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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-0048
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Right arrow Adrenal and Hypertension
The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 8 3097-3105
Copyright © 2008 by The Endocrine Society

A Novel Adrenocorticotropin Receptor Mutation Alters Its Structure and Function, Causing Familial Glucocorticoid Deficiency

Rocío A. Artigas1, Angel Gonzalez1, Erick Riquelme, Cristian A. Carvajal, Andreína Cattani, Alejandro Martínez-Aguayo, Alexis M. Kalergis, Tomas Pérez-Acle and Carlos E. Fardella

Department of Endocrinology (R.A.A., C.A.C., C.E.F.), Pediatrics (A.C., A.M.-A.), and Rheumatology (A.M.K.), Facultad de Medicina, Millennium Nucleus on Immunology and Immunotherapy (E.R., C.A.C., A.M.K., C.E.F.), Departamento de Genética Molecular y Microbiología (A.M.K., C.E.F.), Facultad de Ciencias Biológicas, Centre for Bioinformatics (A.G., T.P.-A.), Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, and Fundación Ciencia para la Vida (A.G., T.P.-A.), 8330074 Santiago, Chile

Address all correspondence and requests for reprints to: Dr. Carlos E. Fardella, M.D., Departamento de Endocrinología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Lira 85, 5° piso, 8330074 Santiago, Chile. E-mail: cfardella{at}med.puc.cl.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Familial glucocorticoid deficiency (FGD) is an autosomal recessive disorder characterized by unresponsiveness to ACTH. In this study, two mutations of the ACTH receptor (MC2R) gene are reported in this FGD clinical case.

Objective: The objective of the study was to characterize a novel MC2R gene mutation in a compound heterozygous patient with FGD phenotype.

Design: This was a clinical case description, biochemical, molecular, and bioinformatics analysis to describe a novel MC2R gene mutation.

Patients: The subject of the study was a male diagnosed with primary adrenal insufficiency. The family history showed nonconsanguineous healthy parents, three healthy siblings, and one brother affected with FGD.

Main Outcome Measures: The mutant MC2R-Ala126Ser showed significantly lower activity when it was stimulated with ACTH-(1–24) than did cells transfected with wild-type MC2R.

Results: The molecular studies demonstrated the presence of an adenine heterozygous insertion (InsA1347) in the MC2R gene (G217fs) in the patient. This insertion was due to a frame shift mutation in one allele and a premature stop codon codifying an aberrant receptor of 247 residues (27.2 kDa). We also found a novel heterozygous mutation alanine 126 by serine. Molecular dynamic simulations showed that serine 126 side chain fluctuates forming a noncanonical intrahelical hydrogen bond in the transmembrane helix 3 of the mutated receptor. This produces a structural rearrangement of the MC2R internal cavities that may affect the ligand recognition and signal transduction throughout the G protein.

Conclusions: We propose a molecular explanation for the reduced activity exhibited by the MC2R alanine 126 by serine mutant.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder in which secretion of cortisol and androgen is deficient and unresponsive to ACTH stimulation (1). Although symptoms of the disease are usually observed during the first year of life, they can also appear during infancy or later childhood with hyperpigmentation, muscle weakness, hypoglycemia, and seizures caused by low cortisol and elevated ACTH levels (2). It has been shown that inactivating mutations in the adrenal receptor for ACTH [melanocortin 2 receptor (MC2R)] are found in about 25% of FGD kindreds (FGD type 1, OMIM 202200) (3). Most mutations are missense and there is no specific hot spot for mutations. The small numbers of nonsense mutations, stop codons, and frameshift mutations occur as compound heterozygotes with a missense mutation in all but a few cases (4). In the remaining cases, no mutations are identifiable in the MC2R gene (FGD type 2). Recent studies (5) identified the gene encoding for a small single transmembrane domain protein known as melanocortin 2 receptor accessory protein (OMIM *609196) as another underlying FGD type 2. This protein functions as an accessory protein for MC2R, contributing in part to promote its expression at the cell’s surface. Mutations in melanocortin 2 receptor accessory protein account for a further 15–20% of all FGD cases, implying that at least half of all FGD cases result from other genes yet to be identified (3, 4, 5).

The MC2R is the smaller member of the class A rhodopsin-like G protein-coupled receptors (GPCRs) family within which it joined the melanocortin 1, 3, 4, and 5 to form the melanocortin receptor (MCR) subgroup (6). In contrast to the other four MCRs, MC2R is activated only by ACTH, whereas the other MCRs can be activated by both ACTH and MSH (7). It is known that the binding of ACTH to MC2R activates the heterotrimeric G protein complex that induces adenylate cyclase to form cAMP, which stimulates steroidogenesis acutely through the action of the steroidogenic acute regulatory protein and chronically through transcriptionally induced accumulation of mRNAs for steroidogenic enzymes (8).

The human MC2R gene is in chromosome 18 and consists of two exons spanning about 1.1 kb. The only coding region is within the second exon, which gives rise to a protein with 297 amino acids (4). The predicted protein for the MC2R had a molecular mass in its unmodified form of 33 kDa. Available evidence supports the notion that all GPCRs share a common fold composed of seven transmembrane helixes (TMHs) of about 25–35 residues long (TMHs 1–7) that span the cellular membrane connected by three extracellular (1, 2, 3) and three cytoplasmatic loops (1, 2, 3) (9). The N-terminal region, which varies in length and function, is located on the extracellular side of the membrane, whereas the C-terminal region is on the intracellular side (10). Despite the overall low sequence identity exhibited by multiple alignments of the class A GPCRs sequences, the existence of highly conserved residues in their seven transmembrane-spanning domains has allowed the development of comparative models based on the crystallographic x-ray data of the bovine rhodopsin as a structural template (11). This approach has been broadly used to gain insights on the biophysical properties of a variety of class A family members and provide support to site-directed mutagenesis experiments. It is also the main source for structural hypotheses tending to characterize the transition between the ensemble of active and the inactive states of these receptors (12, 13). The recent release of two crystallographic structures of the human β2-adrenergic receptor (14, 15), at different resolutions, increases the number of templates available for GPCR comparative modeling. As expected, these new structures have a similar arrangement of transmembrane helices compared with the homologous structure of rhodopsin.

Here we report a novel heterozygous mutation in the MC2R gene that shows reduced activity in comparison with wild type and seems to be responsible of the FGD phenotype. Molecular dynamic (MD) simulations performed in explicit membrane conditions, using comparative models of the wild-type MC2R and the alanine 126 by serine (Ala126Ser) mutant, permitted us to compose a structural hypothesis that could be used to explain this reduced activity.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Clinical case and biochemical profile

We present a male, who was born from nonconsanguineous Chilean parents after an uneventful delivery. He was a term newborn, with a birth weight of 3220 g. During his first month of life, he had generalized hyperpigmentation. His genitals showed normal development for his age, without pubarche. No sign of achalasia or alacrimia were noted. At the age of 2 yr, he had hypoglycemia and pleuropneumoniae associated with shock. During this stress, the endocrinological analysis revealed extremely low basal cortisol less than 1.1 µg/dl (<27.59 nmol/liter) and after 250 µg iv ACTH 1.1 µg/dl (30.35 nmol/liter); with plasma renin activity 2.8 ng/ml·h (normal value 1–2.5 ng/ml·h), ACTH 1047 pg/ml (236.28 pmol/liter), 17-hydroxyprogesterone less than 0.1 ng/dl and testosterone less than 10 ng/dl. Other causes of primary adrenal insufficiency were studied (16, 17): the adrenal antibody, immunofunctional assay less than 1:4 (not detectable), normal concentrations of very long-chain fatty acids, and normal brain magnetic resonance imaging. With the diagnosis of primary adrenal insufficiency, hydrocortisone replacement therapy was introduced (100 mg/m2 during stress and then 10–12 mg/m2·d). At the age of 2 yr, his height was 99 cm (99.9th percentile). The father’s height was 190 cm (96.8th percentile), and the mother’s height was 172 cm (91.0th percentile), and he had an accelerated bone age with a bone age to chronological age ratio of 2. Moreover, his height velocity was 15 cm/yr during the year before the diagnosis. The TSH was 5.57 mIU/ml [normal range (NR) 0.49–6.1 mIU/ml] and normal free T4 1.43 ng/dl (NR 0.8–2.1 ng/dl). The level of IGF-I was 158 ng/ml (NR 17–248), and IGF binding protien-3 was 3.6 µg/ml (NR 0.9–4.1). The parents had ACTH, basal and post-ACTH cortisol, and plasma renin activity (PRA) within the normal ranges and were not affected with FGD. The youngest brother (6 months old) was studied because of a generalized hyperpigmentation. He presented a low basal cortisol at 0.4 µg/dl (<27.59 nmol/liter) with elevated ACTH plasma levels (1250 pg/ml) associated with normal PRA (11.3 ng/ml·h).

Analysis of genomic DNA

Genomic DNA was isolated from peripheral blood mononuclear cells obtained from the index case, his parents, and siblings. DNA purification protocol was carried out using DNAzol reagent (Invitrogen Corp., San Diego, CA). The entire coding region of MC2R gene was amplified by conventional PCR using previously reported primers, MC2R-S and MC2R-AS primers (18), and procedures (19) (see supplemental Materials and Methods, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org). PCR fragments were purified by a Qiaquick gel extraction purification kit (QIAGEN, Valencia, CA) and directly sequenced in an ABI Prism 377 DNA genetic analyzer (Applied Biosystem, Foster City, CA). The MC2R gene sequence was compared using BLASTn (20) with the nucleotide database available in the GenBank (21). As a result, the complementary region that span from positions 13.894.624 to 13.875.517 nt of the chromosome locus 18p11.2 (GenBank accession no. NM_000529) was obtained. This sequence was globally aligned to our MC2R gene sequence using ClustalW program (22) while looking for genetic alterations.

Site-directed mutagenesis and transient transfection

pcDNA3.1-MC2R plasmid (generously donated by Professor Walter L. Miller, University of California, San Francisco, San Francisco, CA) was used to perform the expression studies and generate the mutant MC2R cDNA (Ala126Ser) vector (8) by means of a site-directed mutagenesis system (Stratagene, La Jolla, CA) and oligonucleotides designed following the manufacturer’s instructions. The parental methylated wild-type cDNA was digested with 10U DpnI at 37 C for 90 min, and the remaining unmethylated mutagenized cDNA plasmid was used to transform Escherichia coli DH5 cells. The resulting mutagenized plasmids were sequenced in an automatic analyzer to confirm the presence of the required mutation. CHO cells, which do not express MC2R gene by themselves, were cultured in 24-well plates 48 h before transfection at approximately 80% confluence. Before transfection, the cells were maintained in a medium, supplemented without antibiotic. Cells were incubated overnight with DNA-Lipofectamine 2000 complexes containing vectors expressing wild-type or mutant MC2R cDNA or an equal mass of empty pcDNA3 plus a cAMP-responsive luciferase (Luc) reporter plasmid (pCREluc) that contained 16 cAMP response element units. At the end of the incubation, the DNA-Lipofectamine 2000 complexes were removed, and the cells were incubated in a fresh medium for 36 h to allow gene expression before stimulation with synthetic ACTH-(1–24) (Sigma, St. Louis, MO). Forty-eight hours after the transfection and before the incubation with ACTH, some wells were removed to evaluate receptor expression wild-type or mutant MC2R on the cells transfected surface. This was assessed through flow cytometry by staining it with rabbit antimouse MC2R polyclonal antibody, unconjugated (H-70; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by goat antirabbit IgG-PE (Pierce, Rockford, IL). The remaining wells were then incubated with 10–7 M ACTH-(1–24) for 18 h at 37 C in 5% CO2. As positive control, we used 1 mM 8-bromoadenosine-cAMP because pCREluc is activated by cAMP. Cells were lysed and assayed for Luc activity using the luciferase assay kit (Stratagene). A cotransfection of β-galactosidase reporter vector was used as the transfection efficiency control and the results were expressed as activity Luc/activity β-gal.

Real-time PCR (qPCR) of MC2R

Total RNA was extracted with TRIZOL LS reagent (Invitrogen). RNA (2 µg) was reversely transcribed using the StrataScipt RT (Stratagene) and random primers. For qPCR, 2 µl of total cDNA were amplified with QuantiSyg (Quantimix Easy SYG kit, BioTools; B&M Labs, S.A., Madrid, Spain) and gene-specific primers (Q-MC2R-S and Q-MC2R-AS; see supplemental Materials and Methods). Reaction conditions were 3 min at 95 C followed by 35 cycles of 15 sec at 95 and 30 sec at 60 in a Rotor-Gene 6000 (Corbett, Sydney, Australia). qPCR data were obtained during the extension phase, and threshold cycle values were obtained at the log phase of each gene amplification. PCR product quantification was performed by the relative quantification method (23) and standardized against human glyceraldehyde-3-phosphate dehydrogenase or 18S RNA. Efficiency for each primer pair was assessed using serial dilutions from a reverse transcription product.

Sequence alignment and MC2R comparative modeling

To produce a MC2R molecular model, the highest-resolution 2.2 Å crystal structure of bovine rhodopsin (PDBid: 1U19) was used as a template for comparative modeling (24). MC2R and rhodopsin sequences were aligned using the available information of highly conserved residues shared within the GPCRs (10) (see supplemental Fig. I, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org). This alignment was used as an input to MODELLER version 8 (25) to develop a total of 100 comparative models that were ranked with the highest level of quality (26, 27). The best evaluated structure was selected for further refinement. An Ala126Ser mutant model was produced using the BuildMutant module available within MODELLER version 8, being subsequently energy optimized using the loop optimization procedure available in this program.

Molecular dynamic simulations in explicit membrane environment

The MC2R wild-type and Ala126Ser mutant models (see supplemental Fig. II) were independently embedded in a preequilibrated lipid bilayer consisting of 288 molecules of 1,2-dipalmitoylphosphatidylcholine (DPPC) (28). Insertion of the {alpha}-helical bundle into the lipid core was adjusted to obtain both the TMH4 and the cytosolic half of TMH6 perpendicular to the membrane plane (29, 30). Protein-overlapping lipids were subsequently removed. Final systems dimension resulted in a periodic box of 98 x 98 x 112 (Å) with a total of 98.418 atoms (see Fig. 1Go). Simulations were carried out using the NAMD version 2.6 MD package (31) using the TIP3 water model and the CHARMM22 all-hydrogen parameter file for proteins (32) and the CHARMM27 all-hydrogen force field for lipid parameter (33). Long-range electrostatic interactions were calculated using the particle mesh Ewald method to get a uniform charge density average area during simulation (34). The Lennard-Jones and short-range neighbor list for Coulombic interactions were set at 12 Å. Simulations were performed under the isothermal-isobaric ensemble at 1 Atm using a Langevin piston. To compensate the net charge of the protein-membrane systems, Na+ and Cl– ions were added to reach an ionic concentration of 0.1 mol/liter. The temperature was controlled by a 310K temperature bath using a Langevin damping algorithm. Energy minimization, to reduce close contacts, was achieved through the steepest-descent algorithm. The energy minimized, systems were then preequilibrated (0.3 nsec), being subsequently subjected to a 5 ns MD simulation at 310K with a 1fsec time step. Trajectory frames were saved each 10 psec.


Figure 1
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  		FIG. 1. DPPC-MC2R molecular system [98 x 98 x 112] (Å) with a total of 98.418 atoms. The thickness of the water layer was 30 Å on both sides of the lipid bilayer. Lateral (A) and extracellular (B) views of the lipid receptor system after 5 ns MD simulation. Wild-type MC2R appears in golden ribbons and lipids in gray licorice. The blue surface points represent water molecules in A but were excluded in B. Ions are displayed in red.

 
Statistical analysis

Statistical analyses were performed using a one-way ANOVA test, followed by a Bonferroni’s multiple comparison test. Values were considered statistically significant for P ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Clinical studies

The diagnosis of FGD was suspected on the basis of a low or undetectable plasma cortisol, a markedly elevated plasma ACTH, and normal electrolytes with a normal renin and aldosterone (35). The other laboratory studies supported the exclusion of adrenoleukodystrophy, congenital adrenal hyperplasia, and autoimmune Addison’s disease. The triple A syndrome was excluded by the absence of achalasia, alacrimia, and unexplained neurological defects (36). Because the index case is a boy presenting prepubertal genitalia, normal PRA, and tall stature, we focused our attention on the MC2R receptor gene.

Molecular studies

Sequencing analysis of the MC2R gene from affected child revealed two different heterozygous mutations in the coding sequence: a heterozygous insertion of an adenine at position 1347 InsA1347Het (G217fs) and an alanine-to-serine substitution at position 126. The insertion would produce a frame shift mutation in one allele, and a premature stop codon codifying an aberrant receptor of 247 amino acids (27.2 kDa), instead of the 297 amino acids normal receptor (32.7 kDa). This mutation was also present in the index case’s affected brother and also in their mother (Fig. 2AGo). The second mutation corresponds to a guanine to thymidine change. This is a novel mutation that results an amino acid change: Ala126Ser, Het(GCG/TCG). This substitution was also present in the affected brother and their father’s DNA (Fig. 2BGo). Either father or mother carries one of the heterozygous mutations, Ala126Ser and InsA1347Het (G217fs), respectively, without symptoms of glucocorticoid deficiency. We found that the index case in our study and his affected younger brother, who manifest the glucocorticoid deficiency, present both mutations together in their DNA.


Figure 2
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  		FIG. 2. A, Sequence analysis and identification of the MC2R insertion (InsA1347). DNA sequences of controls, heterozygous not affected (mother) and compound heterozygous affected patients (the index case and his youngest brother) are shown. B, Sequence analysis and identification of the MC2R mutation Ala126Ser. DNA sequences of controls, heterozygous not affected (father), and compound heterozygous affected patients (the index case and his youngest brother) are shown. F, Father; M, mother.

 
Expression studies

To determine the functional consequences of the Ala126Ser substitution, we constructed expression vectors to produce the normal and mutant MC2R proteins in suitable mammalian cells. These vectors were used to transfect CHO cells, which do not express MC2R. qPCR analysis indicated similar a expression of MC2R RNA from both constructs (data not shown). We then evaluated the surface expression of both wild-type and Ala126Ser MC2R through flow cytometry. These results show that in both conditions there is an equivalent amount of MC2R in the cell surface (Fig. 3AGo). To test the activity of the Ala126Ser mutation, CHO cells were transfected with MC2R wild-type, MC2R-Ala126Ser, and cotransfected with pCREluc and incubated with synthetic ACTH-(1–24). CHO cells were cotransfected with pCREluc and the vector for MC2R wild-type had a robust response to 10–7 M ACTH-(1–24), but cells cotransfected with pCREluc and mutated MC2R had a lower activity than the former when stimulated with ACTH-(1–24) (Fig. 3BGo). The empty pcDNA3.1 vector had no response. Furthermore, the activity of luciferase was measured in unstimulated cells or after stimulation with 1 mM 8-bromoadenosine-cAMP that served as the ACTH-independent positive control. We also performed a full dose-response curve and the differences observed previously were maintained throughout the different doses evaluated (supplemental Fig. III).


Figure 3
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  		FIG. 3. A, Analysis receptor expression. The surface expression of both wild-type (WT) and Ala126Ser MC2R was assessed through flow cytometry. B, Luciferase activity assays. CHO cells were transfected with the vector pcDNA3.1 expressing the wild-type MC2R cDNA and mutant MC2R(Ala126Ser) cDNA and cotransfected with the reporter plasmid pCREluc cDNA or with an equal mass of empty pcDNA3.1. Luciferase activity was measured in unstimulated cells or after stimulation with 1 mM 8-bromoadenosine-cAMP or 10–7 M ACTH-(1–24) overnight. Cells transfected with the empty pcDNA3.1 vector served as control. Values are expressed as the mean SEM of three independent transfection experiments, each performed in triplicate. Statistical differences were assessed through a one-way ANOVA followed by Bonferroni’s multiple comparison test. *, P ≤ 0,05; **, P ≤ 0,01; ***, P ≤ 0,001. n.s., Nonsignificant.

 
Molecular modeling and dynamics simulation

The initial global alignment of the MC2R sequence with the rhodopsin template was tailored using highly conserved residues present in all TMHs that are shared by the Class A GPCR family (see supplemental Fig. I). To accomplish this with experimentally obtained suggestions, an S-S disulfide bridge was included between Cys245 and C251 in the comparative modeling step (37). A mutant model was derived from the MC2R wild-type structure to find a putative structural-based explanation to the reduced activity shown by the Ala126Ser substitution (supplemental Fig. II). The mutated residue (Ser126) was located just above the highly conserved DRY motif in the TMH 3 (38) and placed in the i-2 position from the most conserved residue R3.50 [in accordance with Ballesteros notation (39)]. Due to the {alpha}-helix periodicity, its position resulted opposite of the internal receptor vestibule with its polar lateral chain oriented toward the membrane lipid environment, contrasting with the majority of GPCRs that exhibit hydrophobic residues in this region (10). To gain insights into the dynamic effects of this single substitution, both the MC2R wild-type and MC2R-Ala126Ser mutant models were independently embedded in a DPPC preequilibrated lipid bilayer and subjected to MD simulations. Figure 4AGo shows the C{alpha} root mean square deviation (RMSD) computed along the 5 ns trajectory for the two systems. As seen, the main structural variation along the MD simulations corresponds to loop regions, whereas the TMHs remain mostly stable under 3 Å of spatial fluctuation. This result supports the notion that the MC2R wild-type and mutant models resulted in stable structures, suitable to perform further biophysical analyses.


Figure 4
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  		FIG. 4. MD Simulations of MC2R models in explicit lipid environment. A, RMSD of the MC2R wild-type (WT) and Ala126Ser protein backbones during simulation. The RMSD values for TMHs and loops regions are displayed separately. B, Conformational preference calculated for the Ser126 {chi}1 torsion angle along the MD simulation. C, Distance between the Ser126 (O{gamma}) atom and the carbonyl oxygen (O) of Ser122 along the 5 ns (nanoseconds) MD trajectory. The figure also shows a longitudinal view of the TMH3 C-terminal region at 1, 3, and 5 ns of simulation.

 
The behavior of the Ser126 polar lateral chain in a hydrophobic environment was studied by computing the {chi}1 angle fluctuation along the MD simulation, as can be seen in Fig. 4BGo. This analysis revealed a transient shift of the {chi}1 angle that is correlated with the formation of an intrahelical hydrogen bond between the Ser126(O{gamma}) atom and the i-4 carbonyl oxygen of Ser122, as can be seen in Fig. 4CGo. With the aim to reveal the effects of the {chi}1 angle transient shift, we proceeded to compute the volume of the receptor cavities using the average structures of the MC2R wild-type and Ala126Ser mutant, extracted from the MD simulation trajectory (Fig. 5Go, A and B). For comparative purposes, we repeated this calculation using two discrete structures of the wild-type and mutant models, obtained at 3 ns of simulation, where the Ser126(O{gamma}) atom resulted in the trans orientation, as can be deduced in Fig. 4BGo. As depicted, a comparative analysis between average ensembles of the MC2R wild-type and the Ala126Ser models resulted in significant volumetric variations to the extent that the three cavities available in the wild-type model were transformed into five cavities: two at the extracellular side and three at the intracellular side of Ala126Ser receptor (see Fig. 5Go, A and B). It is interesting to note that the wild-type model obtained at 3 ns of simulation time (Fig. 5CGo), mostly conserves the binding’s cavities configuration available in the average wild-type structure (Fig. 5AGo) showing minor volumetric variations, as seen in Table 1Go. In contrast, the Ala126Ser mutant model, obtained at the same simulation time (Fig. 5DGo), showed significant variations in the number and volume of the available binding cavities, in comparison with the average ensemble of the Ala126Ser mutant (Fig. 5BGo). Table 1Go shows the volume per cavity available in the wild-type and the mutant average models and also for the selected representatives of both models at 3 ns of simulation time. As can be noted, the average ensemble of the Ala126Ser mutant presents a compartmentalized and diminished binding site on both sides of the receptor that is not compensated, even by summing the available volume cavities.


Figure 5
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  		FIG. 5. Comparative analyses of cavities between the MC2R wild-type and Ala126Ser molecular models. A and B, Average structures of the 5 ns time span of the trajectories for the wild-type and mutant receptors, respectively. C and D, Snapshots from the molecular dynamics run retrieved at 3 ns of simulation for the wild-type and mutant receptors, respectively. The receptor cavities were identified using Accelrys Discovery Studio Binding Site Tools (Accelrys Inc., San Diego, CA) with a grid resolution 0.5 Å. The corresponding volumetric values for the defined regions are presented in Table 1Go.

 

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  		TABLE 1. Volumes (cubic amstrongs) of the major cavities identified in the MC2R wild-type and Ala126Ser MD representative structures

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In this study we described a novel MC2R gene mutation and predicted the resulting MC2R 3D structure, in a compound heterozygous patient with FGD phenotype due to InsA1347 (maternal) and Ala126Ser (paternal).

Either father or mother carries one of the heterozygous mutations, Ala126Ser and insA1347 (G217fs), respectively, without symptoms of glucocorticoid deficiency confirming an autosomal recessive mode of inheritance and indicating that mutations of the ACTH receptor are the cause of the disorder within this family. Most of the clinical features in FGD can be explained by MC2R gene mutations (40). A MC2R knockout mice model has been recently published, which shown that disruption of MC2R in these pups leads to neonatal lethality in 75% of the cases, and those surviving to adulthood resemble many features observed in FGD patients such as undetectable levels of corticosterone (cortisol) despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia (41).

However, the mechanism underlying the increased growth reported in several patients with MC2R mutations is unclear and is not associated with abnormalities in the GH/IGF-I axis. Tall stature is specifically associated with MC2R mutations and is not found in other forms of Addison’s disease. However, tall stature is also found in patients with MC4R mutations typically presented with severe obesity (body mass index >40 kg/m2) and normal adrenal function (42).

The first mutation reported in this study, already described, corresponding to InsA 1347 (G217fs), producing a frame-shift loss, generating a premature stop codon and an aberrant receptor (43). The InsA1347mutation lies in the third cytoplasmic loop near the sixth transmembrane helix (7). The premature termination introduced by this mutation would eliminate the entire TMH7 of the receptor, including the highly conserved NPxxY-motif that plays an essential role in the GPCRs structure and function (44). Thus, a complete lost of activity for this mutation is expected. The second mutation is a novel mutation, corresponding to a guanine to thymidine change, converting Ala126 to Ser causing the substitution of an apolar (alanine) for a polar (serine) amino acid. This mutation is located at the cytoplasmatic end of TMH3.

In our study, the functional consequences of the novel mutation Ala126Ser were evaluated in an assay activity of MC2R in vitro, suggesting that the novel mutation of Ala126Ser is impairing the functionality of the receptor by approximately 40%. This loss of functionality is not due to a lower surface expression as can be seen in Fig. 3AGo.

To date, more than 30 MC2R mutations have been described: missense, benign polymorphisms, and nonsense or frame shift mutations, many of which are included in the Human Gene Mutation Database (www.hgmd.cf.ac.uk) (45). The functional consequences of a number of these mutations have been studied in vitro and include a loss of ligand binding or affinity, truncated receptors (resulting from a frame-shift mutation), disruption of TMHs, and a loss of signal transduction (7).

Based on our modeling and simulation results, we propose that the lateral side chain of the Ser126 residue becomes exposed to the lipid environment in the MC2R mutant. As seen in Fig. 4BGo, a transient shift of the Ser126 {chi}1 angle can be depicted along the MD simulation. This shift is correlated with the formation of an intrahelical hydrogen bond between the Ser126 hydroxyl group and the carbonyl oxygen of Ser122 (Fig. 4CGo). Similar Ser {chi}1 (trans-gauche) shift variations have been described before, where serine is able to act as a hinge residue that affects the conformation of an {alpha}-helix via an intrahelical hydrogen bond between the O{gamma} atom and the i-3 or i-4 carbonyl oxygen of the helix backbone (46). This shift is particularly important for other GPCRs in which serine substitutions could induce perturbations in the TMHs (47) and lead to constitutive activity for some receptors (48). In this case, the mutation proximal to the conserved TMH3 DRY motif become particularly relevant if we consider the available evidence that supports the important role of TMHs 3 and 6, in the activation of various class A GPCRs (49, 50, 51, 52). Thus, it is expected that perturbations in the structure of TMHs, close to a functionally conserved region as DRY, conforms a scenario in which some conformational changes could compromise the activation process (53). In searching for these changes, a binding site analysis was performed using MD representative structures of both wild-type and Ala126Ser mutant MC2R models, results that can be reviewed in Fig. 5Go, A–D and also in Table 1Go. As depicted, there are important differences in the volume, number, and disposition of the internal cavities between the wild-type and mutant average models (Fig. 5Go, A and B). These differences become less significant when comparing the wild-type and mutant model at 3 ns in which the Ser126 {chi}1 angle exhibits a trans orientation (Fig. 5Go, C and D). These data as a whole suggest that the set of observed changes could be related to the Ser126 {chi}1 angle (trans-gauche) shift produced along the fluctuation dynamics of the mutant MC2R. Moreover, due to the transient nature of the {chi}1 angle shift (Fig. 4BGo), it is expected that the MC2R Ala126Ser mutant could fluctuate within a set of intermediate states in which some functionality is retained. In summary, we report a case of FGD associated with a novel Ala126Ser mutation that impairs the functionality of the ACTH receptor. The MC2R structure modeling suggests that the Ala126Ser mutation induces a perturbation in TMH 3 that could be related to the observed changes in the number and disposition of the receptor internal’s cavities. These observations, as a whole, propose a suitable molecular explanation for the reduced activity exhibited by the MC2R Ala126Ser mutant.


   Footnotes
 
This work was supported by Fondo Nacionale de Desarrollo Cientifico y Ttechnológico de Chile 1040834-1070876 (to R.A.A., C.A.C., C.E.F.), Millennium Nucleus on Immunology and Immunotherapy Grant P04/030-F (to C.A.C., C.E.F., E.R., A.M.K.), and Chilean Foundation for Cellular Biology and the Fundación Ciencia para la Vida, Chile (to T.P.-A., A.G.). A.G. and E.R. are recipients of a Comisión Nacional de Investigación Científica y Tecnólogica de Chile (CONICYT) doctoral fellowship.

The authors have nothing to declare.

First Published Online May 20, 2008

1 R.A.A. and A.G. contributed equally to this work. Back

Abbreviations: Ala126Ser, Alanine 126 by serine; DPPC, 1,2-dipalmitoylphosphatidylcholine; FGD, familial glucocorticoid deficiency; GPCR, G protein-coupled receptor; Luc, luciferase; MCR, melanocortin receptor; MC2R, ACTH receptor; MD, molecular dynamic, NR, normal range; PRA, plasma renin activity; qPCR, real-time PCR; RMSD, root mean square deviation; TMH, transmembrane helix.

Received January 8, 2008.

Accepted May 14, 2008.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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