MC2R was observed at the cell membrane of 293/FRT/MRAP{alpha} and of 293/FRT/MRAPß cell lines, with a 1.9 ± 0.4-fold (P < 0.05) and 2.9 ± 0.3-fold increase, respectively, over Myc-MC2R basal expression in 293/FRT cells (P < 0.001, n = 4) (Fig. 5CGo). In agreement with results from Fig. 5AGo, cell surface Myc-MC2R expression was increased by 1.5-fold in 293/FRT/MRAPß cells compared with MRAP{alpha} cells (P < 0.05). In individual MRAP isoform stable isogenic cells, the level of membrane detection of all MRAP constructions was low, nearing the limit of detection. However, the level of MRAP{alpha}-Flag was higher than Flag-MRAP{alpha} (P < 0.05). MRAPß-Flag was also higher than Flag-MRAPß (P < 0.001) and higher than MRAP{alpha}-Flag when isogenic cell lines are compared (P < 0.05) (Fig. 5DGo). Together, these results indicate that MRAP isoforms enhance Myc-MC2R cell surface expression compared with its basal level and that MRAP{alpha} is less potent than MRAPß in this process. Furthermore, lower expression of proteins in MRAP isoform stable isogenic cells did not reveal the same pattern of cell surface expression for MRAP isoforms as in transiently transfected cells. Confirming previous observations (23, 24), in stable isogenic conditions, MRAP{alpha} and MRAPß could be detected only in intracellular N termini and extracellular C termini topology. Thus, as previously described in other models (30), lower expression in stably transfected cells (or stable isogenic cell lines herein) appears to be a more physiological and suitable model in comparison with transient overexpression in a small percentage of cells. Therefore, to avoid artifactual misinterpretation due to the overexpression of MC2R or MRAP isoforms in transiently transfected cells, the following studies were conducted using stable bicistronic isogenic cell lines. All the data described in Fig. 5Go were validated by immunofluorescence observations and ELISA measurements (supplemental data 2 published as supplemental data on The Endocrine Society’s Journals Online web site).

MC2R and MRAP Isoforms Are in Close Apposition in Isogenic Cell Lines
To determine whether MC2R and MRAP isoforms were colocalized in our isogenic cell lines, immunofluorescence images were acquired from permeabilized 293/FRT/MycMC2R/MRAP{alpha}-Flag (Fig. 6Go, A–H) and 293/FRT/MycMC2R/MRAPß-Flag cells (Fig. 6Go, I–P). Cells were examined at the plane of the nuclei to visualize the border of the cell membrane and the cytoplasm (Fig. 6Go, panels A–D and I–L) and at the plane of cell adhesion to the coverslip to delineate an area in close proximity to the cell membrane (Fig. 6Go, panels E–H and M–P). At both planes, MC2R appeared both in the cytoplasm and at the plasma membrane (Fig. 6Go, panels A, E, I, and M). Comparison of panels A and E with panels I and M reveal that expression of MC2R was more intense at the cell membrane when combined with MRAPß expression rather than with MRAP{alpha}. These latter results are consistent with the data reported in Fig. 5Go, A and D, and are supported by cell surface expression of the proteins quantified in ELISA experiments (Fig. 6QGo). However, as shown in the merged images and magnification insets, red and green fluorescent labels were not superimposed (Fig. 6Go, C and G; and magnification of the merged images, Fig. 6Go, panels D, H, L, and P, arrows). These immunofluorescence data were also supported by ELISA measurements. As shown in Fig. 6RGo, MRAP C termini were hardly present at the cell membrane; however, as in Fig. 5DGo, MRAPß appeared more abundant than MRAP{alpha}. Thus, whereas MC2R was found in high concentration at the cell surface, MRAP isoforms were expressed at low levels and appeared in the vicinity of MC2R, rather than superimposed with MC2R.


Figure 6
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  		Fig. 6. Immunofluorescence Labeling of MC2R and MRAP Isoforms Expressed in Double Stable Isogenic Cell Lines

293/FRT/Myc-MC2R/MRAP{alpha}-Flag (A–H) or 293/FRT/Myc-MC2R/MRAPß-Flag cells (I–P) were processed for immunofluorescence labeling as described in Materials and Methods. MC2R and MRAP were labeled with anti-c-Myc antibody and with anti-Flag, respectively, and detected using secondary antibodies coupled to Alexa-Fluor488 for Myc-MC2R (green) and to Alexa-Fluor594 for MRAP (red). Green and red signals were acquired at the nuclear plane to visualize both the outside and cytoplasmic border of the cell membranes (A–D and I–L) or at the plane of cell adhesion to the coverslip to visualize only the area in close proximity to the cell membrane (E–H and M–P). Blue emissions (DAPI; nuclei) are represented in their respective planes only. Images are representative illustrations of more than 100 cells from three experiments. D, H, L, and P, Inset magnifications (4-fold) representing a portion of the cell membrane of merged images from panels C, G, K, and O. Scale bar, 10 µm or 60 µm for insets. Q–R, Cell surface ELISA detection of Myc-MC2R (Q) or Flag epitopes (MRAP) (R) in double-isogenic 293/FRT/Myc-MC2R/MRAP{alpha}-Flag and 293/FRT/Myc-MC2R/MRAPß-Flag cells. Results represent the mean ± SEM of three experiments, each performed in triplicate. *, P < 0.05, comparison between cell lines. Scale bar, 10 µm for all panels, 3.6 µm for inset 1, and 1.8 µm for inset 2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Using N-terminal-c-Myc-tagged human MC2R, N- and C-terminal Flag- or 6xHis-tagged MRAP{alpha} and MRAPß constructions, together with Flp recombinase-mediated homologous recombination in HEK293-related cells (293/FRT cell line), we were able to generate isogenic populations expressing both Myc-MC2R with either of the appropriately tagged MRAP{alpha} or MRAPß isoforms. Results demonstrate that although Myc-MC2R was inserted at the plasma membrane without MRAP assistance, functionality i.e. ACTH-induced cAMP production, was only observed in the presence of either MRAP isoform. In contrast, the MC2R-GFP fusion protein was impaired both in cell surface expression and cAMP production, with or without the presence of MRAP isoforms, as compared with untagged- and Myc-MC2R. In addition, we provide evidence that MRAP{alpha} and MRAPß differentially regulate the cell surface density of MC2R as well as ACTH binding and coupling to cAMP production.

Herein, immunofluorescence and ELISA studies conducted on unpermeabilized Myc-MC2R expressing 293/FRT cells, exempt of endogenous MCRs, clearly indicate that the MC2R is located at the cell surface, in the absence of MRAP isoforms. The comparison between Myc-MC2R and MC2R-GFP indicates that although the C-terminal GFP fusion approach has been used successfully for several other GPCRs, including MC3R, MC4R, and ß2-adrenergic receptors (26, 31, 32), it does not appear appropriate for the smallest human GPCR, MC2R. As previously published and confirmed in the present study, fusion of GFP in the C terminus of MC2R impairs trafficking and signaling (10, 27, 31). It is known that the integrity of MC1R’s putative dileucine-like motif located in its C-terminal tail is especially important for cell surface expression (33). Mutational analysis of this motif in MC4R also confirmed its importance for cell surface expression and function (34). A similar motif, methionine-290 and isoleucine-291 in the small C-terminal tail of MC2R, may be encumbered in the MC2R-GFP fusion protein. Indeed, short N-terminal epitope tagging of MC1R, MC2R, or MC4R does not affect receptor trafficking or function (13, 19, 35).

Transfection of human MC2R with human MRAP isoforms induces cAMP production after ACTH stimulation, as previously described with mouse Mrap by Metherell et al. (23). In their study, Metherell et al. showed an interaction between Mrap and Mc2r-GFP and the functional consequence of these interactions in terms of ACTH responsiveness. Functionality was obtained in the SK-N-SH cell line, which expresses endogenous MCRs, with a single ACTH concentration (1 µM) and in the presence of IBMX (10 µM), both of which were 10-fold higher than the concentrations used in the present study. In a recent study, using M3 cells transfected with Myc-MC2R, we obtained EC50 values ranging from 7.6 nM to 11.9 nM, with maximal stimulation ranging from 20.9 ± 0.7 to 24.7 ± 0.7-fold increase over basal cAMP levels, which are also low compared with in vivo or in vitro situations with primary adrenocortical cells (36, 38, 39). Thus, importantly, the present results are the first to reproduce dose-responses curves of ACTH-induced cAMP production exhibiting properties similar to that previously described in primary cultures of human glomerulosa and fasciculata adrenocortical cells (36). Indeed, in isolated or cultured adrenocortical cells, ACTH stimulation is initiated at a low physiological threshold (pM range), with high maximal stimulation (≤60-fold over control) (37); for review see Refs. 4 and 5). Thus, the reconstituted cell model described herein is able to reproduce a physiological system.

Measurements of membrane expression by ELISA, together with immunofluorescence studies, corroborate ACTH binding capacities and indicate that MRAP isoforms confer small but significantly different densities of MC2R at the plasma membrane. Furthermore, binding studies indicate that MRAP{alpha} provides slightly higher affinity (2-fold) for ACTH binding to MC2R than MRAPß. As a result, dose-response curves reveal greater sensitivity (4-fold) to ACTH stimulation in MRAP{alpha}-expressing cells comparatively to MRAPß-expressing cells, whereas maximal responses are higher in MRAPß-expressing cells (1.4-fold). Although the difference in EC50 values and maximal responses from cAMP experiments were modest, our data provide evidence that MRAP isoforms differentially modulate MC2R cell-surface expression and ACTH binding, hence affecting cAMP response both in amplitude and sensitivity.

In contrast to MC2R, MRAP isoforms C termini were present at limited levels at the plasma membrane in isogenic conditions. Moreover, MC2R is not in a functional state unless it is in the vicinity of MRAP{alpha} or MRAPß. These results suggest that, in addition to promoting greater MC2R density at the surface of cells, MRAP isoforms may also promote cell surface expression of proteins essential for coupling, such as guanyl nucleotide-binding regulatory protein subunits ß and {gamma}, which are involved in ACTH responsiveness and adenylyl cyclase type 2 or 4 expression (40, 41). MRAP isoforms may also be components of a scaffold complex. For example, components of lipid rafts regulate adenylyl cyclase type 5 or 6 activity (42), which are major adenylyl cyclases involved in ACTH/MC2R signaling pathways and adrenal steroidogenesis (43, 44). As reported for other receptors (30, 45, 46), molecular determinants within MC2R, including allosteric interaction and/or regulation with accessory proteins (47), are probably essential for cell surface expression and signal transduction of MC2R (48).

The advantages of the novel model described herein as a human expression system of Myc-MC2R and MRAP isoforms, compared with the M3 cell line for example, can be summarized as follows: 1) the model eliminates possible cross-regulation by endogenous Mc1r receptors expressed in M3 cells, especially because MC1R is known to heterodimerize (19); 2) it eliminates the risk of discrepancies observed when using cell lines of murine origin (i.e. Y6 and M3 cells) in which endogenous mouse Mrap may not be as efficient as human MRAP isoforms in enabling human MC2R function; 3) it allows flexible control over transient vs. stable expression of either MC2R, MRAP{alpha}, MRAPß, or other genes of interest; 4) the model is adaptable to polycistronic vectors and allows the integration of at least two genes at a single and transcriptionally active genomic locus which, in the case of MC2R and MRAP isoforms, is necessary to achieve functional expression; and 5) Flp-In cell line generation takes no more than 2 wk, achieving rapid and efficient stable isogenic expression.

In summary, the present results clearly indicate that although MC2R is inserted at the plasma membrane without MRAP assistance, human MRAP{alpha} and -ß isoforms differentially modulate the functional properties of ACTH receptors in a fashion that appears isoform dependent. In addition, careful consideration should be taken when using MCR carboxy terminus modifications such as MC2R-GFP. From a physiological standpoint, the slight but significant differences observed in EC50 values for either MC2R/MRAP{alpha}- or MC2R/MRAPß-expressing cells may reflect an indirect regulation of ACTH-induced steroidogenesis during adrenal gland adaptation to various situations or hormonal stimulations. Indeed, the sensitivity of the MC2R system to raise cAMP levels in response to ACTH is a critical step in the initiation of steroidogenesis by adrenal cells (reviewed in Ref. 9). MC2R/MRAP{alpha} and MC2R/MRAPß combinations may regulate steroidogenesis differently at low physiological (pM range) or high (nM range) ACTH concentrations, such as in stressful situations (3).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
The human MC2R cDNA (GenBank accession no. X365633) was provided by Dr. Roger D. Cone (Vollum Institute, Oregon Health and Science University, Portland, OR) and the MC2R-GFP fusion protein by Dr. Helgi B. Schiöth (Department of Neuroscience, Uppsala University, Uppsala, Sweden). Cell culture reagents used in this study were purchased as follows: high-glucose DMEM, fetal bovine serum, GlutaMAX, and Zeocin from Invitrogen Life Technologies (Burlington, Ontario, Canada). The RNAqueous-4PCR system and DNase I were from Ambion (Austin, TX). Superscript II RNase H reverse transcriptase, primers, antibiotics, the Flp-In system, 293/FRT cells, Lipofectamine PLUS reagent, and the pcDNA3 vector were from Invitrogen. Deoxynucleotide triphosphates were obtained from GE Healthcare (Baie d’Urfé, Quebec, Canada) and the Expand High FidelityPLUS PCR System polymerase from Roche Applied Sciences (Laval, Quebec, Canada). Restriction endonucleases, modifying enzymes, Standard Taq DNA polymerase, and endoglycosidase H were purchased from New England Biolabs (Ipswich, MA); plasmid DNA purification kits and gel extraction kits were from QIAGEN, Inc. (Mississauga, Ontario, Canada). The pGEM T-easy kit was purchased from Promega Corp. (Madison, WI) and pEGFP from CLONTECH Laboratories, Inc. (Mountain View, CA). ACTH (1–24) was purchased from Organon (Toronto, Ontario, Canada) and ACTH (1–39) from Novartis (Boucherville, Quebec, Canada). IBMX (3-isobutyl-1-methylxanthine), cAMP, ATP, forskolin, and NDP-MSH were from Sigma (Oakville, Ontario, Canada); [3H]adenine (25 Ci/mmol) was from PerkinElmer (Boston, MA). [125I]iodotyrosyl23-ACTH (1–39) (2000 Ci/mmol) was from GE Healthcare (Buckinghamshire, UK). The mouse monoclonal anti-Myc antibody clone 9E10 was obtained from Dr. Michel Bouvier (Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, Montréal, Québec, Canada); the anti-6xHis rabbit polyclonal antibody (H15 probe) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and the anti-Flag rabbit polyclonal antibody from Sigma. Goat antimouse or antirabbit coupled to Alexa-Fluor488 or Alexa-Fluor594 as well as DAPI (4',6-diamidino-2-phenylindole) were purchased from Molecular Probes, Inc. (Eugene, OR) and Vectashield from Vector Laboratories, Inc. (Burlington, Ontario, Canada); secondary antibody coupled to horseradish were from GE Healthcare (Baie d’Urfé, Quebec, Canada). All other chemicals were of grade A purity.

MRAP{alpha} and MRAPß Expression, Cloning, and Plasmid Constructions
Human adrenal glands were prepared as previously described (36, 49). Both fetal and adult projects were approved by the Human Subject Review Committee of our institution. Total RNA, from a fetal adrenal gland (18 wk old) and from zona glomerulosa and zona fasciculata from a 46-yr-old donor, were extracted and treated with DNAse I using the RNAqueous-4PCR system, followed by first-strand cDNA synthesis using Superscript II RNase H reverse transcriptase as recommended by the manufacturers.

Total RNA from human fetal adrenal glands was used for cloning human MRAP {alpha} and ß isoforms. The entire coding sequences were amplified by PCR with the Expand High FidelityPLUS PCR System polymerase using a common forward primer and isoform-specific reverse primers. The PCR products were cloned in pGEM, pcDNA3, and pcDNA5/FRT (Flp recombinase target) vectors. The Expand High FidelityPLUS PCR System was used to generate different N- and/or C-terminal epitope-tagged MRAP{alpha} and -ß constructions. Two different small epitope tags, Flag and 6xHis, were used. SpeI-Kozak-epitope-MRAP-XbaI or SpeI-Kozak-MRAP-epitope-XbaI cDNA sequences were amplified from pcDNA3/MRAP{alpha} and pcDNA3/MRAPß native templates, respectively. The PCR products were purified, digested, and cloned into pcDNA5/FRT, thus yielding constructions with MRAP{alpha} or ß fused at either N or C terminus to the Flag (NYDDDDKC) or 6xHis (GGSHHHHHH) epitope tags with preceding initiation codon (M: methionine) when required. Double tagged constructions with MRAP{alpha} or -ß fused at N terminus to 6xHis tag and at C terminus to Flag tag were also produced. mRNA expression of MRAP isoforms in 293/FRT cells and in human fetal and adult adrenal glands was assessed by classical RT-PCR analysis using Standard Taq DNA polymerase with the same primers as for cloning.

The human MC2R cDNA was provided by Dr. Roger D. Cone (Vollum Institute, Oregon Health and Science University) in pcDNA1. This cDNA and our previously described pcDNA3/c-Myc-hMC2R construction (13) were cloned from pcDNA3 to pcDNA5/FRT using restriction endonucleases and named pcDNA5/FRT/untagged-MC2R and pcDNA5/FRT/Myc-MC2R, respectively. The human MC2R-GFP cDNA was provided in the pCEP4 vector by Dr. Helgi B. Schiöth (Department of Neuroscience, Uppsala University, Uppsala, Sweden). The bicistronic pcDNA5/FRT/Myc-MC2R/MRAP{alpha}-Flag and pcDNA5/FRT/Myc-MC2R/MRAPß-Flag vectors encode two proteins of interest, Myc-MC2R and one or the other of the MRAP isoforms tagged in C terminus with the Flag epitope. They were created using DNA primers designed to amplify SphI-TATA box-cytomegalovirus-GOI-polyA-SphI sequences by PCR with a high fidelity DNA polymerase (Expand High FidelityPLUS) from the pcDNA5/FRT-MRAP{alpha}-Flag and pcDNA5/FRT/MRAPß-Flag vectors as templates. The PCR products were digested and cloned into pcDNA5/FRT/Myc-MC2R cut at the SphI site located past the poly-A signal. All plasmid constructions were confirmed by DNA sequencing (Service de Séquençage, IPS, Faculté de Médecine, Université de Sherbrooke). All primer sequences are found in Table 1Go and MRAP constructions are summarized in Table 2Go. In general, DNA/protein sequences, names of genes, constructions, plasmids or cell lines cited are always read from 5'/N terminus to 3'/C terminus. For example, the name of an epitope tag precedes the name of a gene of interest (GOI)/protein when tag is located in 5'/N terminus and it follows the name of the GOI/protein when tag in 3'/C terminus.


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  		Table 1. Primers Used for MRAP Cloning, MRAP Tagging, and Bicistronic Vectors

 
Cell Culture, Transfection, and Isogenic Cell Lines
The Flp recombinase-mediated homologous recombination system (Flp-InTM) was used to generate cell lines stably expressing Myc-MC2R and/or either one of the appropriately tagged MRAP isoforms. The 293/FRT cell line (HEK293 cell line with single genome-integrated FRT) was maintained in high-glucose DMEM with 7% fetal bovine serum, 2 mM GlutaMAX, and 100 µg/ml Zeocin. Unless otherwise stated, cells were transfected with 0.5 µg of plasmid DNA per dish using Lipofectamine and PLUS reagent. To maintain similar transcriptional and translational activity when required, transfection of pEGFP was used instead of an empty vector.

The production of the various stable isogenic Flp-In cell lines was carried out as previously described (50) and as recommended by the manufacturer (28, 29). Briefly, 3 x 106 293/FRT cells placed in 100-mm dishes without Zeocin were transfected 24 h later with 0.5 µg pcDNA5/FRT/GOI (expression vector for the GOI) together with 4.5 µg pOG44 (Invitrogen’s expression vector for Flp recombinase). The next day, cells were washed and were positively selected with 100 µg/ml Hygromycin B. The complete media was changed every 3–4 d to remove dead cells. After 2 wk, colonies were pooled as isogenic populations expressing the gene of interest and kept under constant Hygromycin B selection. Cells were also negatively selected with 100 µg/ml Zeocin to confirm insertion into the genomic FRT site only. Flp recombinase-mediated homologous recombination ensures the integration of a single pcDNA5/FRT/GOI plasmid at the genomic FRT site of native 293/FRT cells to yield 293/FRT/pcDNA5/FRT/GOI isogenic cell populations, enabling stable and isogenic expression in positively selected cells. Cell lines created for this study were named as follows: 293/FRT (native cells from the supplier); 293/FRT/Myc-MC2R; 293/FRT/MRAP{alpha}, 293/FRT/MRAPß, 293/FRT/Flag-MRAP{alpha}, 293/FRT/MRAP{alpha}-Flag, 293/FRT/Flag-MRAPß, and 293/FRT/MRAPß-Flag (stably expressing the indicated GOI); 293/FRT/MycMC2R/MRAP{alpha}-Flag and 293/FRT/MycMC2R/MRAPß-Flag (stably expressing the two indicated GOIs introduced with bicistronic vectors). Cell lines with MRAP isoforms tagged with 6xHis as well as with Flag and 6xHis epitope tags, at N and/or C terminus, were also generated. Functionality and epitope tag detection of each modified cDNA construction and associated cell lines were confirmed by cAMP assays, ELISA, and immunofluorescence labeling, in experiments such as provided in supplemental data 1 and 2. As recommended, 293/FRT recombinant cell lines were not maintained for more than 60 d (50). Cells were tested negative for the presence of mycoplasm.

Immunofluorescence Microscopy
Cells were grown onto poly-L-lysine coated coverslips placed into 35-mm dishes 1 d before transfection. Twenty-four hours after transfection with 0.5 µg DNA (when required), cells were fixed on ice with 1.85% formaldehyde in PBS for 15 min. Alternatively, cells were also fixed and permeabilized using 100% methanol followed by an acetone wash. Mouse monoclonal anti-c-Myc antibody (1:500) (13) and/or rabbit polyclonal anti-Flag (1:200) or anti-6xHis (1:200) in blocking solution were incubated for 1 h at room temperature. Secondary antibodies coupled to Alexa-Fluor488 and Alexa-Fluo594 (1: 500) were used to detect primary antibodies. DAPI (300 mM) was used to stain nuclei. Slides were mounted with Vectashield mounting medium, and images were acquired with a Hamamatsu, ORCA-ER digital camera and examined under a Nikon Eclipse 2000 inverted fluorescence microscope (Nikon, Mississauga, Ontario, Canada) equipped for epi-illumination. For MC2R-GFP detection, cells were fixed as above, and green fluorescence was detected using UV illumination. Images were acquired using a x100 objective. Images were processed with Metamorph (version 4.6r10) software (Universal Imaging Corp., West Chester, PA). All images were acquired using identical camera settings for contrast and brightness.

Cell Surface ELISA Procedures
Cells were seeded into poly-L-lysine-coated 24-well plates at 1 x 105 cells per well and transfected with 0.125 µg DNA/ well. After 24 h, cells were washed with PBS, fixed on ice for 15 min with 1.85% formaldehyde, and then incubated with anti-c-Myc (1:666), anti-Flag (1:200) and/or anti-6xHis (1:200) for 1 h at room temperature, followed by incubation with appropriate antimouse or antirabbit antibodies (1:1000) coupled to horseradish peroxidase. The enzyme-immune complexes were incubated with 300 µl O-phenylenediamine (1 µg/ml) in citrate/phosphate buffer (0.5 M each), pH 5.0, for 10 min. Reactions were halted by the addition of an equal volume of H2SO4 (2 M), after which 200 µl of the reaction medium was transferred to 96-well plates for absorbance reading at 492 nm using a Benchmark microplate reader (Bio-Rad Laboratories, Mississauga, Ontario, Canada). Negative controls included empty wells, Myc-MC2R, or Flag-tagged MRAP-expressing cells incubated without primary or secondary antibody. Corrected OD refers to background-subtracted data at 492 nm wavelength. Linearity of the assays was verified using varying DNA concentrations at various time points.

cAMP Measurements
Transient transfections were performed, or not, 24 h after initial seeding of 4 x 105 cells in 35-mm dishes by transfecting 0.5 µg of plasmid DNA. Intracellular cAMP production was determined 24 h later by measuring the conversion of [3H]ATP into [3H]cAMP, as previously described (13, 38). Cells (at a density of 1 x 106 cells per 35-mm dish) were incubated for 2 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), 130 mM NaCl, 3.5 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 2.5 mM NaHCO3, 5 mM HEPES, supplemented with 1 g/liter glucose, and then stimulated by addition of 10-fold ACTH concentrations, ranging from 0.1 pM to 100 nM for 15 min at 37 C. Separation of [3H]cAMP from [3H]ATP was obtained after chromatography on Dowex and alumina columns, and cAMP formation was calculated as follows: percent conversion = [3H]cAMP/ ([3H]cAMP + [3H]-ATP) x 100 per 15 min. The % of [3H]cAMP conversion to [3H]ATP (% cAMP/ATP) was transformed to fold increases over basal values and to 0–100% normalized responses using GraphPad Prism 4.0. (GraphPad Software, Inc., San Diego, CA).

Binding Assays
Binding assays were performed in 24-well plates containing approximately 0.27 x 106 cells per well 24 h after transfection (all data were normalized to that cell number) with slight modifications of the method described previously (37). Cells were washed with HBS containing 1% glucose and then incubated for 30 min at 20 C with 45 pM [125I]iodotyrosyl22-ACTH(1–39) and increasing concentrations of unlabeled ACTH(1–24) in HBS containing 0.1% BSA in a total volume of 200 µl. Nonspecific binding was determined with 1 µM ACTH(1–24). The binding reaction was stopped on ice after two rapid washes with HBS. Cells were harvested with 0.5 N NaOH, 0.4% sodium deoxycholate. Radioactivity was counted in a Beckman 9000 {gamma}-counter (Beckman Coulter, Inc., Fullerton, CA). Specific binding was 5.5 ± 0.1 and 5.6 ± 0.2% of total radioactivity, with nonspecific binding representing 0.5% of total binding, for MycMC2R/MRAP{alpha}-Flag and MycMC2R/MRAPß-Flag transiently expressing cells, respectively.

Data Analysis
The mean of the replicates from each individual experiment was used for data compilations and SEM calculations. The results are presented as mean ± SEM unless stated otherwise. GraphPad Prism 4.0 was used for all nonlinear curve fittings. EC50, IC50, 95% CI, maximal effects, and other parameters describing curve fittings are calculated by Prism 4.0 using log-scaled ACTH concentrations to minimize the sum-of-squares. For dose-response curve analyses, P values were obtained with the extra sum-of-squares F test performed on dose-response curve fittings (constant slope, no constraint) using the log (EC50) for EC50 analysis and the top value for maximal stimulatory effects. EC50 and IC50 values are given with 95% confidence intervals as follows: mean [lower limit, higher limit]. SigmaPlot 8.02 was used for the other data representations. Statistical analyses such as ANOVA were performed using the Sigma Stats software. Homogeneity of variance was assessed by Bartlett’s test, and P values were determined using Tukey’s post test for significant difference. A P value < 0.05 was considered statistically significant.


   ACKNOWLEDGMENTS
 
We thank Dr. Roger D. Cone (Vollum Institute, Oregon Health and Science University, Portland, OR) for providing us with the human MC2R cDNA; to Dr. Helgi B. Schiöth (Department of Neuroscience, Uppsala University, Uppsala, Sweden) for the MC2R-GFP fusion protein; and to Dr. Michel Bouvier (Canada Research Chair in Signal Transduction and Molecular Pharmacology, Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, Montréal, Québec, Canada) for providing us with the anti-Myc antibody. Special thanks to Lucie Chouinard for technical assistance and to Zuzana Kilianova for assistance and helpful discussions. We also thank Claude Roberge and Dr. Marcel D. Payet, from the Department of Physiology and Biophysics, for their invaluable stimulating discussions regarding this project.


   FOOTNOTES
 
This work was supported by Grant MOP-10998 from the Canadian Institutes for Health Research (to N.G.-P.). M.R. was a recipient of a fellowship from the Fondation pour la recherche médicale (France) in 2004–2005. N.G.-.P. is the recipient of the Canada Research Chair in Endocrinology of the Adrenal Gland.

Author Disclosure Summary: S.R., M.R., and N.G.-P. have nothing to declare. The authors have declared that no conflict of interest exists.

First Published Online April 24, 2007

Abbreviations: DAPI, 4',6-Diamidino-2-phenylindole; FRT, Flp recombinase target site; 293/FRT, HEK293 cell line with single genome-integrated FRT site; GFP, green fluorescent protein; GOI, gene of interest; GPCR, G protein-coupled receptor; HEK293, human embryonic kidney cell line; HBS, Hank’s buffered saline; IBMX, 3-isobutyl-1-methylxanthine; M3, mouse Cloudman melanoma S91 cell line; MCR, melanocortin receptor; MC1R, MC2R, MC3R, and MC4R, human melanocortin type 1, 2, 3, and 4 receptors, respectively; Mc1r, mouse melanocortin type 1 receptor; Mc2r, mouse MC2R; MRAP, human melanocortin 2 receptor accessory protein, isoform {alpha} or ß; Mrap, mouse MRAP; NDP-MSH, (Nle4, D-Phe7) {alpha}-MSH.

Received for publication January 22, 2007. Accepted for publication April 18, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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