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Molecular Endocrinology 18 (9): 2151-2165
Copyright © 2004 by The Endocrine Society

New Naturally Occurring Missense Mutations of the Human Mineralocorticoid Receptor Disclose Important Residues Involved in Dynamic Interactions with Deoxyribonucleic Acid, Intracellular Trafficking, and Ligand Binding

Paola Sartorato, Françoise Cluzeaud, Jérôme Fagart, Say Viengchareun, Marc Lombès and Maria-Christina Zennaro

Institut National de la Santé et de la Recherche Médicale (INSERM) U478, Faculté de Médecine Xavier Bichat, 75870 Paris Cedex 18, France

Address all correspondence and requests for reprints to: Maria-Christina Zennaro, M.D., Ph.D., Institut National de la Santé et de la Recherche Médicale (INSERM) U478, Faculté de Médecine Xavier Bichat, B.P. 416, 16, rue Henri Huchard, 75870 Paris Cedex 18, France. E-mail: zennaro{at}infobiogen.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have investigated the functional consequences of three naturally occurring amino acid substitutions of the human mineralocorticoid receptor (hMR). These mutations are located in the DNA-binding domain and the ligand-binding domain (LBD) and are associated with autosomal dominant or sporadic type I pseudohypoaldosteronism. All mutant receptors bound specifically to glucocorticoid-responsive elements but presented modified transcriptional properties. The DNA-binding domain mutant G633R, which possesses a normal affinity for a glucocorticoid-responsive element, displayed altered interaction with, and a reduced dissociation rate from, DNA. Its intracellular localization in the absence of hormone was predominantly nuclear in comparison with predominant cytoplasmic location of hMR. Hormone-dependent nuclear cluster formation was comparable to wild-type hMR. These results and the three-dimensional modeling of the interaction of R633 with DNA suggest that altered interaction dynamics with DNA as well as modified intracellular localization may be responsible for submaximal transcriptional potency of hMR. Two LBD mutations, Q776R and L979P, were also investigated. Our data confirm the fundamental role of amino acid Q776 for anchoring the C3 ketone group of steroids in the ligand-binding pocket. Analysis of LBD conformation of mutant P979 demonstrates the relevance of hydrophobic interactions in the extreme C-terminal tail of the hMR for the correct ligand-binding competent state of the receptor. Our data underline the importance of studying naturally occurring mutants to identify crucial residues involved in hMR function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SALT AND WATER balance are regulated in a major way by aldosterone acting through the mineralocorticoid receptor (MR) in the distal part of the nephron (1). Disruption of this signaling cascade and MR mutations lead to type I pseudohypoaldosteronism (PHA1) or hypertension (2). PHA1 (OMIM177735) is a rare inherited form of mineralocorticoid resistance typically presenting in the newborn with renal salt wasting and failure to thrive (3). Associated features include hyponatremia, hyperkalemia, and metabolic acidosis. Two different forms of the disease result from distinct genetic abnormalities. In autosomal dominant PHA1, different heterozygous loss-of-function mutations in the human MR (hMR) gene have been identified and characterized, including frameshift, nonsense and missense mutations, and gene deletions (4, 5, 6, 7, 8, 9).

The MR belongs to the nuclear receptor superfamily [NR3C2 (10)], which includes the other steroid hormone receptors, the thyroid hormone, retinoic acid, and vitamin D receptors. In the absence of hormone, MR is associated with a multiprotein complex composed of heat shock proteins and immunophilins (11). Hormone binding is followed by a change in receptor conformation, dimerization, nuclear translocation, and binding to specific hormone-responsive elements located in regulatory regions of aldosterone-regulated genes. Nuclear receptors are modular proteins harboring different conserved domains (12). Some receptor functions are localized to isolated motifs that can be transferred from the receptor to a heterologous protein, whereas other functions require multiple receptor domains. The N-terminal part is the less conserved region among nuclear receptors, both in size and sequence, and represents almost half of the MR protein. This region contains a ligand-independent activation function 1, which has been shown to be unstructured in solution, and which is important for interaction with transcriptional coregulators (13, 14) and for intramolecular interactions with the ligand binding domain [LBD (15)]. The centrally located DNA binding domain (DBD) is the most conserved region of the receptor (16). It folds into two zinc fingers, in which one zinc atom is tetracoordinated by four cysteines. The core DBD contains two {alpha}-helices; the first one, or recognition helix, binds to the major groove of DNA, making contacts with specific bases. This domain also contains segments involved in receptor homo-and heterodimerization. Putative nuclear localization signals are localized in the C-terminal part of the DBD and the beginning of the hinge region. Finally, the LBD is located in the C-terminal part of the receptor (16). This domain is complex, as it harbors regions involved in formation of the ligand-binding pocket, interaction with heat shock proteins, dimerization, and a ligand-dependent activation function 2, which interacts with transcriptional coregulators (12).

Crystal structures of several unliganded and liganded nuclear receptor LBD, including the progesterone (17), androgen (18, 19), and glucocorticoid (20, 21) receptors, have been solved in recent years, allowing development of three-dimensional homology models of the hMR (22, 23, 24). Based on these models, the hMR LBD is organized as a three-layer sandwich fold consisting of 11 helices (H1, H3–H12) and four short ß-strands forming two ß-sheets. The hMR ligand-binding pocket is delineated by helices H3, H5, H7, H11, and H12, the first ß-sheet, and the loops H6–H7 and H11–H12. Binding of transcriptional coregulators occurs through a hydrophobic structure composed of different helices in the LBD. Importantly, the second ß-structure, consisting of one strand located between H8 and H9 and another in the middle of the C-terminal extension of the LBD is likely to be important in stabilizing the positioning of this part of the receptor.

The hMR gene spans approximately 400 kb and is composed of 10 exons (25). Exon 2 contains the translation start site and codes for the amino-terminal part of the receptor. Exons 3 and 4 code for the DBD, whereas the LBD is encoded by exons 5–9. Two 5'-untranslated exons are alternatively transcribed to generate two mRNA isoforms, hMR{alpha} and hMRß, which are coexpressed in aldosterone target tissues (26). Two alternative promoters allow for tissuespecific and differential hormonal regulation of hMR expression (27, 28). Multiple hMR isoforms generated through alternative splicing exist, which diverge in their DBD (29) or LBD (30, 31). One of them, hMR{Delta}5,6, resulting from an alternative splicing event skipping exons 5 and 6 of the hMR gene, displays ligand-independent transcriptional activity and might play a role in modulating mineralocorticoid and glucocorticoid effects in target tissues (31).

We have previously reported six new hMR mutations found in families affected by autosomal dominant or sporadic PHA1 (7). Three of them were missense mutations. Substitution G633R is located in the DBD, whereas Q776R and L979P are localized in the LBD. In this report we have investigated the functional consequences of these naturally occurring mutations on hMR function. Our data reveal the importance of dynamic receptor-DNA interactions for maximal transactivation and the role of G633 for subcellular receptor localization. Furthermore, we confirm the importance of the C-terminal end of the 3-keto-steroid receptors, which plays a major role in ligand binding and receptor activation through stabilization of H12 in the active conformation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of hMR Mutations on Transactivation by hMR
Mutations G633R, Q776R, and L979P were recreated into mammalian expression vectors to elucidate molecular mechanisms underlying the physiopathology of autosomal dominant PHA1. All mutations affected residues that are highly conserved among members of the steroid receptor subfamily of nuclear receptors (Fig. 1Go, A and B). In previous studies we have shown that mutant R633 retained the aldosterone binding capacity of the wild-type hMR and displayed the same affinity for aldosterone, whereas LBD mutant R776 displayed only 30% of maximal aldosterone binding with a Kd for aldosterone that was approximately 6-fold higher than that of the wild-type receptor (7) (Table 1Go). These two mutations reduced maximal aldosterone-dependent transactivation of reporter genes to different extents, depending on the promoter context (Ref. 8 and Table 1Go). Furthermore, mutation Q776R induced a shift in the aldosterone doseresponse curve toward higher hormone concentrations (Ref. 7 and Fig. 2AGo). In contrast, substitution of the highly conserved leucine 979 by proline completely abolished aldosterone binding and transactivation from a mouse mammary tumor virus (MMTV) promoter, even in the presence of very high aldosterone concentrations (Ref. 7 and Table 1Go).



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  		Fig. 1. Schematic Representation of hMR Mutations and Alignment with Other Steroid Receptor Proteins

A, The hMR gene is represented with its intron/exon structure. Exons 2–9 code for the functional domains of the hMR protein (exon numbers are indicated on the bottom of each exon). hMR mutations investigated in our study are depicted on the gene. The functional domains of the receptor are indicated on the amino acid sequence. N-ter, N-terminal domain; ATG, translation initiation site; TGA; translation stop codon. B, Alignment of protein sequences for hMR (MCR_HUMAN), human glucocorticoid receptor (GCR_HUMAN), human progesterone receptor (PRGR_HUMAN), and human androgen receptor (ANDR_HUMAN). Amino acids corresponding to hMR mutations found in PHA1 are boxed.

 

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  		Table 1. Summary of hMR Mutations Examined (from Refs. 7 and 8 )

 


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  		Fig. 2. Dose-Response Curves of Transcriptional Activation by Wild-Type and Mutated hMRs

Rabbit renal RCSV3 cells were transiently transfected with expression vectors for wild-type or mutated hMRs together with an MMTV-luciferase reporter plasmid. A ß-galactosidase expression vector was also transfected to normalize for transfection efficiency. Cells were incubated 24 h after transfection with fresh medium containing the indicated doses of hormone. A, Aldosterone dose-response curves. Induction observed with wild-type hMR in the presence of 10 nM aldosterone was set as 100%. B, Cortisol dose-response curves. Induction observed with wild-type hMR in the presence of 10 nM cortisol was set as 100%. Results represent the mean ± SEM of three independent experiments performed in triplicate and are the ratio of luciferase activity over ß-galactosidase activity.

 
Aldosterone and glucocorticoids bind the hMR with the same affinity; however, hMR possesses an intrinsic discriminating property due to different off-rates of the two hormones from the receptor (32). It is believed that a more stable ligand-receptor interaction may influence subsequent steps, such as nucleocytoplasmic shuttling, DNA binding, and interaction with the transcriptional machinery. This functional preference of hMR for aldosterone over glucocorticoids is reflected by the higher doses of glucocorticoids required to achieve maximal transcriptional activation. To further analyze the functional properties of the mutant receptors, we investigated whether mutations G633R and Q776R differentially affect the aldosterone- or glucocorticoid-induced transcriptional activity of hMR. Transient transfection assays were performed in rabbit renal RCSV3 cells. Expression vectors for wild-type or mutant receptors were transfected together with an MMTV-luciferase reporter plasmid in the presence of increasing concentrations of aldosterone (10–11–10–7 M) and cortisol (10–10–10–7 M). As shown in Fig. 2BGo, cortisol increased transactivation by the wild-type hMR in a dose-dependent manner, with a plateau reached at 10–8 M. Maximal luciferase activity induced by R633 and R776 was 50% of that obtained with hMR at 10–8 M cortisol. For R633, a plateau in the dose-response curve was reached at 10–9 M, whereas no plateau was observed for R776. In comparison with what was observed in the presence of aldosterone (Fig. 2AGo), there was no difference in the ED50 of the cortisol dose-response curve with R633 (~7 x 10–10 M), compared with wild-type hMR, whereas a shift of at least 2 orders of magnitude toward higher hormone concentrations was observed with mutant R776.

Our results indicate that mutations G633R and Q776R reduce both aldosterone- and cortisol-induced transactivation from the MMTV promoter. Whereas the dose-response curves of the mutant receptors display a similar pattern in the presence of the two hormones, maximal transactivation appears more severely affected in the presence of cortisol.

DNA Binding of hMR Mutants
Next we investigated the DNA binding properties of wild-type hMR and hMR mutants by EMSA. Equal amounts of in vitro-translated wild-type and mutated hMR proteins were assayed for binding to a consensus glucocorticoid-responsive element (GRE) sequence. In agreement with previous results (31), receptors produced in the reticulocyte lysate system displayed a consistent DNA-binding activity even in the absence of hormone. hMR specifically bound to a consensus 32P-labeled GRE oligonucleotide (Fig. 3Go, line 2 and 3). R633, R776, and P979 all bound to the GRE in a specific manner, because binding was efficiently competed for by excess amounts of unlabeled GRE (Fig. 3Go, lanes 3–9).



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  		Fig. 3. Binding of Wild-Type and Mutant hMRs to a Consensus GRE Sequence

Equal amounts of in vitro-translated wild-type and mutated hMR proteins were analyzed for binding to a double-stranded consensus GRE oligonucleotide. After a 15-min incubation of receptors with 20,000 cpm of radiolabeled GRE, protein-DNA complexes were separated on nondenaturing 4.5% polyacrylamide gel and detected by autoradiography. Arrowheads indicate position of specific MR-GRE complexes. For competition experiments, 100 ng unlabeled GRE were used. Rec, Receptor; Comp, competitor; F, free probe.

 
Mutation G633R is localized in the DBD and, although aldosterone binding is not affected, mutant R633 presented a reduced maximal transactivation of two different reporter genes (7, 8). We therefore investigated whether this mutation affected the affinity of the receptor for a GRE oligonucleotide by incubating recombinant hMR or R633 with increasing concentrations of unlabeled competitor. As shown in Fig. 4AGo, binding of wild-type and mutant receptor was competed for by increasing concentrations of unlabeled oligonucleotide, as indicated. Quantification of receptor-GRE complexes by InstantImager showed that GRE binding of hMR and R633 was progressively reduced by increasing amounts of competitor to 5% and 25% for R633 and hMR, respectively. No difference was observed in the IC50 of binding of R633 to the GRE compared with the wild-type receptor (~5 ng of competitor), which corresponds to a relative Kd of approximately 10–8 M, indicating that mutation G633R does not affect the affinity of hMR for a consensus GRE binding site.



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  		Fig. 4. Affinity of Binding of Wild-Type hMR and R633 to a GRE

A, In vitro-translated wild-type hMR and R633 were incubated at room temperature with radiolabeled GRE (20,000 cpm) in the absence or presence of increasing amounts (1–50 ng) of cold competitor (Comp) as indicated and analyzed for binding to the consensus GRE. The arrow indicates the migration of the receptor-GRE complex. B, Receptor-DNA complexes were quantified by InstantImager software, and GRE binding was plotted as a function of the amount of competitor. GRE binding in the absence of competitor was set as 100%, and the IC50 is indicated for each receptor. The figure shows a representative experiment that was repeated three times. F, Free probe.

 
Because transcriptional activation is a dynamic event in which receptors undergo cycles of DNA binding and release (33, 34), we wondered whether mutant R633 displayed an altered dissociation kinetics from a consensus GRE. We therefore incubated in vitro-translated wild-type hMR and R633 with radiolabeled GRE for 15 min. Dissociation was studied by adding an excess of unlabeled GRE (50 ng) for different times (0–40 min). Surprisingly, whereas hMR dissociated from the GRE oligonucleotide in the presence of competitor (Fig. 5AGo, lanes 2–8), R633 remained nearly completely bound after 32 min of incubation (Fig. 5AGo, lanes 9–15). Dissociation rates, indeed, were very different between the two receptors, as quantified by InstantImager (Fig. 5BGo). Whereas wild-type hMR displayed rapid dissociation from DNA with increasing competitor incubation times, with a t1/2 estimated at about 30 min, approximately 80% of R633 remained bound to GRE all over the time lapse of the experiment. Comparison of the slopes of the dissociation curves showed a significant difference between hMR and R633, with the regression slope of R633 not being statistically different from zero. When in vitrotranslated receptors were incubated with aldosterone, the receptor-DNA dissociation rate was accelerated for both hMR and R633; also in the presence of hormone, the dissociation rate of R633 from the GRE was slower than that of hMR (data not shown).



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  		Fig. 5. Kinetic Studies of the Interaction of Wild-Type hMR and R633 with a GRE

Interaction kinetics of wild-type and mutated hMR were determined by analyzing the dissociation from (panels A and B) and the on rate of the receptors on DNA (panels C and D). A, In vitro-translated wild-type hMR and R633 were incubated with radiolabeled GRE (20,000 cpm) for 15 min. Dissociation was studied at 20 C by adding an excess of cold GRE (50 ng) for different times (0–40 min) as indicated. The arrow indicates the migration of the receptor-GRE complex. The figure shows a representative experiment that was repeated three times. B, Receptor-DNA complexes were quantified by InstantImager, and percentage of reduction of GRE binding was plotted against time of incubation with cold competitor. GRE binding in the presence of competitor at time 0 was set as 100. Results represent the means of three independent experiments. Linear regression equations, calculated using Instat version 2.01 (GraphPad Software, San Diego, CA), were as follows: hMR: y = –1.20x + 81.54; r2 = 0.82; P < 0.0001; R633: y = –0.42x + 86.78; r2 = 0.14. For R633 the regression slope was not statistically different from zero. C, In vitro-translated wild-type hMR and R633 were incubated with radiolabeled GRE (20,000 cpm) at 4 C for different times (0–50 min) as indicated. Complex 1 and an additional complex 2 formed by R633 are indicated by arrows. The figure shows a representative experiment that was repeated twice. D, Receptor-DNA complexes 1 and 2 were quantified by InstantImager. The upper panel represents quantification of complex 1 formed by hMR; GRE binding at the end of the experiment was set as 100, and percentage increase in GRE binding was plotted against time of incubation. In the lower panel, complexes 1 (open squares) and 2 (solid circles) formed by R633 are depicted. Complex 1 is presented as above; percentage decrease of complex 2 is represented compared with time 0 (100%). Results represent the means of two independent experiments. F, Free probe; P, probe only.

 
Because the affinity is proportional to the ratio of on rate/off rate and the affinity of R633 for DNA was unchanged (see Fig. 4Go), we investigated also the on rate of wild-type and mutant receptors on the GRE (Fig. 5Go, C and D). For this purpose, in vitro-translated wild-type hMR and R633 were incubated with radiolabeled GRE at 4 C for increasing times (0–60 min). Even in experimental conditions known to slow down interaction kinetics (i.e. at 4 C), approximately 50% of receptors were bound to DNA at time 0, therefore preventing detection of major differences in on rate between hMR and R633 (Fig. 5CGo and D, complex 1). However, it is interesting to note that an additional R633-DNA complex displaying faster migration was observed (Fig. 5CGo, complex 2, lanes 9–15), which disappeared progressively with increasing incubation times (Fig. 5DGo, lower panel). This complex, which was also formed in the presence of aldosterone (data not shown), is suggestive of monomeric receptor-DNA complexes, which would progressively be replaced by dimeric complexes, further supporting altered interaction of R633 with DNA. Altogether, these data indicate that, although DNA binding affinity of hMR is not affected by the mutation G633R, association of the R633 with DNA is modified and the dissociation rate is strongly slowed down, possibly playing a role in the altered transcriptional activity of the mutant receptor.

Effect of Mutation L979P on hMR Conformation
The drastic loss of ligand binding and transactivation induced by hMR substitution L979P prompted us to analyze more extensively this mutation. To define its role in the conformation of the C-terminal part of the hMR LBD, we performed a conformational study by limited proteolytic digestion. Equivalent amounts of in vitro-translated and 35S-labeled hMR and PQ79 were incubated with increasing concentrations of trypsin for 10 min. The digestion products were analyzed by SDS-PAGE (Fig. 6Go). The wild-type and mutant receptors were synthesized at the same level (Fig. 6Go, lanes 2 and 8). One major fragment, corresponding to a molecular mass of 41 kDa, was generated from the wild-type hMR. Two minor bands, corresponding to fragments of 30 and 27 kDa, were also detected (lanes 4–7). The 30-kDa fragment corresponds to hMR region Glu716–Lys984, containing the C-terminal part of the hinge region and the entire LBD (35). The 41-kDa fragment has the same C terminus, whereas the amino terminus is one of the three amino acids, Ser591, Pro599, or Ile602 (35). The nature of the 27-kDa fragment is not known, but it does not encompass the N-terminal domain of hMR (36). These fragments were progressively degraded with increasing concentrations of trypsin. Tryptic digestion of mutant P979 also produced the 41-, 30-, and 27-kDa fragments (lanes 10–13). However, their relative abundance differed, with the 30- and 27-kDa fragments being largely prevalent. The 41- and 30-kDa fragments were rapidly digested with increasing concentrations of trypsin, whereas the 27-kDa fragment was more resistant. Our results reveal an increased sensitivity to proteolysis due to L979P substitution, suggesting a change in receptor compaction and the exposure of sites sensitive to tryptic digestion.



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  		Fig. 6. Limited Proteolytic Digestion of Wild-Type hMR and Mutant P979

35S-labeled, in vitro-translated wild-type hMR and P979 were incubated for 10 min at 20 C without or with increasing amounts of trypsin (5–90 µg/ml) as indicated (lanes 2–7 and 8–13). An equal volume of water was added for the undigested controls (lanes 2 and 8). Digestion products were analyzed by electrophoresis on a 12% SDS-PAGE gel and autoradiographed. Molecular weight markers are indicated. Experiments were performed twice with reproducible results.

 
Intracellular Trafficking of hMR Mutants
To determine the influence of hMR mutations on subcellular receptor distribution, we examined nucleocytoplasmic trafficking of wild-type and mutated hMR fused to green fluorescent protein. We first studied the transcriptional activities of the fusion proteins to verify that their functional characteristics were conserved. In transient transfection experiments in RCSV3 cells, enhanced green fluorescent protein (EGFP)-hMR gave strong activation of an MMTV-luc reporter gene in the presence of 10 nM aldosterone, which corresponded to 80% of that observed with wild-type hMR at the same hormone concentration (data not shown). Relative hormone-dependent transcriptional induction observed with hMR mutants fused to EGFP at 10–8 M aldosterone was comparable to their nonchimeric counterparts (see Ref. 7) (EGFP-R776: 29% of EGFP-hMR vs. R776: 30% of hMR; EGFP-P979: 4% of EGFP-hMR vs. P979: 6% of hMR activity), except for mutant EGFP-R633, which activated transcription significantly, but slightly less than its nonfused counterpart (21% of EGFP-hMR vs. R633: 50% of hMR activity at 10–8 M aldosterone).

We then examined the cellular distribution of wild-type and mutant hMR fusion proteins in the absence or presence of aldosterone. RCSV3 cells were transiently transfected with EGFP-hMR, EGFP-R633, EGFP-R776, or EGFP-P979. In the absence of hormone, hMR was found prevalently either in the cytoplasm or both in the nucleus and the cytoplasm (Fig. 7Go, A and B). After incubation with aldosterone, there was a time-dependent accumulation of receptors in the nucleus, which was complete in the majority of cells after 30 min. In contrast to wild-type hMR, mutant R633 was found almost entirely either in the nucleus or both in nucleus and cytoplasm even in the absence of hormone. As soon as 5 min after incubation with aldosterone, 50% of R633 was localized in the nucleus, and nuclear entry was complete after 15 min of hormone incubation. Also in this case, nucleoli were excluded from receptor occupation. Subcellular distribution of mutants R776 and P979 in the absence of hormone was very similar to that of wild-type receptor. According to hormone binding data (7), R776 shifted very slowly into the nucleus and, after 30 min incubation with aldosterone, the mutant was nuclear in only 34% of cells. Results observed with mutant P979 in the presence of aldosterone were somewhat intriguing. Although this mutant had no affinity for aldosterone as revealed in binding studies, incubation with hormone induced a partial shift of the mutant receptor from the cytoplasm to the nucleus. This may be due to partial heterodimerization with endogenous wild-type MR or glucocorticoid receptors, which are present in very low amounts in RCSV3 cells (27).



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  		Fig. 7. Intracellular Trafficking of Wild-Type and Mutant Receptors in Living Cells

RCSV3 cells were transiently transfected with chimeric receptor proteins EGFP-hMR, EGFP R633, EGFP-R776, and EGFP-P979 and grown in steroid-free medium. Intracellular localization of fluorescent proteins was then observed in the absence (no Aldo) or presence of 10 nM aldosterone for various periods of time (0–60 min). A, Nuclear translocation of fluorescent wild-type receptor and mutant proteins after 30 min incubation with 10 nM aldosterone. B, Quantification of intracellular localization of wild-type and mutant receptors at different time points as indicated. Between 20 and 60 cells were examined at each time point for each receptor, except for P979 (10–20 cells). Results are represented as percentage of the total number of cells observed. Experiments were repeated at least three times. N, Nuclear; NC, nuclear and cytoplasmic; C, cytoplasmic.

 
The intracellular localization of mutant R633 was further investigated. For this purpose, human H5 renal collecting duct cells were stably transfected with an EGFP-R633 fusion protein, and nucleocytoplasmic shuttling of R633 was compared with that of wild-type hMR stably expressed in the same H5 cell line (37) by confocal microscopy (Fig. 8AGo). In our experimental conditions, i.e. at 37 C in the presence of 5% CO2, hMR was cytoplasmic in the absence of hormone in nearly 75% of cells (Fig. 8BGo). Nucleocytoplasmic redistribution was visible as early as 5 min after incubation with aldosterone and after 15 min, 60% of cells presented the receptor in the nucleus. Nuclear localization was complete after 30 min. According to previous studies (38), MR accumulated into discrete clusters, whereas nucleoli remained excluded from receptor occupation (Fig. 8CGo). As observed in transient transfection experiments, in stable transfected cells R633 was found predominantly (64%) in the nucleus or in both the nucleus and cytoplasm in the absence of hormone (vs. 25% for hMR), with only 37% of cells displaying cytoplasmic localization of the mutant receptor (Fig. 8AGo). After hormone addition, the remaining cytoplasmic receptor rapidly shifted into the nucleus, and nearly 90% of cells presented a nuclear localization after only 10 min of hormone incubation (vs. 35% of hMR, Fig. 8BGo). Interestingly, in the absence of hormone, R633 was distributed rather uniformly in the nucleus. After hormone addition, accumulation into subnuclear clusters occurred, which were indistinguishable from those seen with wild-type receptor in the presence of aldosterone (Fig. 8CGo). These results indicate that mutation of glycine 633 into arginine affects intracellular localization of hMR in the absence of hormone. However, this residue is not involved in hormone-dependent cluster formation.



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  		Fig. 8. Nuclear Localization of Wild-Type hMR and R633 in Living Cells

The human collecting duct cell line H5 was stably transfected with a plasmid coding for a chimeric EGFP-R633 protein. Parallel experiments were performed using a H5 cell line stably expressing wild-type hMR. Cells were seeded in Lab-Tek culture chambers at a density of 60,000 cells and cultured in steroid-free medium. After addition of 10 nM aldosterone, intracellular fluorescence was observed using confocal microscopy at constant temperature and pH. Three independent experiments were performed in triplicate. A, Representative experiment showing the intracellular localization of EGPF-hMR and EGFP-R633 before and at different time points after addition of aldosterone. B, Quantification of the intracellular localization of hMR and R633. N, Nuclear; NC, nuclear and cytoplasmic; C, cytoplasmic. Between 30 and 60 cells were examined at each time point, and results are represented as percentage of the total number of cells observed. Experiments were repeated at least three times in triplicate. C, Hormone-induced nuclear cluster formation by wild-type hMR and R633 in the absence and presence of 10 nM aldosterone.

 
Three-Dimensional Models of hMR Mutants
To investigate the incidence of mutations G633R, Q776R, and L979P on hMR protein structure, we introduced the three substitutions in the hMR homology model. The three-dimensional model of the hMR-DBD was generated on the basis of the crystal structure of the hGR-DBD (39). The DBD of the hMR adopts the typical organization observed for the other members of the nuclear receptor superfamily. It is composed of two modules, each one being nucleated by a zinc coordination center followed by an amphipathic {alpha}helix. The N-terminal module is involved in protein-DNA contacts. Its amphipathic helix contains the P box that interacts with the bases in the major groove (see Fig. 9AGo). The second module is involved in protein-protein interactions and harbors the dimerization interface located in the D box. The G633 is located at the junction of both subdomains in the loop between the P and D boxes (Fig. 9AGo). From the hMR-DBD model it appeared that an additional hydrogen bond can be formed with a DNA phosphate when an arginine is substituted for the G633 (Fig. 9AGo). As suggested by our EMSA experiments on the dissociation kinetics of hMR from a consensus GRE, the three-dimensional model indicates that mutant R633 is more tightly associated with DNA than the wild-type receptor.



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  		Fig. 9. Three-Dimensional Homology Models of the hMR DBD and LBD

A, Picture showing the binding of the DBD P box within the DNA major groove. Only residues K624, V625, and R629 involved in the specific recognition of the GRE and the mutant R633 are depicted. The hydrogen bonds between the DBD side chains and the DNA bases were visualized as dots. B, Close-up view of the aldosterone A ring-anchoring site of the hMR LBD. Depicted are the Q776 and R817 residues that anchor the C3-ketone function together with the R776 mutated residue. C, Close up view of the ß-sheet that anchors the C-terminal extension. The hydrogen bonds that stabilize the ß-sheet are depicted as dots. D, Close up view of the ß-sheet harboring the L979P mutation.

 
The hMR-LBD is composed of 11 helices and four short ß-strands forming two ß-sheets (22). The first sheet is located between the H5 and H6 helices and is part of the ligand-binding pocket. The second one consists of one strand located between the H8 and H9 helices and the other in the middle of the C-terminal extension. Mutagenesis studies have shown that aldosterone is anchored by four polar residues, distributed at both extremities of the ligand-binding cavity. Q776 and R817 form two hydrogen bonds with the C3-ketone function of aldosterone (Fig. 9BGo) (22). The C18 and C21-hydroxyl moieties interact with N770, and the C20 ketone group is anchored by T945. Substituting an arginine for the Q776 altered the anchoring of the A ring. Indeed, such a substitution introduced a long side chain at the vicinity of the ligand that was unable to establish a hydrogen bond with Q776.

The L979 residue is located in the middle of the C-terminal extension of the LBD and is part of the second ß-sheet of the domain (Fig. 9CGo). Substituting this residue by a cyclic proline removed one of the hydrogen bonds that form the ß-structure (Fig. 9DGo). Moreover, the cyclic side chain of proline created a steric hindrance with the main chain of the second ß-strand. Thus, the L979P substitution notably destabilized the positioning of the C-terminal extension and likely the compaction of the receptor, confirming the results observed in limited proteolysis experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this work, we have evaluated the functional consequences of three hMR point mutations found in autosomal dominant PHA1. These residues lie in the DBD and the LBD and are conserved among the human MR, glucocorticoid (GR), androgen (AR) and progesterone receptor (PR). G633 is located at the end of the DNA recognition helix of the DBD between the two zinc fingers. Substitution of glycine by arginine did not affect ligand binding; however, maximal hormone-induced transactivation was reduced both from an MMTV promoter and a compound promoter containing two consensus GREs upstream of a TATA box (7, 8). Interestingly, whereas R633 bound a consensus GRE oligonucleotide with an affinity comparable to wild-type hMR, its association with DNA was modified; its strongly reduced dissociation rate indicates that the receptor is more tightly bound to DNA. This is in agreement with the three-dimensional model of interaction of R633 with a response element. Indeed, the model revealed that an additional hydrogen bond can be formed between the R633 side chain and a phosphate of the DNA main chain. Such a hydrogen bond could stabilize the DNA-DBD complex, thereby reducing its interaction kinetics.

Several lines of evidence suggest that dynamic interactions within the nucleus and steroid receptor recycling are essential for full transcriptional response (33, 34, 40). Receptor movement in the nucleus is highly dynamic, with the receptors undergoing constant exchange between genomic regulatory elements, multiprotein complexes with other transcription factor partners, and subnuclear structures. Indeed, recent studies have shown early and late events in hormone-regulated gene transcription (41). Agonist-bound estrogen receptor (ER) and recruited coactivators show a biphasic association with chromatin at an estrogen-responsive promoter during chronic hormone exposure. ER and coactivators recruited to the promoter within 15–30 min of hormone treatment are released after an additional 60-min hormone exposure and are reassembled onto the promoter for an additional 30–45 min. Thus, receptors and coactivators cycle continuously on the promoter during persistent hormone exposure. Interestingly, the coactivator complex formed after recycling does not contain the same components as during transcription initiation. Furthermore, experiments using different photobleaching (34) and ultrafast laser cross-linking (40) techniques have shown that steroid receptors interact dynamically with specific sequences within chromatin templates. Response elements on target promoters are occupied in a transient way, after a "hit-and-run" model of interaction, providing evidence of a continuous exchange of the receptor between chromatin and the nucleoplasmic compartment. In this context, our data concerning the interaction of R633 with DNA suggest that altered association/dissociation dynamics on a hormone-responsive element could be responsible for the reduced maximal transactivation capacity observed with this mutant.

Steroid hormone receptors are shuttling proteins that circulate rapidly between the nucleus and the cytoplasm (42, 43). In the absence of hormone, MR has been shown to be both nuclear and cytoplasmic in many cell types, although it is excluded from the nucleoli. After hormone exposure, the remaining cytoplasmic MR accumulates in the nucleus, and an alteration in nuclear organization is visible: receptors form distinct clusters, each containing approximately 10–50 MR molecules that remain excluded from the nucleolus (38). Cluster formation requires either N- or C-terminal sequences to be present and was suggested to represent higher order receptor complexes organized on hormone-responsive element multimers (44). In our experiments, R633 was predominantly localized to the nucleus in the absence of hormone, both in transient and in stable transfections. Computer analysis using the PredictNLS server (45) indicated that mutation G633R does not introduce a novel nuclear localization signal into the hMR protein. Interestingly, DNA binding has been shown to facilitate the concentration of liganded GR in the nucleus by decreasing the pool of GR available for export (46). Cytoplasmic unliganded GRs were shown to be in a dynamic equilibrium between nucleus and cytoplasm, although this equilibrium is expected to be in favor of a cytoplasmic localization (47). By analogy, it is likely that unliganded MR may also constitutively shuttle between nucleus and cytoplasm, being in a dynamic equilibrium between the two compartments. Altered interaction of R633 with DNA may promote retention of R633 in the nucleus, favoring a nuclear localization of the shuttling receptor. Alternatively, abnormal nuclear localization may be due to disruption of a nuclear export signal. Whereas nuclear export of PR has been related to a nuclear localization signal localized to the DBD and the hinge region of the receptor (48), the steroid receptor DBD has been shown to act as nuclear export signal for different nuclear receptors, including GR, AR, thyroid hormone receptor, and vitamin D receptor (49). The important residues involved in this function are two phenylalanines lying between the two zinc fingers of the DBD. However, mutation of the glycine residue corresponding to G633, together with the neighboring glutamine, had no effect on the export activity of the GR DBD. Further experiments are required to elucidate whether glycine 633 is involved in nuclear export of hMR. Interestingly, as observed for the estrogen receptor {alpha} (50), mutant R633 was distributed uniformly throughout the nucleus in the absence of ligand, whereas in hormone-treated cells, the receptor adopts a focal distribution. Because cluster formation has been correlated to the activated state of MR (44), our results suggest that, although differently localized in the absence of hormone, R633 undergoes normal activation upon hormone binding.

Amino acids Q776 and L979 lie within the LBD of hMR. Residue Q776 is located in helix H3 at the extremity of the hydrophobic ligand-binding pocket. Using a hMR homology model, based either on the human retinoic acid receptor-{gamma} (22) or human PR crystal structure (24, 51) it has been shown that 19 residues line the hMR ligand-binding pocket. Thirteen residues contribute to the hydrophobic nature of the cavity, whereas five polar residues are located at the two ends. Q776, which is located on one end of the cavity, is essential for ligand docking by anchoring the C3-ketone group of the steroid. Previous studies have shown that mutation of Q776 to alanine resulted in a 13-fold reduction in ligand-binding affinity for aldosterone and a shift in the ED50 of transactivation on an MMTV promoter of 4 orders of magnitude toward higher hormone concentrations (22). Accordingly, we have previously shown a 6-fold reduced binding affinity of R776 for aldosterone and reduction in the ED50 of aldosterone-induced transactivation of at least 2 orders of magnitude (7). Interestingly, whereas mutation Q776R affected aldosterone- and cortisoldependent transactivation in a comparable way, mutation of the same residue to alanine completely abolished transcriptional activation by cortisol (22). Indeed, due to the presence of two free hydroxyl groups at the C11 and C17 positions, cortisol is not nicely accommodated into the hMR ligand-binding pocket as compared with aldosterone (51). The Q776A substitution drastically reduces the occupied volume at the vicinity of the ligand. Such a modification may induce a local compaction of the domain, resulting in a smaller ligand cavity unable to accommodate cortisol. In contrast, the Q776R mutation still presents a side chain near the ligand and could therefore maintain the volume of the ligand-binding cavity unchanged, thereby allowing a correct accommodation of cortisol.

The LBD of hMR contains two ß-structures that participate in ligand docking. The second one consists of one strand located between helices H8 and H9 and the other in the middle of the C-terminal extension of the LBD. This ß-sheet, which is also observed in the crystal structures of PR (17), AR (18, 19), and GR (20, 21) was suggested to be important in stabilizing the positioning of the C-terminal extension of the LBD in a ligand-competent state. Accordingly, it has been shown that deletion of the last four amino acids of the hMR completely abolished aldosterone binding, although the interaction with heat shock protein 90 was not affected (35). Limited proteolysis analysis has shown that deletions at the C-terminal part of hMR severely affect receptor conformation. Accordingly, in our study the mutation of leucine 979 to proline drastically affects receptor compaction. These results were also confirmed by studies investigating the role of the extreme C-terminal end of the hAR in ligand-binding function. Indeed, mutation of I914 of hAR, which corresponds to L979 of hMR, resulted in a 5-fold reduction in ligand-binding affinity and a shift of the ED50 of ligand-dependent transactivation of 3 orders of magnitude toward higher hormone concentrations (52). Limited proteolytic digestions showed that, in the presence of high hormone concentrations, mutant I914A was able to bind agonists but induced a conformational change corresponding to the inactive conformation of the receptor. Indeed, a correct ligand-binding conformation of hAR requires the correct positioning of the extreme C-terminal end and the presence of hydrophobic residues, including I914, favoring hydrophobic interactions in this area. Structural analysis of the C-terminal extension of hMR showed that L979 is part of the ß-sheet establishing a hydrogen bond with S879. Substitution of a proline for L979 not only eliminates this hydrogen bond but also introduces a steric hindrance with the second ß-strand. Thus, the presence of a proline at this position, which destabilizes the interaction between both ß-strands, destroys the secondary structure and alters the LBD conformation, thus destroying the ligand binding-competent conformation of the unliganded receptor.

Altogether our results underscore the importance of investigating naturally occurring hMR mutations to detect important residues involved in receptor function. This may allow not only investigation of structure-function relationships, but also detection of interactions with other signaling pathways, opening interesting perspectives for steroid receptor biology and the development of new therapeutic strategies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Site-Directed Mutagenesis
Each mutation was created using the QuikChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA) on the recombinant pcDNA3-hMR plasmid containing a 3-kb hMR XmaIII-AflII fragment corresponding to the published hMR sequence (16) inserted into pcDNA3 (Invitrogen, Paisley, Scotland, UK) (53). The following sense primers were used together with their corresponding antisense oligonucleotides:

G2119A-S 5'-CTTCAAAAGAGCAGTGGAAAGGCAACACAACTATTTATG-3'

A2549G-S 5'-CGCTTAGCAGGCAAACGGATGATCCAAGTCGTG-3'

T3158C-S 5'-GGGAACGCCAAGCCGCCCTACTTCCACCGGAAG-3'

The desired mutations were identified by direct sequencing. After identification of mutated clones, these were entirely sequenced to check for the absence of random mutations. hMR fragments were subsequently excised with HindIII and NotI and subcloned into a new pcDNA3 expression vector.

For chimeric EGFP-hMR proteins, wild-type and mutated hMRs were excised from the original pcDNA vector by KpnI and ApaI and inserted into the corresponding restriction sites of the pEGFP-C1 vector (CLONTECH, Palo Alto, CA).

Cell Culture and Transfection Procedures
Rabbit RCSV3 cells derived from kidney cortical collecting duct (54) were kindly provided by Dr. P. Ronco (Hôpital Tenon, Paris). Cells were grown in a defined medium composed of DMEM-Ham’s F12 supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin, 20 mM HEPES, 50 nM sodium selenate, 50 nM dexamethasone, and 2% charcoal-stripped fetal calf serum. The cells were seeded in six-well plates at a density of 3 x105 cells per well at least 6 h before transfection in fresh medium without any added steroid. For all transfection experiments, RCSV3 cells were used between passages 30 and 40.

Cells were transfected by the calcium phosphate method with 0.3 µg of plasmids pcDNA3-hMR, pcDNA3-R633, pcDNA3-R776, and pcDNA3-P979, coding for the wild-type and mutated hMRs, in the presence of 0.8 µg of a mouse mammary tumor virus (MMTV)-luciferase reporter construct (pF31luc, gift of Dr. H. Richard-Foy), as previously described (31). Cotransfection of 0.16 µg pSVßgal (CLONTECH), a plasmid encoding for ß-galactosidase, was performed to normalize for transfection efficiencies. The day after transfection, cells were rinsed with PBS and steroids were added for 24 h. The cells were rinsed twice with cold PBS and lysed in 25 mM glycyl-glycine, pH 7.8; 1 mM EDTA; 1 mM dithiothreitol; 8 mM MgSO4; 1% Triton X100; 15% glycerol. Cellular extracts were assayed for luciferase and ß-galactosidase activities (31). Results were standardized for transfection efficiency and expressed as the ratio of luciferase activity over ß-galactosidase activity in arbitrary units. Aldosterone and cortisol were purchased from Sigma (St. Louis, MO).

For transient transfection experiments investigating chimeric EGFP-hMR receptors, cells were seeded in six-well plates at a density of 4 x 104 cells per well 12 h before transfection in fresh medium without any added steroid. The next day, cells were transfected using Lipofectamine (Invitrogen, Paisley, Scotland) in serum-free Opti-MEM medium (Invitrogen). After 5 h, Opti-MEM was replaced by fresh medium without steroids. On d 3, cells were incubated with 10 nM aldosterone for various periods of time (0–60 min) and observed on an inverted DM IRB microscope (Leica Corp., Rueil-Malmaison, France) using an N2.1 filter (515- to 560-nm band-pass filter).

For stable transfections, the human collecting duct cell line H5 (55), provided by Dr. P. Ronco, was stably transfected using Lipofectamine as described above with plasmid pEGFP-C1-R633 coding for a chimeric protein consisting of EGFP N terminal of mutant hMR R633. Cells were grown as RCSV3 cells. Clones were selected with 200 µg/ml geneticin G418 (Invitrogen). Four different clones were isolated. For subsequent experiments, clone 8_12 was used. The stable H5 cell line expressing wild-type hMR was described previously (37). For the study of intracellular localization of fluorescent proteins, cells were seeded at a density of 60,000 cells in self-enclosed chambered cover slips (Lab-Tek, Nalge Nunc International, Naperville, IL) and grown in medium without any added steroid. Experiments were done three times in triplicate. Fluorescence was observed by confocal microscopy before and after addition of 10 nM aldosterone. Cells were kept at 37 C in the presence of 5% CO2 and imaged on a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY). EGFP was excited with the 488-nm line from an argon laser. For quantification, cells were counted and classified into three different categories: C for exclusive cytoplasmic staining, NC when nuclear and cytoplasmic staining were of comparable intensities, and N for unique nuclear staining.

In vitro Transcription and Translation
In vitro transcription and translation were accomplished using the TNT Quick Coupled Transcription/Translation system (Promega Corp., Madison, WI) following the manufacturer’s protocol. Recombinant plasmids coding for wild-type or mutated hMR were used as a template for transcription with T7 polymerase followed by translation with [35S]methionine (1000 Ci/mmol, Amersham, Les Ulis, France). Cold methionine was used for translation of proteins used in EMSAs.

Limited Proteolytic Digestion
Wild-type hMR and mutant hMRP979 (1 µg) were synthesized in vitro in the presence of [35S]methionine. One seventh of the reaction volume was incubated with increasing concentrations of trypsin (15–90 µg/ml) for 10 min at 20 C. After addition of protein loading buffer, aliquots of the digestion product (10 µl) were denatured for 5 min at 95 C and analyzed by migration on a 12% SDS-PAGE gel. Gels were fixed in 10% acetic acid/30% methanol, soaked in Amplifiy solution (Amersham), dried, and exposed to autoradiography for 48 h.

EMSAs
For EMSA, 1 µg recombinant wild-type or mutant hMR was in vitro translated in the presence of cold methionine, as described above. Aliquots of translated product were used in gel mobility shift assays, which were performed essentially as described elsewhere (31). Purified oligonucleotides were annealed and labeled with [{alpha}32P]dCTP (Amersham Pharmacia Biotech, Little Chalfont, UK) using the Klenow fragment of DNA polymerase (United States Biochemical Corp., Cleveland, OH) to a specific activity of approximately 108 cpm/µg of DNA; 20,000 cpm were used for each experiment. Unlabeled oligonucleotides were used as competitors. Oligonucleotides used in the gel mobility shift experiments are as follows:

GREcon: 5'-AGCTGCTCAGCTAGAACACTCTGTTCTCTACT-3' and 5'-AGCTAGTAGAGAACAGAGTGTTCTAGCTAGC-3'

Protein-DNA complexes were allowed to interact for 15 min and were separated from free DNA by electrophoresis on nondenaturing 4.5% polyacrylamide gel in 0.25x Tris-borate EDTA buffer at 200 V for 1 h. Gels were dried and exposed to x-ray film at –80 C. For quantification, receptor-DNA complexes were analyzed on an InstantImager (Packard Instruments, Meriden, CT).

Model Building
The DBDs of the hMR and hGR are characterized by a sequence identity of 98% corresponding to three divergent residues. The three-dimensional model of the hMR-DBD was generated by substituting in the O package (56) the residues Y497, R498, and E508 of the crystal structure of the hGR-DBD (protein data bank code 1GLU, chain A) by those of the hMR (L660, Q661, and G671). The G633R mutation was generated using the same procedure. The mutations Q776R and L979P were introduced in the previously described hMR-LBD homology model (23) in a way similar to that developed for the DBD. For all substituted residues, the orientation of the side chain was adjusted by using the rotamer library of the O package.


   ACKNOWLEDGMENTS
 
We thank Dr. P. Ronco for the gift of RCSV3 cells and Dr. M.-E. Oblin for helpful discussion.


   FOOTNOTES
 
This work was supported by the Institut National de la Santé et de la Recherche Médicale and by Ministero dell’Istruzione, dell’Università e della Ricerca 40% 1999.

Present address for P.S.: Department of Medical and Surgical Sciences, Division of Endocrinology, Via Ospedale 105, 35100 Padova, Italy.

Abbreviations: AR, Androgen receptor; DBD, DNA-binding domain; EGFP, enhanced green fluorescent protein; ER, estrogen receptor; GR, glucocorticoid receptor; GRE, glucocorticoid-responsive element; LBD, ligand-binding domain; MMTV, mouse mammary tumor virus; MR, mineralocorticoid receptor; PHA1, type I pseudohypoaldosteronism; PR, progesterone receptor.

Received for publication October 21, 2003. Accepted for publication June 1, 2004.


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 DISCUSSION
 MATERIALS AND METHODS
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