Replacement of short segments within transmembrane domains of MC2R disrupts retention signal

in Journal of Molecular Endocrinology
View More View Less
  • 1 Latvian Biomedical Research and Study Centre, Department of Neuroscience, Ratsupites 1, LV-1067 Riga, Latvia

The proteolysis of the pro-opiomelanocortin precursor results in the formation of melanocortins (MCs), a group of peptides that share the conserved -H-F-R-W- sequence, which acts as a pharmacophore for five subtypes of MC receptors (MCRs). MC type 2 receptor (MC2R; also known as ACTHR) is the most specialized of all the MCRs. It is predominantly expressed in the adrenal cortex and specifically binds ACTH. Unlike other MCRs, it requires melanocortin receptor accessory protein 1 (MRAP) for formation of active receptor and for its transport to the cell membrane. The molecular mechanisms underlying this specificity remain poorly understood. In this study, we used directed mutagenesis to investigate the role of various short MC2R sequence segments in receptor membrane trafficking and specific activation upon stimulation with ligands. The strategy of the study was to replace two to five amino acid residues within one MC2R segment with the corresponding residues of MC4R. In total, 20 recombinant receptors C-terminally fused to enhanced green fluorescent protein were generated and their membrane trafficking efficiencies and cAMP response upon stimulation with α-MSH and ACTH(1–24) were estimated during their stand-alone expression and coexpression with MRAP. Our results indicate that both the motif that determines the ligand-recognition specificity and the intracellular retention signal are formed by a specific extracellular structure, which is supported by the correct alignment of the transmembrane domains. Our results also indicate that the aromatic-residue-rich segment of the second extracellular loop is involved in the effects mediated by the second ACTH pharmacophore (-K-K-R-R-).

Abstract

The proteolysis of the pro-opiomelanocortin precursor results in the formation of melanocortins (MCs), a group of peptides that share the conserved -H-F-R-W- sequence, which acts as a pharmacophore for five subtypes of MC receptors (MCRs). MC type 2 receptor (MC2R; also known as ACTHR) is the most specialized of all the MCRs. It is predominantly expressed in the adrenal cortex and specifically binds ACTH. Unlike other MCRs, it requires melanocortin receptor accessory protein 1 (MRAP) for formation of active receptor and for its transport to the cell membrane. The molecular mechanisms underlying this specificity remain poorly understood. In this study, we used directed mutagenesis to investigate the role of various short MC2R sequence segments in receptor membrane trafficking and specific activation upon stimulation with ligands. The strategy of the study was to replace two to five amino acid residues within one MC2R segment with the corresponding residues of MC4R. In total, 20 recombinant receptors C-terminally fused to enhanced green fluorescent protein were generated and their membrane trafficking efficiencies and cAMP response upon stimulation with α-MSH and ACTH(1–24) were estimated during their stand-alone expression and coexpression with MRAP. Our results indicate that both the motif that determines the ligand-recognition specificity and the intracellular retention signal are formed by a specific extracellular structure, which is supported by the correct alignment of the transmembrane domains. Our results also indicate that the aromatic-residue-rich segment of the second extracellular loop is involved in the effects mediated by the second ACTH pharmacophore (-K-K-R-R-).

Introduction

The melanocortins (MCs) are a group of peptides that originate from the proteolysis of the pro-opiomelanocortin precursor and all share the same conserved MC motif -H-F-R-W- sequence, which acts as the pharmacophore for their receptors (Eberle 1988). There are five subtypes of MC receptors (MCRs), which act predominantly through the Gsα subunit (Chhajlani & Wikberg 1992, Mountjoy et al. 1992, Gantz et al. 1993a,b, 1994). These receptors are expressed within various organs, ranging from the CNS to the skin (Roselli-Rehfuss et al. 1993, Gantz et al. 1994, Mountjoy et al. 1994, Beuschlein et al. 2001, Gantz & Fong 2003). In some cases, the expression of a specific receptor type in a certain tissue correlates clearly with the regulation of a specific function. This is the case for the MC type 2 receptor (MC2R), also known as the adrenocorticotropic hormone receptor (ACTHR), and its expression in the adrenal cortex (Mountjoy et al. 1992). However, the full extent of the regulatory functions of some of these receptors is still unclear.

Systematic directed mutagenesis has provided valuable knowledge regarding the molecular mechanisms underlying the signal transduction of the MCRs. It is well established that the binding pocket of the MC4R, and probably other MCRs, is formed by amino acid residues located on the extracellular side of the transmembrane (TM) domains and consists of two elements: the negatively charged pocket (formed by TMIIE100, TMIIID122, and TMIIID126) and the aromatic pocket (formed by TMIVF184, TMVF202, TMVIF261, TMVIF262, and TMVIIF284), which interact with the positively charged arginine residue and aromatic phenylalanine and tryptophan residues of the pharmacophore respectively (Yang et al. 2000, Haskell-Luevano et al. 2001, Fleck et al. 2005, Ignatovica et al. 2011).

MC2R is the most specialized of all the MCRs. Its expression is limited to the adrenal cortex and adipocytes (Mountjoy et al. 1992, Boston & Cone 1996, Beuschlein et al. 2001) and it specifically binds ACTH. The second -K-K-R-R- pharmacophore, which is located in the central part of ACTH, is required to activate this receptor (Schwyzer 1977, Kovalitskaia et al. 2008). In the adrenal cortex, MC2R affects glucocorticoid production (Clark & Cammas 1996) and synthesis of its own mRNA (Naville et al. 1999). Research with mouse adipocytes has shown that MC2R participates in lipolysis (Boston 1999) and the production of both leptin (Norman et al. 2003) and interleukin 6 (Jun et al. 2010), but its functions in human adipocytes are still unclear. Genetic defects in the MC2R gene cause familial glucocorticoid deficiency, a rare autosomal recessive disorder that is characterized by low plasma cortisol levels and excessive ACTH (Penhoat et al. 2002).

MC2R must be coexpressed with melanocortin receptor accessory protein 1 (MRAP) to become functionally active and localize to the cell surface (Metherell et al. 2005). At present, two subtypes of this protein are known in humans: MRAPα and MRAPβ. Both of them can couple to all MCRs, but, unlike MC2R, this coupling inhibits their functional activities (Chan et al. 2009), although results presented in one recent report have indicated that this pattern might be more complicated (Kay et al. 2013). Structurally, MRAPs are single TM proteins. MRAPα and MRAPβ are expressed from one gene, as the products of alternative splicing, and have identical N-termini and TM domains, but distinct C-termini (Metherell et al. 2005). In the cell, MRAP exists as a unique, very stable, SDS-resistant, antiparallel homodimer and the region located adjacent to the N-termini of TM region is important for its antiparallel dimerization. The TM domain itself is responsible for its specific coupling to the MC2R and the region located in the N-terminal tail is crucial for MC2R ligand recognition (Sebag & Hinkle 2007, 2009, Cooray et al. 2008, Webb et al. 2009).

Studies of the structural determinants of MC2R are lagging behind the advances in MRAP research, probably because of the greater complexity of the protein. However, truncation of the C-terminus of MC2R is known to cause the loss of signal transduction (Hirsch et al. 2011). In our previous study, we identified two regions of MC2R that are responsible for retention of the receptor within the interior of the cell: the N-terminus of the receptor, which seems to lack sufficient number of glycosylation sites to form a membrane export signal, and the TM3–TM4 region, which contains an active retention signal (Fridmanis et al. 2010). Similar research was also carried out by Hinkle et al. (2011). Analysis of the membrane expression and activation of the chimeric receptors engineered by this group indicated that the retention signal probably is the conformation of the receptor as a whole, that TM1 is very important for the formation of the retention signal, and that the N-terminus is crucial for ligand recognition (Hinkle et al. 2011). The latest results in the field have indicated that serine and threonine residues located within the second intracellular loop of MC2R play a major role in the formation of a functionally active receptor and in the internalization and desensitization of the receptor. Two amino acid residues with a particularly great influence are T143 and T147 (Roy et al. 2011).

The aim of this study was to investigate the TM3–TM5 region of MC2R in detail and to identify the amino acid residues involved in the formation of the intracellular retention signal and in the ligand specificity of the receptor.

We used a well-established approach involving the generation of chimeric MC2R/MC4R and fluorescent marker fusion proteins, followed by the functional analysis and confocal visualization of these receptors in the presence and absence of MRAPβ.

Materials and methods

Cloning and expression vector

pCEP4-GFP-C, modified from the pCEP4 vector (Invitrogen), was used to express all the recombinant receptors. This modification included the insertion of the gene encoding enhanced green fluorescent protein (EGFP) at the 3′-end of the multiple cloning site. The details of the vector modifications are described by Fridmanis et al. (2010). pcDNA5/FRT Mammalian Expression Vector (Invitrogen) was used to express human MRAPβ.

Generation of coding sequences

The human MC2R and human MC4R coding sequences were inserted into the pCEP4-GFP-C vector by amplifying both receptor genes from plasmids based on pcDNA3.1. The human MRAPβ coding sequence was inserted into the pcDNA5/FRT vector by amplifying its coding sequence from a plasmid based on pUC19. The primers used included HindIII or XhoI restriction sites (the MC2R reverse primer included a coding sequence for a nine amino acid linker, GSGTGSGLE). All chimeric receptors were created with site-directed mutagenesis by overlap extension (Ho et al. 1989) and verified by DNA sequencing using BigDye v3.1 and the ABI Prism 3130xl Genetic Analyzer sequencing system (Applied Biosystems), according to the recommendations of the supplier. The sequences of all the primers used are listed in Supplementary Table 1, see section on supplementary data given at the end of this article.

Expression of receptor clones

During this study, BHK cells were grown in DMEM/F12 medium supplemented with 5% FCS. For the microscopy experiments, the cells were cultured on coverslips, whereas for the cAMP response studies, they were cultured in standard 100 mm cell culture Petri dishes. For both types of experiments, the cells were transiently transfected with the plasmids using TurboFect Transfection Reagent (Fermentas, Vilnius, Lithuania), but for the cells on coverslips, a reverse transfection technique was used. In the case of co-transfection, both plasmids were premixed in equal amounts (mass to mass). Cells attached to coverslips were examined by confocal microscopy after incubation for approximately 24 h and those on Petri dishes were used in the cAMP response studies after incubation for approximately 48 h.

cAMP response studies

The LACNE cAMP 384 Kit (PerkinElmer, Waltham, Massachusetts, USA) was used to determine the cAMP response upon stimulation with human α-MSH or ACTH(1–24) (PolyPeptide Laboratories, Strasbourg, France). The numerical data acquired were used to determine the 50% effective concentrations (EC50) with nonlinear regression, using the PRISM 3.0 Software (GraphPad Software Inc., San Diego, California, USA). The initial concentration of ligand in the dilution series was either 100 or 10 μM, the dilution factor was 10, and seven points were measured during each experiment. All experiments were carried out in duplicate and repeated three times (Supplementary Fig. 1, see section on supplementary data given at the end of this article).

Confocal microscopy studies

To label the membranes of the cells, the coverslips were rinsed in 1× PBS and incubated for 3 min in Alexa Fluor 633-labeled wheat germ agglutinin (AF-WGA; 10 μg/ml in 1× PBS; Molecular Probes–Life Technologies). After incubation, the coverslips were rinsed twice with 1× PBS, fixed for 10 min with 4% formaldehyde (Sigma–Aldrich) in 1× PBS, and rinsed twice with 1× PBS. The coverslips were then placed on microscope slides and inspected under a confocal fluorescence microscope (Leica DM 6000B: Leica Microsystems CMS GmbH, Mannheim, Germany).

During the microscopic analysis, EGFP and AF-WGA fluorescent images acquired in six cells from at least two independent transfections (not more than three cells from each) were used for each receptor construct. Eighteen evenly distributed cell cross-sectional images were captured along the cell-depth axis of each analyzed cell. These images were used to assess the efficiency of transport of the EGFP-tagged receptors to the cell membrane. This was estimated by calculating the EGFP/AF-WGA fluorescence intensity ratio at multiple points on the plasma membrane. Twenty random points on each cell were selected, giving at least 120 points for the analysis of each receptor construct per cell. All measurements were made using the Leica Confocal Software (LCS-Lite v2.61) quantification-profiling tool. The methodology used was described in detail by Fridmanis et al. (2010).

Statistical analysis of confocal microscopy data

All datasets acquired from six cells expressing the same receptor were tested for uniformity using the Kruskal–Wallis test (significance level α=0.05). If the Kruskal–Wallis test showed that the medians of the acquired data varied significantly, then Dunn's multiple comparison test (Dunn 1964) was performed (significance level α=0.05) to determine which of the datasets was different. Datasets that differed significantly (P<0.05) from more than two other datasets were replaced with values acquired from repeated independent transfection experiments and confocal microscopic analysis. Data acquired from different receptors were analyzed by comparing the median values and interquartile ranges, and the significance of differences in the datasets was estimated using Dunn's multiple comparison test (significance level α=0.01). Based on these results, all the receptors analyzed were categorized into three groups. The first group was formed after the exclusion of all receptors for which the EGFP/AF-WGA fluorescence ratio at the cell membrane was significantly lower than the MC4R fluorescence ratio (P<0.01). These were considered to show ‘high membrane export’. The second group of receptors was formed after the exclusion of all receptors for which the EGFP/AF-WGA fluorescence ratio at the cell membrane was significantly higher (P<0.01) than the MC2R fluorescence ratio. These were considered to show ‘no membrane export’. Finally, the third group included all those receptors that had an EGFP/AF-WGA fluorescence ratio at the cell membrane significantly lower than the MC4R ratio (P<0.01) but significantly higher than the MC2R ratio (P<0.01), and were thus considered to have ‘low membrane export’ (Supplementary Fig. 2, see section on supplementary data given at the end of this article). The methodology used has been described in detail by Fridmanis et al. (2010).

Results

All receptors created in this study were C-terminally fused to EGFP via a serine–glycine-rich linker sequence, allowing us to quantify the receptor levels within the membrane with confocal microscopic imaging. All the receptors were tested for their capacity to induce cAMP production upon stimulation with α-MSH and ACTH. To distinguish MC2R-specific retention from nonspecific retention (e.g. attributable to receptor misfolding), both types of experiments were performed using BHK cells expressing a particular receptor alone and coexpressed with human MRAPβ, which was selected over its isoform MRAPα because of its higher efficiency in transporting MC2R to the cell membrane and its ability to facilitate a higher cAMP response when stimulated with ACTH.

To ensure that the results were not confounded by the presence of the N-terminal part of MC2R, which lacks proper membrane trafficking signals, we used a chimeric receptor (Ch2 (Fig. 1)) from our previous study as the core construct for the mutant receptors and as the negative control in the functional characterization. In this receptor, the N-terminal segment is replaced by that of MC4R. All the constructs used in the study are designated ‘Ch’ followed by a number, to avoid any confusion with our previous study.

Figure 1
Figure 1

Schematic of the recombinant receptors Ch16–Ch22. Regions from MC4R are shaded gray and regions from MC2R are shaded black.

Citation: Journal of Molecular Endocrinology 53, 2; 10.1530/JME-14-0169

During analysis of MC2R, MC4R, and Ch2 in the absence of MRAPβ, significant EGFP fluorescence was detected at the cell membrane only for MC4R. However, when coexpressed with MRAPβ, all three receptors were transported to the cell membrane, although the fluorescence levels for MC4R and Ch2 (both coexpressed with MRAPβ) were significantly lower than that for MC4R expressed alone (P<0.01; Supplementary Fig. 2; Figs 2 and 3). The cAMP assays showed that α-MSH induced cAMP production in the presence and absence of MRAPβ; cAMP could only be detected within cells expressing MC4R, and the EC50 values of the α-MSH-induced cAMP response in the presence of MRAPβ were 16.6-fold higher than in its absence and ACTH-induced EC50 values of cAMP response in the presence of MRAPβ were 3.4-fold lower than those in its absence. The EC50 values of ACTH-induced cAMP response in the presence of MRAPβ were 129- and 16-fold higher for Ch2 and MC4R respectively in comparison with the EC50 value for MC2R (Table 1 and Supplementary Fig. 1).

Figure 2
Figure 2

An example set of the confocal fluorescence microscopy images that were acquired during quantification of receptor transportation to the cell membrane. Membranes of receptor–EGFP (green) expressing BHK cells were stained with AF-WGA (red). A full colour version of this figure is available at http://dx.doi.org/10.1530/JME-14-0169.

Citation: Journal of Molecular Endocrinology 53, 2; 10.1530/JME-14-0169

Figure 3
Figure 3

Graph representing the medians, interquartile, and min/max ranges of the EGFP/AF-WGA fluorescence intensity ratios at the plasma membrane for MC4R, MC2R, Ch2, and Ch16–Ch22. Bars that are labeled with ↑ or ↑↑ represent receptors with low or high levels of membrane export respectively. Bars that are labeled with ⊥ represent receptors that are retained. Symbols * and ° represent the significance of difference in rank sum between specific receptor and MC2R and MC4R respectively. Three symbols represent P<0.001 and two symbols represent P<0.01.

Citation: Journal of Molecular Endocrinology 53, 2; 10.1530/JME-14-0169

Table 1

Effects of α-MSH and ACTH(1–24) on cAMP production by BHK cells transfected with MC2R and MC4R expression constructs. Ch2 and Ch16–Ch22 with and without MRAPβ (means±s.e.m.). EC50 values were obtained from cAMP response curves using α-MSH and ACTH(1–24) as stimulators

MC2RMC4RCh2Ch16Ch17Ch18Ch19Ch20Ch21Ch22
−MRAPβ
 EC50±s.e.m. α-MSH (nM)ND358.0±227.3NDNDNDNDNDNDNDND
 EC50±s.e.m. ACTH (nM)ND194.2±39.1NDNDNDNDNDNDNDND
 Membrane++/−++++
+MRAPβ
 EC50±s.e.m. α-MSH (nM)ND5950.0±5935.0NDNDNDNDNDNDNDND
 EC50±s.e.m. ACTH (nM)3.6±1.057.9±56.2464.7±187.216.2±9.218.9±6.4ND861.5±784.458.3±2.0NDND
 Membrane++/−+/−+/−+++

+, Receptors with high membrane export; +/−, receptors with low membrane export; −, receptors that are retained; ND, not determined.

Structural domain replacements

To clarify the role of TM5–TM6 in the retention of MC2R, we created two sets of chimeric receptors, replacing these MC2R regions with the corresponding parts of MC4R. The first set comprised Ch16–Ch18, while the second set comprised Ch19–Ch21. The receptors of the second set were identical to the first, except that their MC2R N-terminal segments were replaced with the corresponding region from MC4R (Fig. 1).

Quantification of the EGFP/AF-WGA fluorescence intensity ratio at the cell membrane during the stand-alone expression of the chimeric receptors revealed that only Ch16 and Ch17 were present in the plasma membrane, although Ch16 levels were lower than those of MC4R (Fig. 3 and Table 1). The cAMP assays performed on these receptors showed that none of them was activated when stimulated with α-MSH or ACTH (Table 1 and Supplementary Fig. 1). The coexpression of Ch16–Ch18 with MRAPβ greatly altered their patterns of membrane expression. The presence of MRAPβ reduced the fluorescence ratio at the plasma membrane for both Ch16 and Ch17, whereas that of Ch18 was slightly increased. MRAPβ coexpression did not induce cAMP accumulation after any of the chimeric receptors was stimulated with α-MSH, but activated cAMP accumulation in cells expressing Ch16 and Ch17 when stimulated with ACTH. Thus, the EC50 values for Ch16 and Ch17 were only 4.5- and 5.3-fold higher, respectively, than that for MC2R (Table 1 and Supplementary Fig. 1).

The localization and functional activity assays of Ch19–Ch21 revealed that when expressed without MRAPβ, all three receptors were effectively transported to the cell surface (Fig. 3 and Table 1). However, none of them induced cAMP production when stimulated with either α-MSH or ACTH (Table 1 and Supplementary Fig. 1). The coexpression of these receptors with MRAPβ did not cause any significant change in the membrane transportation of Ch19 and Ch20, which are analogs of Ch16 and Ch17 respectively, but effectively prevented the membrane trafficking of Ch21, which is an analog of Ch18 (Fig. 3 and Table 1). None of the receptors were activated when stimulated with α-MSH, but stimulation with ACTH showed that Ch19 and Ch20 were functional, with EC50 values 239.3- and 16.2-fold higher, respectively, than that of MC2R, and 1.8-fold higher and eightfold lower, respectively, than that of Ch2 (Table 1 and Supplementary Fig. 1).

The results from our previous study have indicated that the integrity of the TM1–TM3 region is critical for the functional activity of the receptor. Therefore, an additional chimeric receptor, Ch22, was created to evaluate whether the replacement of the sole TM3 domain influences the membrane transport of the receptor and whether this receptor retains its functionality (Fig. 1). Localization analysis showed that Ch22 was not transported to the membrane when expressed alone, but appeared in the cell membrane when coexpressed with MRAPβ (Fig. 3 and Table 1), thus displaying MC2R-like membrane expression. However, Ch22 was functionally inactive, and no cAMP accumulation was detected when it was stimulated with ACTH or α-MSH in the presence or absence of MRAPβ (Table 1).

Amino acid motif replacements

To further investigate the TM3–TM5 region, we replaced two to five mismatching amino acid residues within the TM3–TM5 region of the Ch2 sequence with the corresponding residues from MC4R based on an alignment of Ch2 and MC4R. The number of amino acid residues replaced within each receptor constructed depended on the number of mismatches between MC4 and Ch2 within the stretch of amino acid residues coded by 12–18 nucleotides, which is the most suitable length for PCR-based mutagenesis. Consequently, 13 receptors were created.

First, we explored the functionality of TM3 creating four mutant receptors: Ch23–Ch26 (Fig. 4 and Supplementary Table 2, see section on supplementary data given at the end of this article). When expressed alone, Ch23, Ch25, and Ch26 were detected at high levels in the cell membrane, whereas the level of Ch24 in the membrane did not differ from that of MC2R (P>0.01; Table 2 and Fig. 5). cAMP assays revealed that despite their effective transport to the membrane, none of these receptors induced a signal molecule response when stimulated with α-MSH or ACTH (Table 2, Fig. 6 and Supplementary Fig. 1). Coexpression of these receptors with MRAPβ showed the following: the membrane localization of Ch23 decreased to a level significantly lower than that of MC4R (P<0.01) but significantly higher than that of MC2R (P<0.01); the membrane localization of Ch24 increased to a level similar to the MC4R level; the presence of Ch25 at the cell membrane was significantly reduced, to the level of MC2R; and the transportation of Ch26 to the cell membrane remained unaltered (Table 2 and Fig. 5). Measurement of the cAMP response revealed that when stimulated with ACTH, all the receptors (except Ch24) were active and the EC50 value of Ch23 was 2.3-fold higher than that of Ch2 and 298-fold higher than that of MC2R; the EC50 value of Ch25 was 14.5-fold lower than that of Ch2 but 8.9-fold higher than that of MC2R; and the EC50 value of Ch26 was 12.3-fold lower than that of Ch2 but 10.5-fold higher than that of MC2R. None of the receptors were activated when stimulated with α-MSH (Table 2, Fig. 6 and Supplementary Fig. 1).

Figure 4
Figure 4

Schematic of recombinant receptors Ch23–Ch35. (Upper panel) Snake-like plot. Regions from MC4R are shaded gray, regions from MC2R are shaded black, and substituted amino acid residues are shaded according to their membrane transport efficiency. Circles shaded in green represent substitutions that facilitated high levels of membrane export, those in yellow represent low levels of membrane export, and those in red represent intracellular retention. The left side of the circle represents the membrane export level during stand-alone expression, and the right side represents the membrane export level during coexpression with MRAPβ. The symbols on the left side of receptor name indicate the effect of MRAPβ coexpression on the membrane export efficiency of the corresponding receptor. ‘↑’ Represents increased export efficiency; ‘↓’ represents reduced export efficiency; ‘_’ unaltered high export efficiency; and ‘_’ unaltered retention. (Lower panel) Alignment of Ch2 and MC4R amino acid sequences. Shaded rectangular fields indicate transmembrane domain boundaries predicted using the TMPRED tool (Hofmann & Stoffel 1993). The positions of the substituted amino acids within recombinant receptors Ch23–Ch35 are marked with ‘+ − − +’ and the corresponding receptor number above. A full colour version of this figure is available at http://dx.doi.org/10.1530/JME-14-0169.

Citation: Journal of Molecular Endocrinology 53, 2; 10.1530/JME-14-0169

Table 2

Effects of α-MSH and ACTH(1–24) on cAMP production by BHK cells transfected with Ch23–Ch35 expression constructs with and without MRAPβ (means±s.e.m.). EC50 values were obtained from cAMP response curves using α-MSH and ACTH(1–24) as stimulators

Ch23Ch24Ch25Ch26Ch27Ch28Ch29Ch30Ch31Ch32Ch33Ch34Ch35
−MRAPβ
 EC50±s.e.m. α-MSH (nM)NDNDNDNDNDNDNDNDNDNDNDNDND
 EC50±s.e.m. ACTH (nM)NDNDNDNDNDNDNDNDNDNDNDNDND
 Membrane+++++/−+/−+++/−+
+MRAPβ
 EC50±s.e.m. α-MSH (nM)NDNDNDNDNDNDNDNDNDNDNDNDND
 EC50±s.e.m. ACTH (nM)1073.0±462.1ND32.0±17.637.9±21.0292.7±170.7209.3±53.4257.0±49.7324.0±144.0ND1237.0±521.923.0±3.871.7±18.738.9±10.6
 Membrane+/−+++++++/−++

+, Receptors with high membrane export; +/−, receptors with low membrane export; −, receptors that are retained; ND, not determined.

Figure 5
Figure 5

Graph representing the medians and interquartile ranges of the EGFP/AF-WGA fluorescence intensity ratios at the plasma membrane for Ch23–Ch35. Bars that are labeled with ↑ or ↑↑ represent receptors with low or high levels of membrane export respectively. Bars that are labeled with ⊥ represent receptors that are retained. Symbols * and ° represent the significance of difference in rank sum between specific receptor and MC2R and MC4R respectively. Three symbols represent P<0.001 and two symbols represent P<0.01.

Citation: Journal of Molecular Endocrinology 53, 2; 10.1530/JME-14-0169

Figure 6
Figure 6

Snake-like plot of the TM3–TM5 region of MC2R showing the effects of ACTH(1–24) on cAMP production by cells cotransfected with recombinant receptors Ch23–Ch35 and MRAPβ. Substituted amino acid residues of receptors whose EC50 values were within the range of 23.0–71.7 nM are shaded in green, within 209.3–324.0 nM are shaded in yellow, and within 1073.0–1237.0 nM are shaded in orange. Substitutions that produce inactive recombinant receptors are shaded in red. A full colour version of this figure is available at http://dx.doi.org/10.1530/JME-14-0169.

Citation: Journal of Molecular Endocrinology 53, 2; 10.1530/JME-14-0169

We also introduced amino acid replacements into the TM4–TM5 region. In total, nine mutant receptors (Ch27–Ch35) were created in this experiment (Fig. 4 and Supplementary Table 2). When expressed alone, Ch29, Ch32, Ch33, and Ch35 were effectively transported to the cell membrane. Ch30, Ch31, and Ch34 were also present in the cell membrane but in significantly lower amounts (P<0.01) than MC4R, whereas the ratios for Ch27 and Ch28 did not differ significantly from that for MC2R (P>0.01; Table 2 and Fig. 5). As before, none of these constructs were activated during stand-alone expression when stimulated with α-MSH or ACTH (Table 2, Fig. 6 and Supplementary Fig. 1). The coexpression of these mutant receptors with MRAPβ resulted in the following: Ch27, Ch30, Ch31, Ch32, Ch34, and Ch35 were effectively transported to the cell membrane; Ch33 was transported to the membrane, but in significantly lower amounts (P<0.01) than MC4R; and the levels of Ch28 and Ch29 were the same as that of MC2R (Table 2 and Fig. 5). Tests for functional activity revealed that eight of these nine receptors triggered a cAMP response when stimulated with ACTH, whereas Ch31 induced no response. The results of the cAMP assays allow the receptors to be subdivided into three groups. The first group comprises Ch33, Ch34, and Ch35, which displayed the EC50 values 6.4- to 20.2-fold lower than that of Ch2 and 6.4- to 19.9-fold higher than that of MC2R. The second group comprises Ch27, Ch28, Ch29, and Ch30, which displayed the EC50 values 1.4- to 2.2-fold lower than that of Ch2 and 58.1- to 90-fold higher than that of MC2R. Finally, the EC50 value of Ch32 was 2.7-fold higher than that of Ch2 and 343.6-fold higher than that of MC2R. None of the receptors were activated when stimulated with α-MSH (Table 2, Fig. 6 and Supplementary Fig. 1).

Discussion

Several notable observations were made during the functional testing of the control receptors. It has been observed that the EC50 value of the cAMP response after MC4R stimulation with α-MSH was 16.6-fold higher in the presence of MRAPβ than in its absence, which corresponds to the observations of Chan et al. (2009). We also observed 3.4-fold higher cAMP response sensitivity when MC4R was coexpressed with MRAPβ and stimulated with ACTH than when MC4R was expressed alone (Table 1), which has been previously reported by Josep Agulleiro et al. (2013) to be also the case for the zebrafish MC4R. Taken together, these findings indicate that MRAPβ not only selectively reduces the amount of MC4R in the cell membrane, but also negatively affects the receptor's ability to respond to stimulation with α-MSH, while leaving intact or even increasing its ability to respond to stimulation with ACTH. In a broader sense, this might mean that the role of MRAPβ in the MC system actually goes beyond the regulation of receptor export to the membrane. The true biological function of this effect must be thoroughly evaluated.

We have previously suggested that the MC2R arrest signal is formed by the interaction between its TM3 and TM4 regions (Fridmanis et al. 2010). This more detailed investigation using a set of chimeras, Ch16–Ch22, has demonstrated the critical role of the extracellular loops (ELs) in membrane transport, based on the observation that the only structural difference between Ch17 (present in the cell membrane) and Ch18 (absent from the cell membrane) is the presence/absence of the second EL (EL2) of MC2R (Fig. 1). Therefore, the arrest of the receptor occurs only when both EL1 and EL2 of MC2R are present (Figs 1 and 2). This hypothesis is further supported by Ch22. The importance of the ELs corresponds well to our previous results, which showed that all chimeric receptors containing both EL1 and EL2 from MC2R (Ch4 and Ch13) were retained in the ER, whereas chimeras with only EL2 (Ch5) or EL1 (Ch12) from MC2R were present in the cell membrane (Fridmanis et al. 2010). Interestingly, when coexpressed with MRAPβ, both Ch16 and Ch17, which contain EL2 from MC4R, acted in an MC4R-like manner, while at the same time retaining MC2R-like functional properties (Table 1 and Fig. 2). As expected, additional replacement of the N-terminal segment with that from MC4R increased the membrane expression of all three receptors (Ch19–Ch21). Surprisingly, the coexpression of Ch19 or Ch20 with MRAPβ increased their presence in the membrane, in contrast to their counterparts Ch16 and Ch17 respectively (Table 1, Figs 1 and 2), whereas it reduced the membrane expression of Ch21. Therefore, it seems that the disruption of both retention-facilitating elements alters the MRAPβ-induced effects on these receptors. It must be noted that Ch18 and Ch21 were functionally inactive, which can be attributed to changes within the intracellular sides of the receptors, their membrane levels, or the disruption of the binding pocket. As shown previously, ACTH contains two pharmacophores (Kovalitskaia et al. 2008). As the amino acid residues involved in the formation of the -H-F-R-W- binding pocket are located outside the TM4–TM5 region, it is possible that in Ch18 and Ch21, the -K-K-R-R- binding pocket is disrupted. This finding is consistent with the observation from our previous study that TM4–TM5 is the region that determines the ligand specificity of MC2R (Fridmanis et al. 2010).

It is also interesting and worth noting that when Ch16 and MRAPβ were coexpressed, we observed cAMP accumulation, despite the fact that the statistical analysis of the microscopy data indicated that the surface expression of the receptor was similar to that of MC2R during its stand-alone expression. Thus, it seems clear that Ch16 in the presence of MRAPβ is transported to the cell surface, at least at low levels. Perhaps, the most plausible explanation of this phenomenon lies in the fact that the cAMP response involves not just signal transduction across the cell membrane but also its amplification, hence the cAMP assays might be more sensitive than florescent imaging techniques. The results for Ch25, Ch28, and Ch29 coexpressed with MRAPβ were similar, supporting this conclusion.

The results discussed thus far highlight the significant role of the extracellular elements: the N-terminal segment, EL1, and EL2. Based on the knowledge that the extracellular part of bovine rhodopsin forms a compact lid-like structure over the receptor-binding pocket (Palczewski et al. 2000), we can speculate that a similar structure, formed by the N-terminal segment, EL1, EL2, and possibly EL3, might also be in place within MC2R. However, in this case, it can undergo conformational changes, during which the -H-F-R-W- binding pocket is opened. Our results also indicate that this structure is responsible for the formation of an arrest signal. It is possible that this structure is also a part of the -K-K-R-R- binding pocket, which is only formed when MC2R is coexpressed with MRAPβ. Alternatively, the -K-K-R-R- binding pocket might be formed by the amino acid residues located within MRAPβ, and the region that we have identified is responsible for the interaction between MRAPβ and MC2R.

To test this proposition in detail, we created 13 receptors that contained small-scale replacements within the TM3–TM5 region. Most of these replacements increased the membrane export efficiency (except for Ch24, Ch27, and Ch28), and for seven of them, this efficiency was similar to that of MC4R (Table 2, Figs 2 and 5), indicating that even slight changes within the native structural domains can disrupt the overall structure responsible for the formation of the arrest signal. This is contrary to the results obtained with other replacements, where the structures of separate domains (i.e. TM helices or loops) remained intact. MC2R is the smallest GPCR identified; therefore, the structure of this receptor must be very compact, and it is clear that if the previously described compact lid-like structure responsible for the arrest of the receptor in the ER has to remain stable, the integrity of these individual structural elements is of major importance.

To understand as to how the replacements within the TM regions or regions proximal to them alter the α-helix and consequently its adjacent segments, we performed a TM region prediction analysis using the WEB-based TMPRED tool (http://www.ch.embnet.org/software/TMPRED_form.html; Hofmann & Stoffel 1993). This analysis indicated that, in the majority of receptors with replacements located within TM5 and its proximal regions, TM5 boundaries were altered (Fig. 7). These changes can thus explain the consequent distortion of the arrest signal, resulting in MRAPβ-independent membrane transportation. To assess the plausibility of this speculation, we performed an additional TM region prediction analysis using three other WEB-based tools HMMTOP (http://www.sacs.ucsf.edu/cgi-bin/hmmtop.py; Tusnady & Simon 2001), TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/; Krogh et al. 2001), and SOSUI (http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html; Hirokawa et al. 1998). Predictions acquired from two of these tools HMMTOP and TMHMM reported TM5 length and location alterations; HMMTOP predicted these changes within one receptor while TMHMM predicted them within three receptors. The exact TM domain boundaries that were predicted by all four tools did not match; however, they supported the speculation.

Figure 7
Figure 7

Amino acid sequence alignment of the TM4–TM5 regions of Ch2, MC4R, and Ch23–Ch35. ‘+ − − +’ and vertical dashes mark transmembrane domain boundaries, predicted using the TMPRED tool (Hofmann & Stoffel 1993).

Citation: Journal of Molecular Endocrinology 53, 2; 10.1530/JME-14-0169

To analyze the potential effects of these substitutions on other receptor constructs, we compared the properties of the original and substituted amino acid residues (Supplementary Table 2). This analysis revealed that each of these receptors carried at least one substitution that resulted in an alteration of hydrophobicity, polarity, charge, or aromaticity, thus suggesting that the properties of these amino acids substituted within are of most importance for the maintenance of MC2R conformation in the case of Ch25, Ch26, Ch29, and Ch33.

One of the main tests of the structural and functional integrity of all the receptors was their coexpression with MRAPβ, which allowed us to verify that the observed cellular arrest was not attributable to receptor misfolding. It must be emphasized that none of the receptors were inactive in all of the tests performed, including their membrane expression or cAMP activation in either the presence or absence of MRAPβ, indicating that the effects observed in this study did not result from the overall misfolding of any construct.

We observed that receptors with efficient membrane transportation during their stand-alone expression were significantly inhibited by their coexpression with MRAPβ. However, there were some exceptions: Ch26, Ch32, and Ch35 retained high membrane concentrations, whereas Ch28 remained at low levels within the membrane even in the presence of MRAP1β (Figs 2 and 5). Taken together, these results indicate again that even the slightest change in the MC2R structure can lead to various functional changes. It is possible that the substituted segments in the receptors, at least in the cases of Ch26, Ch32, Ch35, and Ch28, might be responsible for their interactions with the accessory protein.

Although this study mainly focused on identifying the mechanisms responsible for the localization specificity of MC2R, we also acquired information regarding the structures involved in determining the specificity of receptor–ligand recognition. The most interesting were the functionally inactive mutant constructs. The loss of function of Ch24 can be explained by the fact that the substitutions made were structurally located under the counterpart of MC4R D126 residue, which is one of the residues involved in the formation of the -H-F-R-W- binding pocket (Yang et al. 2000), and any changes within the immediate vicinity of this residue can result in impaired binding. However, none of the residues in Ch31 have been reported to be involved in the formation of the -H-F-R-W- binding pocket. Therefore, this segment must either be involved in the formation of the specific -K-K-R-R- binding pocket or participate in the functional interaction between MC2R and MRAPβ, which mediates the conformational changes that unlock the -H-F-R-W- binding pocket. Comparison of the properties of the original and substituted amino acid residues in Ch31 revealed that H170→D and H171→S (Supplementary Table 2) are substitutions resulting in alterations of most amino acid properties. Owing to its positive charge and aromatic nature, histidine is commonly involved in the formation of functionally active structures. Therefore, it is plausible that these two residues are actually the main players in the functional interaction between MC2R and MRAPβ.

In summary, the results of this study indicate the existence of an elaborate extracellular structure that acts as both an arrest signal and a region that determines the ligand-recognition specificity of MC2R. It is also clear that the ‘fine-tuned’ overall structure of MC2R is critically important, as has also been suggested by Hinkle et al. (2011). Based on the results from this study, we also propose that the aromatic-residue-rich segment of EL2 within MC2R either forms part of the -K-K-R-R- binding pocket or interacts directly with MRAPβ during ligand recognition, thus ensuring that the -H-F-R-W- binding pocket is unlocked.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/JME-14-0169.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was supported by the Latvian Council of Science grant 364/2012, the Latvian State Research Program 4VPP-2010-2/2.1, and the European Regional Development Fund project no. 2010/0311/2DP/2.1.1.1.0/10/APIA/VIAA/069.

Author contribution statement

D F contributed to experimental planning, generation of recombinant constructs, data analysis, interpretation of acquired results, and generation of visual materials. R P performed cultivation of cell cultures, receptor expression, cAMP assays, and interpretation of acquired results. D P performed confocal microscopy and interpretation of acquired results. H B S contributed to experimental planning and interpretation of acquired results. J K contributed to experimental planning, interpretation of acquired results, generation of visual materials, and editing of the manuscript.

Acknowledgements

The authors thank Aija Ozola for assistance with confocal microscopy during the times of urgent necessity.

References

  • Beuschlein F, Fassnacht M, Klink A, Allolio B & Reincke M 2001 ACTH-receptor expression, regulation and role in adrenocortial tumor formation. European Journal of Endocrinology 144 199206. (doi:10.1530/eje.0.1440199).

    • Search Google Scholar
    • Export Citation
  • Boston BA 1999 The role of melanocortins in adipocyte function. Annals of the New York Academy of Sciences 885 7584. (doi:10.1111/j.1749-6632.1999.tb08666.x).

    • Search Google Scholar
    • Export Citation
  • Boston BA & Cone RD 1996 Characterization of melanocortin receptor subtype expression in murine adipose tissues and in the 3T3-L1 cell line. Endocrinology 137 20432050. (doi:10.1210/endo.137.5.8612546).

    • Search Google Scholar
    • Export Citation
  • Chan LF, Webb TR, Chung TT, Meimaridou E, Cooray SN, Guasti L, Chapple JP, Egertová M, Elphick MR & Cheetham ME 2009 MRAP and MRAP2 are bidirectional regulators of the melanocortin receptor family. PNAS 106 61466151. (doi:10.1073/pnas.0809918106).

    • Search Google Scholar
    • Export Citation
  • Chhajlani V & Wikberg JE 1992 Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA. FEBS Letters 309 417420. (doi:10.1016/0014-5793(92)80820-7).

    • Search Google Scholar
    • Export Citation
  • Clark AJ & Cammas FM 1996 The ACTH receptor. Baillière's Clinical Endocrinology and Metabolism 10 2947. (doi:10.1016/S0950-351X(96)80282-5).

  • Cooray SN, Almiro Do Vale I, Leung KY, Webb TR, Chapple JP, Egertova M, Cheetham ME, Elphick MR & Clark AJ 2008 The melanocortin 2 receptor accessory protein exists as a homodimer and is essential for the function of the melanocortin 2 receptor in the mouse y1 cell line. Endocrinology 149 19351941. (doi:10.1210/en.2007-1463).

    • Search Google Scholar
    • Export Citation
  • Dunn OJ 1964 Multiple comparisons using rank sums. Technometrics 6 241252. (doi:10.1080/00401706.1964.10490181).

  • Eberle AN 1988 The Melanotropins. Chemistry, Physiology and Mechanisms of Action. Basel: S. Karger Publishers

  • Fleck BA, Chen C, Yang W, Huntley R, Markison S, Nickolls SA, Foster AC & Hoare SR 2005 Molecular interactions of nonpeptide agonists and antagonists with the melanocortin-4 receptor. Biochemistry 44 1449414508. (doi:10.1021/bi051316s).

    • Search Google Scholar
    • Export Citation
  • Fridmanis D, Petrovska R, Kalnina I, Slaidina M, Peculis R, Schioth HB & Klovins J 2010 Identification of domains responsible for specific membrane transport and ligand specificity of the ACTH receptor (MC2R). Molecular and Cellular Endocrinology 321 175183. (doi:10.1016/j.mce.2010.02.032).

    • Search Google Scholar
    • Export Citation
  • Gantz I & Fong TM 2003 The melanocortin system. American Journal of Physiology. Endocrinology and Metabolism 284 E468E474. (doi:10.1152/ajpendo.00434.2002).

    • Search Google Scholar
    • Export Citation
  • Gantz I, Konda Y, Tashiro T, Shimoto Y, Miwa H, Munzert G, Watson SJ, DelValle J & Yamada T 1993a Molecular cloning of a novel melanocortin receptor. Journal of Biological Chemistry 268 82468250.

    • Search Google Scholar
    • Export Citation
  • Gantz I, Miwa H, Konda Y, Shimoto Y, Tashiro T, Watson SJ, DelValle J & Yamada T 1993b Molecular cloning, expression, and gene localization of a fourth melanocortin receptor. Journal of Biological Chemistry 268 1517415179.

    • Search Google Scholar
    • Export Citation
  • Gantz I, Shimoto Y, Konda Y, Miwa H, Dickinson CJ & Yamada T 1994 Molecular cloning, expression, and characterization of a fifth melanocortin receptor. Biochemical and Biophysical Research Communications 200 12141220. (doi:10.1006/bbrc.1994.1580).

    • Search Google Scholar
    • Export Citation
  • Haskell-Luevano C, Cone RD, Monck EK & Wan YP 2001 Structure activity studies of the melanocortin-4 receptor by in vitro mutagenesis: identification of agouti-related protein (AGRP), melanocortin agonist and synthetic peptide antagonist interaction determinants. Biochemistry 40 61646179. (doi:10.1021/bi010025q).

    • Search Google Scholar
    • Export Citation
  • Hinkle PM, Serasinghe MN, Jakabowski A, Sebag JA, Wilson KR & Haskell-Luevano C 2011 Use of chimeric melanocortin-2 and -4 receptors to identify regions responsible for ligand specificity and dependence on melanocortin 2 receptor accessory protein. European Journal of Pharmacology 660 94102. (doi:10.1016/j.ejphar.2010.10.113).

    • Search Google Scholar
    • Export Citation
  • Hirokawa T, Boon-Chieng S & Mitaku S 1998 SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14 378379. (doi:10.1093/bioinformatics/14.4.378).

    • Search Google Scholar
    • Export Citation
  • Hirsch A, Meimaridou E, Fernandez-Cancio M, Pandey AV, Clemente M, Audi L, Clark AJ & Fluck CE 2011 Loss of the C terminus of melanocortin receptor 2 (MC2R) results in impaired cell surface expression and ACTH insensitivity. Journal of Clinical Endocrinology and Metabolism 96 E65E72. (doi:10.1210/jc.2010-1056).

    • Search Google Scholar
    • Export Citation
  • Ho SN, Hunt HD, Horton RM, Pullen JK & Pease LR 1989 Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77 5159. (doi:10.1016/0378-1119(89)90358-2).

    • Search Google Scholar
    • Export Citation
  • Hofmann K & Stoffel W 1993 TMbase – a database of membrane spanning proteins segments. Biological Chemistry Hoppe-Seyler 374 166.

  • Ignatovica V, Petrovska R, Fridmanis D & Klovins J 2011 Expression of human melanocortin 4 receptor in Saccharomyces cerevisiae. Central European Journal of Biology 6 167175. (doi:10.2478/s11535-011-0002-3).

    • Search Google Scholar
    • Export Citation
  • Josep Agulleiro M, Cortes R, Fernandez-Duran B, Navarro S, Guillot R, Meimaridou E, Clark AJ & Cerda-Reverter JM 2013 Melanocortin 4 receptor becomes an ACTH receptor by coexpression of melanocortin receptor accessory protein 2. Molecular Endocrinology 27 19341945. (doi:10.1210/me.2013-1099).

    • Search Google Scholar
    • Export Citation
  • Jun DJ, Na KY, Kim W, Kwak D, Kwon EJ, Yoon JH, Yea K, Lee H, Kim J & Suh PG 2010 Melanocortins induce interleukin 6 gene expression and secretion through melanocortin receptors 2 and 5 in 3T3-L1 adipocytes. Journal of Molecular Endocrinology 44 225236. (doi:10.1677/JME-09-0161).

    • Search Google Scholar
    • Export Citation
  • Kay EI, Botha R, Montgomery JM & Mountjoy KG 2013 hMRAPa increases αMSH-induced hMC1R and hMC3R functional coupling and hMC4R constitutive activity. Journal of Molecular Endocrinology 50 203215. (doi:10.1530/JME-12-0221).

    • Search Google Scholar
    • Export Citation
  • Kovalitskaia YA, Kolobov AA, Kampe-Nemm EA, Iurovskii VV, Sadovnikov VB, Lipkin VM & Navolotskaia EV 2008 Synthetic peptide KKRR corresponding to the human ACTH fragment 15–18 is an antagonist of the ACTH receptor. Bioorganicheskaia Khimiia 34 2935. (doi:10.1134/S1068162008010020).

    • Search Google Scholar
    • Export Citation
  • Krogh A, Larsson B, von Heijne G & Sonnhammer EL 2001 Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of Molecular Biology 305 567580. (doi:10.1006/jmbi.2000.4315).

    • Search Google Scholar
    • Export Citation
  • Metherell LA, Chapple JP, Cooray S, David A, Becker C, Ruschendorf F, Naville D, Begeot M, Khoo B & Nurnberg P 2005 Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familial glucocorticoid deficiency type 2. Nature Genetics 37 166170. (doi:10.1038/ng1501).

    • Search Google Scholar
    • Export Citation
  • Mountjoy KG, Robbins LS, Mortrud MT & Cone RD 1992 The cloning of a family of genes that encode the melanocortin receptors. Science 257 12481251. (doi:10.1126/science.1325670).

    • Search Google Scholar
    • Export Citation
  • Mountjoy KG, Mortrud MT, Low MJ, Simerly RB & Cone RD 1994 Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Molecular Endocrinology 8 12981308. (doi:10.1210/mend.8.10.7854347).

    • Search Google Scholar
    • Export Citation
  • Naville D, Penhoat A, Durand P & Begeot M 1999 Three steroidogenic factor-1 binding elements are required for constitutive and cAMP-regulated expression of the human adrenocorticotropin receptor gene. Biochemical and Biophysical Research Communications 255 2833. (doi:10.1006/bbrc.1998.9891).

    • Search Google Scholar
    • Export Citation
  • Norman D, Isidori AM, Frajese V, Caprio M, Chew SL, Grossman AB, Clark AJ, Michael Besser G & Fabbri A 2003 ACTH and α-MSH inhibit leptin expression and secretion in 3T3-L1 adipocytes: model for a central–peripheral melanocortin–leptin pathway. Molecular and Cellular Endocrinology 200 99109. (doi:10.1016/S0303-7207(02)00410-0).

    • Search Google Scholar
    • Export Citation
  • Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T & Stenkamp RE 2000 Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289 739745. (doi:10.1126/science.289.5480.739).

    • Search Google Scholar
    • Export Citation
  • Penhoat A, Naville D, El Mourabit H, Buronfosse A, Berberoglu M, Ocal G, Tsigos C, Durand P & Begeot M 2002 Functional relationships between three novel homozygous mutations in the ACTH receptor gene and familial glucocorticoid deficiency. Journal of Molecular Medicine 80 406411. (doi:10.1007/s00109-002-0333-7).

    • Search Google Scholar
    • Export Citation
  • Roselli-Rehfuss L, Mountjoy KG, Robbins LS, Mortrud MT, Low MJ, Tatro JB, Entwistle ML, Simerly RB & Cone RD 1993 Identification of a receptor for γ melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system. PNAS 90 88568860. (doi:10.1073/pnas.90.19.8856).

    • Search Google Scholar
    • Export Citation
  • Roy S, Roy SJ, Pinard S, Taillefer LD, Rached M, Parent JL & Gallo-Payet N 2011 Mechanisms of melanocortin-2 receptor (MC2R) internalization and recycling in human embryonic kidney (hek) cells: identification of key Ser/Thr (S/T) amino acids. Molecular Endocrinology 25 19611977. (doi:10.1210/me.2011-0018).

    • Search Google Scholar
    • Export Citation
  • Schwyzer R 1977 ACTH: a short introductory review. Annals of the New York Academy of Sciences 297 326. (doi:10.1111/j.1749-6632.1977.tb41843.x).

    • Search Google Scholar
    • Export Citation
  • Sebag JA & Hinkle PM 2007 Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers. PNAS 104 2024420249. (doi:10.1073/pnas.0708916105).

    • Search Google Scholar
    • Export Citation
  • Sebag JA & Hinkle PM 2009 Regions of melanocortin 2 (MC2) receptor accessory protein necessary for dual topology and MC2 receptor trafficking and signaling. Journal of Biological Chemistry 284 610618. (doi:10.1074/jbc.M804413200).

    • Search Google Scholar
    • Export Citation
  • Tusnady GE & Simon I 2001 The HMMTOP transmembrane topology prediction server. Bioinformatics 17 849850. (doi:10.1093/bioinformatics/17.9.849).

    • Search Google Scholar
    • Export Citation
  • Webb TR, Chan L, Cooray SN, Cheetham ME, Chapple JP & Clark AJ 2009 Distinct melanocortin 2 receptor accessory protein domains are required for melanocortin 2 receptor interaction and promotion of receptor trafficking. Endocrinology 150 720726. (doi:10.1210/en.2008-0941).

    • Search Google Scholar
    • Export Citation
  • Yang YK, Fong TM, Dickinson CJ, Mao C, Li JY, Tota MR, Mosley R, Van Der Ploeg LH & Gantz I 2000 Molecular determinants of ligand binding to the human melanocortin-4 receptor. Biochemistry 39 1490014911. (doi:10.1021/bi001684q).

    • Search Google Scholar
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

Supplementary Materials

 

      Society for Endocrinology

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 443 138 20
PDF Downloads 154 45 4
  • View in gallery

    Schematic of the recombinant receptors Ch16–Ch22. Regions from MC4R are shaded gray and regions from MC2R are shaded black.

  • View in gallery

    An example set of the confocal fluorescence microscopy images that were acquired during quantification of receptor transportation to the cell membrane. Membranes of receptor–EGFP (green) expressing BHK cells were stained with AF-WGA (red). A full colour version of this figure is available at http://dx.doi.org/10.1530/JME-14-0169.

  • View in gallery

    Graph representing the medians, interquartile, and min/max ranges of the EGFP/AF-WGA fluorescence intensity ratios at the plasma membrane for MC4R, MC2R, Ch2, and Ch16–Ch22. Bars that are labeled with ↑ or ↑↑ represent receptors with low or high levels of membrane export respectively. Bars that are labeled with ⊥ represent receptors that are retained. Symbols * and ° represent the significance of difference in rank sum between specific receptor and MC2R and MC4R respectively. Three symbols represent P<0.001 and two symbols represent P<0.01.

  • View in gallery

    Schematic of recombinant receptors Ch23–Ch35. (Upper panel) Snake-like plot. Regions from MC4R are shaded gray, regions from MC2R are shaded black, and substituted amino acid residues are shaded according to their membrane transport efficiency. Circles shaded in green represent substitutions that facilitated high levels of membrane export, those in yellow represent low levels of membrane export, and those in red represent intracellular retention. The left side of the circle represents the membrane export level during stand-alone expression, and the right side represents the membrane export level during coexpression with MRAPβ. The symbols on the left side of receptor name indicate the effect of MRAPβ coexpression on the membrane export efficiency of the corresponding receptor. ‘↑’ Represents increased export efficiency; ‘↓’ represents reduced export efficiency; ‘_’ unaltered high export efficiency; and ‘_’ unaltered retention. (Lower panel) Alignment of Ch2 and MC4R amino acid sequences. Shaded rectangular fields indicate transmembrane domain boundaries predicted using the TMPRED tool (Hofmann & Stoffel 1993). The positions of the substituted amino acids within recombinant receptors Ch23–Ch35 are marked with ‘+ − − +’ and the corresponding receptor number above. A full colour version of this figure is available at http://dx.doi.org/10.1530/JME-14-0169.

  • View in gallery

    Graph representing the medians and interquartile ranges of the EGFP/AF-WGA fluorescence intensity ratios at the plasma membrane for Ch23–Ch35. Bars that are labeled with ↑ or ↑↑ represent receptors with low or high levels of membrane export respectively. Bars that are labeled with ⊥ represent receptors that are retained. Symbols * and ° represent the significance of difference in rank sum between specific receptor and MC2R and MC4R respectively. Three symbols represent P<0.001 and two symbols represent P<0.01.

  • View in gallery

    Snake-like plot of the TM3–TM5 region of MC2R showing the effects of ACTH(1–24) on cAMP production by cells cotransfected with recombinant receptors Ch23–Ch35 and MRAPβ. Substituted amino acid residues of receptors whose EC50 values were within the range of 23.0–71.7 nM are shaded in green, within 209.3–324.0 nM are shaded in yellow, and within 1073.0–1237.0 nM are shaded in orange. Substitutions that produce inactive recombinant receptors are shaded in red. A full colour version of this figure is available at http://dx.doi.org/10.1530/JME-14-0169.

  • View in gallery

    Amino acid sequence alignment of the TM4–TM5 regions of Ch2, MC4R, and Ch23–Ch35. ‘+ − − +’ and vertical dashes mark transmembrane domain boundaries, predicted using the TMPRED tool (Hofmann & Stoffel 1993).