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Functional Characterization of Naturally Occurring Mutations of the Human Adrenocorticotropin Receptor: Poor Correlation of Phenotype and Genotype¹

Molecular Endocrinology Laboratory, Department of Chemical Endocrinology, St. Bartholomew’s and the Royal London School of Medicine and Dentistry (L.L.K.E., G.D.P., A.M., A.J.L.C.), West Smithfield, London, United Kingdom EC1A 7BE; and Children’s Hospital, Technical University Dresden (A.H.), D-01307 Dresden, Germany

Address all correspondence and requests for reprints to: Dr. Adrian J. L. Clark, Department of Chemical Endocrinology, St. Bartholomews Hospital, London EC1A 7BE, United Kingdom. E-mail a.j.clark{at}mds qmw.ac.uk

	Abstract

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Several missense mutations of the ACTH receptor (MC2-R) gene have been associated with the autosomal recessive syndrome of familial glucocorticoid deficiency. Attempts to demonstrate the functional role of these mutations have been confounded by difficulties in expression of the cloned receptor in cells lacking endogenous melanocortin receptors. The Y6 cell line, a mutant derived from the Y1 cell line, lacks any endogenous MC2-R and can be used for this purpose. We demonstrate that several MC2-R mutations associated with familial glucocorticoid deficiency result in an impaired maximal cAMP response (S74I, I44M, R146H) or loss of sensitivity for cAMP generation (D103N, R128C, T159K) compared to the wild-type receptor. Considerable variation in clinical phenotype exists even for patients with identical mutations of the MC2-R, and correlation between the estimated severity of the receptor defect in vitro and the age at clinical presentation and degree of clinical severity, as judged by basal and stimulated plasma cortisol concentration, is poor.

	Introduction

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FAMILIAL glucocorticoid deficiency (FGD), also known as isolated or hereditary glucocorticoid deficiency and hereditary unresponsiveness to ACTH (MIM_*202200), is a rare autosomal recessive syndrome characterized by severe resistance to the actions of ACTH (1, 2). The disease usually presents in childhood with frequent or severe hypoglycemia and/or infective episodes accompanied by excessive skin pigmentation. Investigation reveals subnormal cortisol levels that respond inadequately to exogenous ACTH, whereas the renin-aldosterone axis is normal. Among several causes of the syndrome that have been proposed, a defect in the ACTH receptor is perhaps the most obvious.

Mountjoy et al. reported the cloning of the human ACTH receptor on the basis of its homology to the MC1 (MSH) receptor, its tissue distribution, and limited expression data (3). Thereafter, we and others have identified mutations in this sequence in many patients with FGD (4, 5, 6, 7, 8, 9). These mutations segregated with the disease in informative families and were not found in the normal population.

We reported the transient expression of the normal human ACTH receptor (MC2-R) in COS-7 cells and a right-shifted dose-response curve in a receptor containing a particular missense mutation (10). However, these findings were confounded by the presence of an endogenous melanocortin receptor present in these cells, and it was difficult to distinguish 1) what element of the cAMP signal generated derived from which receptor, and 2) whether the transfected receptor could enhance the expression or responsiveness of the endogenous receptor, a phenomenon that has been recognized to occur in other systems (11).

Naville et al. (8) and, more recently, Wu et al. (9) reported the expression of the normal and the mutant human MC2-R in Cloudman M3 melanoma cells, but once again these results were confounded by the endogenous MC1-R in these cells. It has proved impossible to express the human MC2-R in cells lacking an endogenous melanocortin receptor until recently.

Schimmer et al. characterized two cell lines, Y6 and OS3, derived from the mouse Y1 corticoadrenal tumor cell line that are ACTH resistant, but retain signaling by forskolin and appear to have defective expression of the wild-type MC2-R despite a normal DNA sequence encoding that receptor (12). These cells, which lack any endogenous melanocortin receptor function, appear to be capable of expressing transfected human MC2-R, and we have therefore used them to characterize the functional consequences of a number of naturally occurring MC2-R mutations that we have identified in patients with FGD. We show that although most mutations impair ligand recognition by the receptor, another mutation (I44M) binds ligand with essentially normal affinity, but transduces only a weak signal. Several of these mutations are associated with a degree of constitutive activity.

	Subjects and Methods

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Clinical details

The clinical details of patients with these mutations have been described previously (4, 6, 13; Elias L. L. K., A. Huebner, G. D. Pullinger, A. Mirtella, A. J. L. Clark, manuscript in preparation). The essential features that act as a guide to the severity of the disorder are summarized in Table 1. In all cases the biochemical characterization was performed with the patients’ and/or parents’ consent at the referring center and was determined entirely by clinical needs.

MC2-R mutations were detected as described previously (4, 6). The full-length coding sequence was amplified by PCR from specific patient’s genomic DNA and subcloned into the expression vector pcDNA1or pcDNA3 (Invitrogen). The integrity of the DNA sequence was confirmed by sequencing. Mouse Y6 cells (a gift from Prof. Bernard Schimmer) were grown in DMEM-Ham’s F-10 (vol/vol) with horse serum (15%), FBS (2.5%), and penicillin/streptomycin. Cells were transfected with normal or mutant MC2-R and, in the case of pcDNA1-based expression vectors, ptk.NEO, a neomycin resistance expression vector. Cells were selected in the presence of G418 (200 µg/mL) 15–20 days after transfection, and resistant clones were isolated by ringing. Multiple clones expressing each mutation were further cultured and tested for cAMP generation. Representative clones were used for further characterization by cAMP generation studies and ligand binding.

ACTH stimulation

Y6 cells expressing the normal or mutant MC2-R were seeded into six-well plates and grown until 70–80% confluent. On the day of the experiment, cells were incubated with serum-free medium containing 1 mmol/L 3-isobutyl-1-methylxanthine (0.5 mL/well) with different concentrations of ACTH-(1–24) (10^-12-10^-5 mol/L) for 60 min. After incubation, cells and medium were harvested, boiled for 5 min, and stored at -20 C until cAMP determination by binding protein assay (14).

ACTH binding assay

The binding assay was performed as described by Penhoat et al. (15). Y6 cells expressing normal or mutant human MC2-R were seeded into 12-well plates (2.5 x 10⁵ cells/well), and after 3–4 days, the cells were washed twice with ice-cold 0.9% NaCl (1 mL), once with ice-cold acid glycine (50 mmol/L glycine/100 mmol/L NaCl, pH 3; 1 mL) for 5 min, and twice with ice-cold 0.9% NaCl (0.5 mL). Then the cells were incubated for 60 min at 20 C with increasing concentrations of nonradioactive ACTH-(1–24) and 0.025 pmol [¹²⁵I-iodotyrosyl²³]ACTH-(1–39) (Amersham Pharmacia Biotech, Piscataway, NJ) in Ham’s F-10-DMEM containing 0.5% BSA and 0.1% bacitracin. At the end of the incubation, the medium was removed, and the cells were washed three times with 0.9% NaCl and dissolved in 0.5 mol/L NaOH-0.4% sodium deoxycholate. Specific binding was determined by subtracting from the total binding the radioactivity associated with cells in the presence of 10^-5 mol/L ACTH-(1–24). Binding was analyzed initially using PRISM2 software and applying nonlinear curve fitting to the displacement curves. Determination of the best fit of a one- or two-site model was determined by the method of least squares.

	Results

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Table 1 lists some of the clinical features of patients with FGD bearing the mutations studied in this report. In each case, the genetic defect was identified by DNA sequence analysis of PCR-amplified fragments of patients’ genomic DNA as described previously (4, 6). All mutations were confirmed either by repeat amplification and sequencing of the entire gene and/or by restriction enzyme analysis.

The location of the missense mutations of the MC2-R studied are shown in Fig. 1. A transient transfection approach to functional analysis was attempted, but transfection efficiency of Y6 cells was insufficient to permit this. Therefore, stable cell lines expressing either the wild-type or each of these mutations in the MC2-R were selected by G418, expanded, and further characterized.

View larger version (22K): [in this window] [in a new window]   Figure 1. A two-dimensional model of the human MC2-R to illustrate the locations of the six mutations studied.

View larger version (21K): [in this window] [in a new window]   Figure 2. cAMP responses of Y6 cells transfected with the wild type MC2-R or the variants indicated to various concentrations of ACTH-(1-24). Points represent the mean ± SD for three independent experiments in which each point was determined in duplicate.

View larger version (16K): [in this window] [in a new window]   Figure 3. Comparison of basal cAMP production by the wild type and each of the mutant receptors in the absence of agonist. Results are the mean ± SD from four to seven independent experiments, as indicated below each column, in which each point was determined in duplicate. Results are compared to the wild-type basal response using a nonpaired t test. *, P < 0.05; **, P < 0.01.

View larger version (24K): [in this window] [in a new window]   Figure 4. ACTH binding by wild-type and mutant receptors. Displacement of [125I-iodotyrosyl23] ACTH-(1-39) by ACTH-(1-24) from Y6 cells expressing the wild-type or mutant MC2-R as indicated. Each point represents the mean ± SD of at least three independent experiments in which each point was determined in duplicate.

	Discussion

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These studies provide clear evidence that mutations in the MC2-R are capable of causing the resistance to ACTH that characterizes FGD. Several classes of defect were observed. Most of the mutations cause the receptor to lose its high affinity ACTH binding. Among these, some receptors show evidence of a shift of the dose-response curve to the right (i.e. D103N, R128C, and T159K) to the extent that they fail to reach a plateau response with micromolar concentrations of ACTH, whereas others either reach a plateau with a near-normal EC₅₀ (R146H) and show a marked reduction in maximal activity or show no response at all (S74I). The I44M mutation, which has virtually normal binding parameters, also shows a reduced maximal response. The number of binding sites per transfected Y6 cell (10⁵) exceeds that found in normal adrenal cells from various species, which in most studies is less than 10⁴ sites/cell (15, 16, 17, 18), although these adrenal data are inevitably derived from a heterogeneous population of cells, implying a greater concentration of sites in individual ACTH-responsive cells.

Those mutations that shift the dose response to the right have a defect that can be overcome with greater doses of agonist and thus can be considered quantitative defects. Therefore, the D103N mutation probably fails to bind ACTH with the greatest affinity (as confirmed by the binding analysis), but larger concentrations of ACTH will activate a sufficient number of receptors to produce a sizable activation. D103 lies on the extracellular surface of the receptor, and it is perhaps not surprising that it is associated with impaired ACTH binding.

A similar effect on ligand binding may be the explanation for the defect seen with the T159K mutation. T159 lies in the middle of the fourth transmembrane domain and may conceivably have a role in ligand binding, although existing models for MC1-R binding have not specifically highlighted this region in MSH binding, and according to the Baldwin model of G protein-coupled receptors, this residue (equivalent to IV:15 in this model) would not project into the central binding pocket (19). Alternatively, the introduction of a highly basic lysine residue into this -helix may significantly disrupt the structure of the whole -helix and thereby produce the same effect on ligand binding.

The R128C mutation lies in the DRY sequence, the most conserved element in the entire rhodopsin subclass of G protein-coupled receptors (20). In several members of this family naturally occurring mutations or site-directed mutagenesis of members of this tripeptide usually lead to loss of signaling capacity (21, 22), although in some cases, e.g. the GnRH receptor (23) or the AT1 angiotensin receptor (24), this effect is not as marked. This sequence is usually considered to provide the molecular switch that activates the heterotrimeric G protein in other G protein-coupled receptors (25), and it is conceivable that the cysteine substitution would result in an inefficient switch in the MC2-R. This fails to explain the loss of high affinity binding, and it is highly unlikely that R128 is involved in ligand interaction. Such a loss of high affinity binding, however, may be the result of impaired G protein coupling. It is well recognized that treatment of cell membranes with guanosine triphosphate (GTP)--S or other nonhydrolyzable GTP analogs will uncouple this class of receptors and diminish high affinity ligand binding (26). Thus, if R128 had a role in G protein coupling distinct from switching, then the loss of high affinity binding could be explained.

The other mutations exhibit what could be considered a qualitative defect of the receptor, i.e. a defect that cannot be overcome with greater doses of agonist. This type of defect is exemplified by S74I. Serine 74 is located in the second transmembrane domain and would be predicted to lie in close proximity to the first transmembrane domain according to the Baldwin model of G protein-coupled receptors (S74 lies in position II:18 in this model) (19) and presumably is involved in hydrogen bond formation with an unidentified residue. Disruption of this bond by substitution with isoleucine leads to a receptor with only low affinity for ACTH and no significant signal transducing ability.

The R146H mutation also results in the loss of the high affinity binding and significant loss of maximal ACTH-stimulated activity. This indicates an unsuspected importance of R146, which lies close to the junction of the C-terminal end of the second cytoplasmic loop and the fourth transmembrane domain. As with the R128C mutation, it seems likely that this residue is involved in G protein coupling, and that the histidine substitution in this position results in an insurmountable defect and, consequently, impaired high affinity binding.

Finally, the I44M mutation is unusual. We had originally expected that the I44M mutation would be a benign polymorphism, as this is a conservative substitution, and the bovine MC2-R contains a methionine in this position (27). Indeed, this receptor binds ACTH with virtually normal affinity, but fails to transduce a signal effectively. The explanation for this observation is not clear.

Correlation of these findings with estimates of clinical severity is difficult. The data in Table 1 show that even for a single genotype, S74I, the phenotype may be highly variable. Within this group there are individuals who only just fail to meet the criteria for ACTH resistance (patients I-1 and VII-1) and those with undetectable cortisol production (e.g. patient II-1). This is remarkable considering that in our functional studies this mutation renders the MC2-R effectively inactive.

Among the remaining mutations, phenotype/genotype correlation is only slightly better. The R128C mutation exhibits a good ACTH response with a right-shifted dose-response curve in vitro, and indeed, in conjunction with S74I in patient VIII-1, results in a comparatively mild phenotype with a relatively late age of presentation. Other mutations that markedly reduce the responsiveness of the receptor in vitro are associated with an almost inactive receptor in vivo in the case of the patients with I44M, R146H, and T159K (patients X-1, XI-1, XII-1, XIII-1, and XIV-1). I44M has only been identified as a compound heterozygote with a frameshift mutation that prematurely truncates the receptor (6). This truncated receptor would not be expected to exhibit any significant receptor function, and thus, the phenotype of the patient reflects the markedly impaired function of the I44M receptor.

Although these observations are disappointing, particularly within the S74I patients, they may be indicative of imprecision of the ACTH stimulation test within a population with subnormal responsiveness, and the fact that each of these tests was performed in different centers. Additionally, there may be variation in the individual adaptive response of the adrenal to ACTH resistance, which might involve the efficiency of signal transduction or the use of the MC5 receptor, also known to be expressed in the rodent adrenal cortex (28, 29) as an alternative ACTH receptor.

In addition to defects of ligand binding and signal transduction, some of these mutations also exhibit weak constitutive activity. However, in all cases with these mutations in homozygous form (patients XI-1, XII-1, XIII-1, and XIV-1) or in a compound heterozygote (patient XV-1), basal cortisol was very low or undetectable. Computed tomography scan of the adrenals in one of these cases (case XV-1) revealed atrophic adrenal glands, as is typical of this disorder. It is notable that constitutively active MC2-R have not been found in cases of adrenal hyperplasia or neoplasia (30, 31), although there is a single case report of a patient with a hypersensitive MC2-R associated with C21R and S247G compound heterozygous mutations (32). This latter variant has not been functionally studied.

In summary, these studies show that the Y6 cell expression system is an effective one for characterizing the MC2-R and its mutations and confirm the suspicion that these mutations are the cause of FGD. The functional effects of these mutations are varied, and most result in the loss of high affinity ACTH binding.

	Acknowledgments

 
We are extremely grateful to Prof. Bernard Schimmer and Dr. Roger Cone for helpful advice and for provision of the Y6 cell line.

	Footnotes

 
¹ This work was supported by a Wellcome Trust Travelling Fellowship (to L.L.K.E.), the Fundação de Amparo a Pesquisa do Estado de São Paulo (to L.L.K.E.), the Deutsche Forschungsgemeinschaft (to A.W.), and the Medical Research Council.

Received March 9, 1999.

Revised April 14, 1999.

Accepted April 28, 1999.

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