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Am J Physiol Regul Integr Comp Physiol 293: R1120-R1126, 2007. First published June 27, 2007; doi:10.1152/ajpregu.00240.2007
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APPETITE, OBESITY, DIGESTION, AND METABOLISM

Structural insights into the role of the ACTH receptor cysteine residues on receptor function

Yingkui Yang,1 Min Chen,2 Robert Allen Kesterson, Jr.,3 and Carroll M. Harmon1

Departments of 1Surgery, 2Nutrition, and 3Genetics, University of Alabama at Birmingham, Birmingham, Alabama

Submitted 10 April 2007 ; accepted in final form 25 June 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The ACTH receptor, also known as the melanocortin-2 receptor (MC2R), is critical for ACTH-mediated adrenal glucocorticoid release. Human MC2R (hMC2R) has 10 cysteine residues, which are located in extracellular loops (ELs), transmembrane domains (TMs), and intracellular loops (ILs). In this study, we examined the importance of these cysteine residues in receptor function and determined their involvement in disulfide bond formation. We replaced these cysteines with serine and expressed the mutated receptors in adrenal OS3 cells, which lack endogenous MC2R. Our results indicate that four mutations, C21S in NH2 terminus, C245S, C251S, and C253S in EL3, resulted in significant decrease both in receptor expression and receptor function. Mutation of cysteine 231 in TM6 significantly decreased ACTH binding affinity and potency. In contrast, the five other mutated receptors (C64S, C158S, C191S, C267S, and C293S) did not significantly alter ACTH binding affinity and potency. These results suggest that extracellular cysteine residue 21, 245, 251, and 253, as well as transmembrane cysteine residue 231 are crucial for ACTH binding and signaling. Further experiments suggest that a disulfide bond exists between the residue C245 and C251 in EL3. These findings provide important insights into the importance of cysteine residues of hMC2R for receptor function.

familial glucocorticoid deficiency; melanocortin 2 receptor; melanocortin receptor; G protein-coupled receptor

THE MELANOCORTIN-2 RECEPTOR (MC2R), also known as the ACTH receptor, is a member of G protein-coupled receptor (GPCR) superfamily with seven transmembrane domains. MC2R, primarily expressed in adrenal cortex, plays an important role in normal glucocorticosteroid secretion and stress-related hypersecretion (21). MC2R is characterized by a short NH2-terminal extracellular domain, and a short intracellular COOH-terminal domain (2). ACTH binds to MC2R and stimulates adenylate cyclase and thereby elevates the second messenger, cellular cAMP (20). Mutations of the ACTH receptor gene have been demonstrated in humans with the autosomal recessive disorder, familial glucocorticoid deficiency (FGD), which is a serious disease presented in childhood with failure-to-thrive, weakness, fatigue, and possible adrenal insufficiency crisis (13, 19). Affected individuals are deficient in cortisol, and, if untreated, they are likely to succumb to hypoglycemia or overwhelming infection in infancy or childhood. MC2R also plays a key role in Cushing's syndrome, a disorder resulting from high levels of ACTH and episodic cortisol hypersecretion. Therefore, examining the molecular basis of human melanocortin-2 receptor (hMC2R) structure responsible for receptor function will be crucial for our understanding of normal adrenal physiology and for the development of potential therapeutic interventions for MC2R pathological conditions.

Cysteine residues within GPCR have been identified as important for inducing and maintaining the three-dimensional receptor conformation by forming critical intermolecular and intramolecular disulfide bond (3). The hMC2R contains 10 cysteine residues, including four cysteine residues within ELs (C21, C245, C251, and C253), five cysteine residues within transmembrane domains (TMs) (C64, C158, C191, C231, and C267), and one cysteine residue C267 in COOH terminus. All of these residues are conserved through melanocortin receptors except C191 in TM5 (Fig. 1). Unfortunately, little is known about the molecular basis of cysteine residues on MC2R function. In this study, we systemically examined the role of endogenous cysteine in the hMC2R function. Our results suggest that four extracellular cysteine residues, C21, C245, C251, C253, and one TM cysteine residue, C231, are crucial for high-level receptor expression and function. C245 and C251 in EL3 may form a disulfide bond that is crucial for receptor function.


Figure 1
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  		Fig. 1. Alignment of different species MC2R with the members of human MCR family. Human MC2R (hMC2R) (20) sequence is compared with the chicken MC2R (CMC2R) (25), mouse MC2R (mMC2R) (16), bovine MC2R (bMC2R) (23), and human melanocortin receptors: hMC1R (20), hMC3R (9), hMC4R (10), and hMC5R (11). Conserved cysteines of the hMC2R with other MCRs are indicated by black shading.

 

   METHODS
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 METHODS
RESULTS
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Peptides. ACTH was purchased from Peninsula Laboratories (Belmont, CA).

Site-directed mutagenesis. Single mutations were constructed using the Quick-Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). The entire coding region of the mutated receptors was sequenced by the University of Alabama at Birmingham Sequence Core to confirm that the desired mutation sequences were present and that no sequence errors had been introduced. The mutated receptors are shown in Fig. 2. The mutant receptors were subcloned into the eukaryotic expression vector pCDNA 3.1 (Invitrogen, Carlsbad, CA).


Figure 2
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  		Fig. 2. Two-dimensional representation of the seven TM structure of the hMC2R. The residues mutated in these experiments are denoted by gray or black highlighting. Those residues whose mutation significantly affected ACTH binding as determined are highlighted in black.

 
Cell culture and transfection. The OS3 cells, lacking endogenous MC2R, were cultured in DMEM medium containing 10% FBS, 2% CBS, and HEPES. Cells at 80% confluence were washed twice with DMEM, and the receptor constructs were transfected into cells (5 million) using lipofectamine (Life Technologies, Rockville, MD). Experiments were performed 24 h after transfection. Untransfected OS3 cells exhibit no response to melanocortin stimulation, and therefore there is no significant background.

Binding assays. After removal of media, cells were incubated with various nonradioligand in 0.5 ml MEM (Fisher Scientific, Pittsburgh, PA) containing 0.2% BSA and radioligand. Binding experiments were performed using conditions previously described (19). Briefly, 2 x 105 cpm of 125I-ACTH (Amersham, Arlington Heights, NJ) was used in combination with nonradiolabeled ACTH. Binding reactions were terminated by removing the media and washing the cells twice with MEM containing 0.2% BSA. The cells were lysed with 0.2 N NaOH, and the radioactivity in the lysate was quantified in an analytical gamma counter. Nonspecific binding was determined by measuring the amount of 125I-label bound in the presence of 10–6 M unlabeled ligand. Specific binding was calculated by subtracting nonspecifically bound radioactivity from total bound radioactivity. Data were analyzed using GraphPad Prism (San Diego, CA). Ki values for ACTH were calculated using the equation: Ki = Kd = IC50 – [radioligand] (5).

cAMP assay. cAMP generation was measured using a competitive binding assay (TRK 432; Amersham). Briefly, OS3 cells stably expressing hMC2R were used in these assays (19). Cell culture media were removed, and cells were incubated with 0.5 ml Earle's balanced salt solution, containing ACTH (10–10–10–6 M), for 1 h at 37°C in the presence of 10–3 M isobutylmethylxanthine. The reaction was stopped by adding ice-cold 100% ethanol (500 µl/well). The cells in each well were scraped, transferred to a 1.5-ml tube, and centrifuged for 10 min at 1,900 g, and the supernatant was evaporated in a 55°C water bath with prepurified nitrogen gas. cAMP content was measured according to manufacturer's instructions accompanying the assay kit. Each experiment was performed a minimum of three times with duplicate wells.

Receptor expression. For receptor protein expression studies, a FLAG tag was inserted into the NH2 terminus of hMC2R to characterize receptor protein cell surface expression by flow cytometry (FACS). The FLAG protein is an eight-amino acid peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), which is useful for immunoaffinity purification of fusion proteins. hMC2R-FLAG or mutant receptor-FLAG transfected cells were harvested using 0.2% EDTA and washed twice with PBS. Aliquots of 3 x 106 cells were centrifuged and fixed with 3% paraformaldehyde in PBS (pH 7.4). The cells were incubated with 50 µl of 10 µg/ml murine anti-FLAG M1 monoclonal antibody (Sigma, St. Louis, MO) in incubation buffer for 45 min. Under these conditions, the primary antibody binds only to receptors located at the cell surface. The cells were collected by centrifugation and washed three times with incubation buffer. The cell pellets were suspended in 100 µl of incubation buffer containing CYTM3-conjugated Affinity Pure Donkey Anti-Mouse Ig G (ImmunoResearch Lab, West Grove, PA) and incubated at room temperature for 30 min. Flow cytometry was performed on a fluorescence-activated cell sorter (FACStar plus six-parameter cytometer/sorter with a dual-Argon ion laser; Becton Dickinson, San Jose, CA). The results were analyzed using the software CellQuest (Becton Dickinson).

Statistical analysis. Each experiment was performed at three separate times with duplicated wells. Data are expressed as means ± SE. The mean value of the dose-response data of binding and cAMP production was fit to a sigmoid curve with a variable slope factor using nonlinear squares regression analysis (GraphPad Prism, GraphPad Software, San Diego, CA). Significant differences were assessed by one-way ANOVA, with P < 0.05 considered to be statistically significant.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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Characterization of the wild-type hMC2R. Previous results of MCRs indicate that ligand binding affinity and potency were affected by the level of the mutant receptor expression (12, 30, 31). In this study, we first examined the hMC2R expression level on ACTH binding and signaling. We transfected different amounts of hMC2R-WT DNA into OS3 cells (5 million), which lack endogenous MC2R. Our results indicate that the level of hMC2R-WT expression increased with an increase of the amounts of plasmid DNA. The receptor expressions are 22, 38, 58, 76, and 100% at the DNA concentrations of 0.5, 2, 3, 4, and 5, respectively (Fig. 3A). To determine whether the receptor expression affects ACTH binding affinity, we used competitive binding assays to calculate the IC50 of these receptors. Our results indicate that the binding affinity of ACTH was unaffected at the range between 2 and 5 µg DNA but significantly decreased below 0.5 µg DNA (Fig. 3B). We then examined whether the receptor expression alters ACTH potency. ACTH induced cAMP production was measured at OS3 cells transfected with the different amount of hMC2R DNA. Our results indicate that although the receptor expression varied with the different DNA concentrations, the ACTH potency (EC50) was unaffected except at the 0.5 µg DNA. EC50 was significantly shifted rightward when 0.5 µg DNA was transfected into OS3 cells (Fig. 2C). The EC50 of ACTH at these groups are 0.67 ± 0.07, 0.35 ± 0.01, 0.25 ± 0.04, 0.25 ± 0.03, and 0.24 ± 0.06 nM at the DNA concentrations of 0.5, 2, 3, 4, and 5 µg, respectively.


Figure 3
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  		Fig. 3. Relationship between the receptor expression and receptor function. A: effect of different amount of DNA transfection on receptor expression. B: effect of the different levels of receptor expression on agonist binding affinity. C: effect of the different levels of receptor expression on ACTH potency. Five million OS3 cells were transfected with different amounts (range 0.5 to 5 µg) of receptor plasmid DNA. Agonist binding and potency were only affected after using the lowest amount 0.5 µg. Data points represent the means ± SE of three independent experiments with duplicate wells.

 
Effect of substitutions of the hMC2R extracellular loop cysteine residues on receptor function. To examine the role of the extracellular loop cysteines on hMC2R function, we substituted the extracellular cysteine residues, C21, C245, C251, and C253, with serine. To determine mutant receptor expression, we used FACs to detect FLAG-tagged receptor expression. Our results show that each of the mutant receptors was localized to the plasma membrane, but their expression levels were much lower than that of hMC2R-WT (Fig. 4A). Total ACTH binding was significantly reduced at the mutations of C21S, C245S, C251S, and C253S. Specific ACTH binding at C21S, C245S, C251S, and C253S was only 48%, 56%, 46%, and 38% of that of the hMC2R-WT, respectively (Table 1). ACTH induced cAMP production at C21S, C245S, C251S, and C253S was only 26, 34, 25, 23% of that of the hMC2R-WT, respectively (Table 1). To further characterize ACTH binding affinity at these mutants, cells expressing these mutants were incubated with 125I-labeled ACTH and various concentrations of unlabeled ACTH. As shown in Fig. 4A, unlabeled ACTH dose-dependently displaces 125I-labeled ACTH binding at these mutated receptors, but ACTH binding affinity at these mutated receptors were significantly reduced (Table 1). To determine whether mutation alters ACTH potency, cells expressing these mutations were incubated with various concentrations of ACTH, and cAMP production was measured. Our results indicate that ACTH potency was significantly reduced at these mutations (Fig. 4B). Their EC50 values are shown in Table 1.


Figure 4
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  		Fig. 4. Effects of mutations of the hMC2R extracellular loop residues on total ACTH binding and receptor activity. A: total ACTH binding at these mutants. B: ability of ACTH stimulated cAMP production at these mutants. Data points represent means ± SE of three independent experiments with duplicate wells.

 

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  		Table 1. Effect of the substitutions of the cysteine residues of hMC2R on 125I-labeled ACTH binding and cAMP production

 
Effect of substitutions of transmembrane domain cysteine residues on receptor function. To determine whether TM cysteines are important for hMC2R function, five cysteine residues (C64, C158, C191, C231, and C267) in the TMs of the hMC2R were mutated and examined. Our results show that each of the mutant receptors was localized to the plasma membrane, and their expression levels are shown in Table 1. Mutations of cysteine (C64S, C158S, and C191S) did not significantly alter receptor function, and their binding affinities and potencies are similar to that of the hMC2R-WT. Mutation of C267 had only a minor influence on ACTH binding affinity and potency (Fig. 5A). However, mutation C231S dramatically decreased ACTH binding affinity and ACTH-induced cAMP accumulation (Fig. 5B). Their EC50 values are shown in Table 1.


Figure 5
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  		Fig. 5. Effects of mutations of the hMC2R transmembrane domains cysteine residues on ACTH binding affinity and potency. A: ACTH binding affinity at these mutants. B: ability of ACTH stimulated cAMP production at these mutants. Data points represent the means ± SE of three independent experiments with duplicate wells (see Tables 1 and 3 for actual Ki and EC50 values).

 
Effect of substitutions of cysteine residues in the intracellular loop on receptor function. There is only one cysteine residue (C293) in the COOH terminus of the hMC2R, and this residue is conserved among MCRs. To determine whether this residue is important for receptor function, ACTH binding and signaling were examined. Our results indicate that mutation of C293S did not significantly alter ACTH binding affinity and potency, preserving the wild-type ACTH binding and activation ability. EC50 values of this mutation is shown in Table 1.

C245 and C251 for disulfide bridge formation. To determine whether the extracellular loop cysteine residues of the hMC2R form disulfide bond partners, OS3 cells expressing hMC2RWT were treated with DTT (10 mM), a disulfide bond reducing agent. ACTH binding affinity and potency were examined. Our results indicate that ACTH total binding and potency were significantly reduced by DTT treatment, suggesting that the critical disulfide bonds exist in hMC2R (Fig. 6A). To determine whether the extracellular loop cysteines are involved in disulfide bond, the mutant receptors C21S, C231S, C245S, C251S, and C253S were treated with DTT. Our results indicate that DTT treatment did not significantly affect ACTH binding affinity and potency at mutant receptor C21S, C231S, and C253S, which were already depressed (Table 2). However, incubation of the mutant receptor C245S and C251S with 10 mM DTT significantly and partially restored C245S and C251S functions by increasing ACTH-induced cAMP production (Fig. 6B). These findings support the theory in which the malfunction of C245S and C251S is caused by the formation of an improper disulfide bond rather than by the loss of a disulfide bridge in the absence of Cys-245 or C251.


Figure 6
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  		Fig. 6. Effect of DTT on ACTH binding and ACTH-induced cAMP accumulation at hMC2RWT or mutants. OS3 cells were transfected with MC2R WT or mutants and incubated in the presence of 10 mM DTT. ACTH binding and cAMP levels were determined as described under METHODS. A: effect of DTT on total ACTH binding at these mutants. B: effect of DTT on the ability of ACTH-stimulated cAMP production at these mutants. Data points represent the means ± SE of three independent experiments with duplicate wells.

 

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  		Table 2. Effect of DTT on cAMP production at hMC2R mutants

 
We further used double mutations to examine the disulfide bond between the extracellular loops of hMC2R. Double mutations of the extracellular loop cysteine were generated. These mutant receptors include C21/C245, C21/C251, C21/C253, C245/C251, C245/C253, and C251/C253. Our results show that each of the mutant receptors was localized to the plasma membrane, but their expression levels were much lower than that of hMC2R-WT (Table 3). Total ACTH binding was significantly reduced at these mutant receptors (Table 3). Furthermore, ACTH-induced cAMP production at these mutant receptors was only 2–5% of that of the hMC2R-WT (Table 3). In summary, our results indicate that cysteines in the extracellular loops and TM6 of the hMC2R are crucial for receptor function.


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  		Table 3. Effect of double mutations of cysteine residues of hMC2R on ACTH-induced cAMP production

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we systematically characterized the role of the MC2R endogenous cysteine residues on receptor function. Our results show that four extracellular cysteine residues, C21, C245, C251, C253, and one TM residue 231 are important for ACTH binding and signaling. The extracellular loop C245 and C251 may form a disulfide bond, which is important for MC2R function.

Almost all GPCRs contain a pair of conserved Cys residues in the first and second extracellular domains or a disulfide bridge in the second extracellular loop (EL2) to the third transmembrane domain. These residues have been shown to form a receptor stabilizing disulfide bridge structure. In rhodopsin, this disulfide bond has been confirmed through chemical and crystallographic approaches, which play a key role in the formation of a properly folded, stable receptor (4). In the rat GnRH-R, this disulfide bond was confirmed by mutagenesis (3). In the beta-adrenergic receptor, two disulfide bonds were even identified within the extracellular loop that play important roles in receptor function (6, 22). However, the melanocortin receptor family lacks cysteine residues required to make up a highly conserved disulfide bridge that will connect EL1 and EL2 and connect TM3 with EL2 in most GPCRs. Our results demonstrate that the extracellular loop 3 (residue C245, C251, and C253) of hMC2R may be important for maintaining normal receptor structure and function. These results are similar to that of hMC4R and hMC1R, in which the extracellular loop 3 of hMC1R and hMC4R play important roles in maintaining receptor structure and function (1, 26). Our results also indicate that mutation of C21 in NH2 terminus of hMC2R significantly decreased ACTH binding and signaling. This residue mutation was also identified in FGD (18), and the molecular mechanism for this may be due to the low receptor expression at cell surface.

Cysteine residues in the transmembrane domains of GPCRs also play important roles in receptor function. It was also reported that cysteine mutation of the TM5 of hMC1R abolished receptor signaling (8). hMC2R contains five cysteines in the transmembrane domains, and all of them are conserved among MCRs (Fig. 1). However, C231 in TM6 is identified to be involved in ACTH binding and signaling, suggesting that receptor-mediated signaling of hMC2R is different from that of the hMC1R. Cysteines in the COOH terminus have previously been implicated in receptor signaling in beta2-adrenergic receptor, human follicle-stimulating hormone receptor, and glucagon-like peptide receptor (7, 15, 27, 29). Similar to other GPCR, cysteine 315 in the COOH terminus of hMC1R and cysteine 319 in the COOH terminus of hMC4R were also identified to be involved in receptor signaling (8, 28). However, our results indicate that cysteine in the COOH terminus of hMC2R is not involved in ACTH binding and signaling, suggesting that the pathway of hMC2R signaling is different from that of the other MCRs.

The disulfide bond between the extracellular cysteine residues of the GPCR plays an important role in maintaining receptor structure and function. To determine whether the disulfide bond exists in hMC2R, we used the positively charged, cell-impermeant sulfhydryl-reactive reagent DTT to treat both MC2RWT and the mutated receptors (14, 24). If the important disulfide bonds exist, DTT treatment will break this bond, which may alter receptor function. As we expected, DTT treatment resulted in significantly decreased ACTH binding affinity. Further studies indicate that DTT treatment had minimal impairment of the receptor function at the mutant receptor C21 and C253 but partially restored ACTH binding and signaling at C245S and C251S, implying that disulfide bonds are broken between these conserved Cys residues, in a similar fashion to that observed in the hMC2R WT. These results suggest that hMC2R residues Cys245 and Cys251 may form a disulfide bond that is consistent with previous results from the hMC1R and hMC4R, in which the residues, C267, C273 in hMC1R and C271 and C277 in hMC4R, were identified to be involved in disulfide bond (1, 26). All of these results suggest that cysteine residues in the extracellular loop 3 of melanocortin receptor family play important roles in maintaining normal receptor structure and function.

We further expand our initial efforts using double cysteine mutations that may rescue the loss of the receptor function observed above with single mutation based on the literature (17). This approach may provide functional readout of the receptor structure and allow for direct comparisons to our site-directed mutagenesis results. However, our results do not support this hypothesis. The double mutations resulted in even decreased receptor expression, total ACTH binding and signaling compared with that of single mutation. This is not consistent with the previous finding of secretin receptor, in which double mutation partially restored receptor function (17). Our explanation is that these residues are not only important for receptor structure but also important for receptor expression, and low receptor expression is the main reason for the decreased MC2R function.

In conclusion, we identified that four extracellular cysteine residues, C21, C245, C251, C253, and one TM residue C231 are crucial for ACTH binding and signaling. One disulfide bond (C245/C251) may exist within EL3 of the hMC2R. These results provide important insights into the functional role of cysteines on MC2R expression and signaling.


   GRANTS
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 ABSTRACT
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REFERENCES
 
This work has been supported by National Institutes of Health Grants R03-HD047312-01A1 (Y.-K. Yang).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Yang, Div. of Pediatric Surgery, Univ. of Alabama at Birmingham, 300 ACC, 1600 7th Ave. South, Birmingham, AL 35233 (e-mail: ying-kui.yang{at}ccc.uab.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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