Abstract

The anti-Müllerian hormone type II (AMHRII) receptor is the primary receptor for anti-Müllerian hormone (AMH), a protein produced by Sertoli cells and responsible for the regression of the Müllerian duct in males. AMHRII is a membrane protein containing an N-terminal extracellular domain (ECD) that binds AMH, a transmembrane domain, and an intracellular domain with serine/threonine kinase activity. Mutations in the AMHRII gene lead to persistent Müllerian duct syndrome in human males. In this paper, we have investigated the effects of 10 AMHRII mutations, namely 4 mutations in the ECD and 6 in the intracellular domain. Molecular models of the extra- and intracellular domains are presented and provide insight into how the structure and function of eight of the mutant receptors, which are still expressed at the cell surface, are affected by their mutations. Interestingly, two soluble receptors truncated upstream of the transmembrane domain are not secreted, unless the transforming growth factor beta type II receptor signal sequence is substituted for the endogenous one. This shows that the AMHRII signal sequence is defective and suggests that AMHRII uses its transmembrane domain instead of its signal sequence to translocate to the endoplasmic reticulum, a characteristic of type III membrane proteins.

INTRODUCTION

Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance, is produced by Sertoli cells at the initiation of testicular differentiation and is responsible for the regression of Müllerian ducts, an early sign of phenotypic differentiation in the male fetus. Like other members of the transforming growth factor-β (TGF-β) family, AMH signals through two membrane-bound serine/threonine kinases and the Smad effectors. The primary receptor, paradoxically called receptor type II (AMHRII—anti-Müllerian hormone type II receptor), was cloned in 1994 (1,2). It binds AMH and then activates a type I receptor. This leads to the phosphorylation of an R-Smad protein, its interaction with Smad4 and the translocation of the R-Smad/Smad4 complex to the nucleus where it can promote the transcription of target genes. AMH shares its type I receptors, ALK2, ALK3 and ALK6 (3–7), and its R-Smad effectors, Smad1, Smad5 and Smad8 (3) with the bone morphogenetic proteins (BMPs).

In humans, mutations of the AMH and AMHRII genes lead to a rare form of intersex disorder, the persistent Müllerian duct syndrome (PMDS), characterized by the persistence of Müllerian derivatives, uterus, Fallopian tubes and upper part of the vagina, in otherwise normally virilized genetic males (8). The condition is discovered at surgery prompted by cryptorchidism and/or inguinal hernia. PMDS is transmitted according to a recessive autosomal pattern. In our group, the molecular study of 113 families has shown that 50 have a mutation of the AMH gene and 48 have a mutation of AMHRII gene; the origin of the syndrome remains unknown in 15 families. Serum AMH concentrations are usually low or undetectable in patients with AMH gene mutations and normal in those with receptor mutations. Serum AMH levels are informative only in prepubertal children, since serum AMH levels fall under the influence of testosterone produced at sexual maturation (9). AMH mutations are usually found in patients of Mediterranean or Arab origin, whereas those of AMHRII are more often found in patients from northern Europe or the USA.

The AMHRII gene contains 11 exons spread over 8 kb and maps to chromosome 12q13 (10). The first three exons encode the extracellular domain (ECD) which binds the ligand, the fourth exon encodes the single transmembrane domain and the last seven exons encode the intracellular serine/threonine kinase domain. AMHRII is usually classified as a type I membrane protein (11) because of the orientation of its N-terminus to the outside of the cell and the presence of a signal sequence, thought to be responsible for translocation to the endoplasmic reticulum (ER). Up to 2009, 38 different mutations of AMHRII have been identified in our PMDS patients, including missense and splice mutations, insertions and deletions found along the entire length of the gene. One mutation in exon 10, del6331–6357, is extremely frequent (12), occurring in the homozygous or heterozygous condition, in 53% of our patients with receptor mutations.

Of these many AMHRII mutations, only three have been studied in detail. One is a splice mutation that destroys the invariant nucleotide at the 5′ end of the second intron and generates two abnormal mRNAs: one missing the second exon and the other incorporating the first 12 bases of the second intron (10). Both short and long mutants are unable to reach the cell surface and thus do not bind AMH when expressed in COS cells, because they are retained in the ER (13). The other two are mutations of the kinase domain, and their dominant negative effect has been investigated in an AMH-responsive cell line. One is a deletion of a single base leading to a stop codon, causing receptor truncation after the transmembrane domain (del1692), and the other is a missense mutation in the putative substrate-binding site of the kinase domain (Arg406Gln). Like similar receptors of the TGF-β family, these AMHRII mutant receptors are dominant negative in vitro when overexpressed (14). However, when these mutations occur in vivo, only the siblings carrying mutations on both alleles are affected by PMDS, because in clinical conditions, as opposed to in vitro situations, mutant and wild-type (WT) genes are presumably expressed at a one-to-one ratio.

In this paper, we have investigated the effects of 10 mutations identified in patients with PMDS, including del6331–6357, Arg406Gln and del1692, on AMHRII biosynthesis, structure and function. Eight of the mutant receptors are expressed at the cell surface, but either cannot bind AMH or are incompetent at transducing an AMH signal. Molecular models of the extracellular and the intracellular domains are presented, which provide insight into how the mutations could affect structure and function. Interestingly, two soluble receptors truncated upstream of the transmembrane domain are not secreted, unless the TGF-β type II receptor signal sequence is substituted for the endogenous one. This shows that the AMHRII signal sequence is not functional and suggests that AMHRII uses its transmembrane domain instead of its signal sequence to translocate to the ER, a characteristic of type III membrane proteins.

RESULTS

Expression of mutant receptors

Four mutations affecting the ECD were studied: two missense mutations Arg54Cys and Gly142Val, and Arg80Stop and Arg97Stop, which give rise to receptors lacking the transmembrane domain (Fig. 1). We also chose six mutations affecting the kinase domain: del1692, Arg406Gln, Asp426Gly, Asp491His, Arg504Cys and the del6331–6357 mutation, which is extremely frequent. These mutations were reproduced by site-directed mutagenesis of the WT AMHRII human cDNA and tagged with the hemagglutinin (HA) epitope of the influenza virus. We also generated two artificial mutant receptors: Ile145Stop, which represents the entire ECD of AMHRII, and Lys230Arg whose ATP-binding site is destroyed. The mutated cDNAs were transiently transfected in COS cells, and their expression was studied by western blotting using an anti-HA monoclonal antibody (mAb). All the mutant receptors are detected 3 days after the transfection in cell lysates at the expected size (Fig. 2A and B), although a longer exposure is needed to detect Arg80Stop, Arg97Stop and Ile145Stop. These truncated receptors are also detected after in vitro translation (Fig. 2C). Attempts to detect them in the culture medium by western blotting or immunoprecipitation were unsuccessful (not shown), indicating that they are not secreted as would be expected for a soluble receptor and that there is a problem with the intracellular trafficking of these mutant proteins.

Figure 1.

Location of AMHRII mutations. The Ile145Stop artificial mutation, which truncates the receptor just upstream of the transmembrane domain, and the Lys230Arg artificial mutation, which affects the ATP-binding site, are boxed. The extracellular, transmembrane and intracellular domains are indicated. The location of regions II, VIII, IX, X and XI within the kinase domain (hatched) are also indicated.

Figure 1.

Location of AMHRII mutations. The Ile145Stop artificial mutation, which truncates the receptor just upstream of the transmembrane domain, and the Lys230Arg artificial mutation, which affects the ATP-binding site, are boxed. The extracellular, transmembrane and intracellular domains are indicated. The location of regions II, VIII, IX, X and XI within the kinase domain (hatched) are also indicated.

Figure 2.

Synthesis of mutant AMHRII proteins. (A) Western blotting analysis of cell lysates of COS cells transiently transfected with mutant AMHRII cDNAs. Three days after transfection, western blot analysis of cell lysates was performed with a 7.5% SDS–PAGE and a monoclonal anti-HA antibody. (B) Western blotting analysis of receptors truncated upstream of the transmembrane domain. COS cell lysates were analyzed using a 4–20% SDS–PAGE. (C) Analysis of in vitro translation products produced by truncated receptors. In vitro translation products were analyzed using a 4–20% SDS–PAGE.

Figure 2.

Synthesis of mutant AMHRII proteins. (A) Western blotting analysis of cell lysates of COS cells transiently transfected with mutant AMHRII cDNAs. Three days after transfection, western blot analysis of cell lysates was performed with a 7.5% SDS–PAGE and a monoclonal anti-HA antibody. (B) Western blotting analysis of receptors truncated upstream of the transmembrane domain. COS cell lysates were analyzed using a 4–20% SDS–PAGE. (C) Analysis of in vitro translation products produced by truncated receptors. In vitro translation products were analyzed using a 4–20% SDS–PAGE.

Cell-surface expression and ligand binding of mutant receptors

The cell-surface expression of mutant receptors was studied by biotinylation of membrane proteins after the transfection of mutated cDNAs into COS cells. Cell lysates were immunoprecipitated with an anti-HA mAb, followed by western blotting with streptavidin conjugated to horseradish peroxidase. All mutant receptors tested in Figure 3A are expressed at the cell surface, including the del1692 mutant, which migrates with an apparent molecular weight of 30 kDa. We then examined their ability to bind AMH. Transfected COS cells were incubated with AMH, followed by a rabbit anti-AMH antibody and then a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG antibody (Fig. 3B). Results are shown for two mutant receptors: del6331–6357, which is comparable to WT AMHRII in its ability to bind AMH, and Arg54Cys, which does not bind AMH. All other mutant receptors (i.e. those tested in Fig. 3A) showed binding similar to WT AMHRII (data not shown). No binding of AMH was seen with mock-transfected cells (Fig. 3B).

Figure 3.

Expression at the cell surface and AMH binding of mutant receptors. (A) Cell-surface expression of mutant AMHRII in COS cells. Two days after the transfection, COS cells were biotinylated, immunoprecipitated with an anti-HA antibody, resolved onto a 4–20% SDS–PAGE, transferred to a nitrocellulose membrane and probed with horseradish peroxydase-conjugated streptavidin as described in Materials and Methods. All mutant receptors were expressed at the cell-surface membrane. (B) AMH binding. Three days after transfection with mutant receptors, COS cells were successively incubated for 4 h with 3.5 nm human recombinant AMH, 2 h with 3 µg/ml of a rabbit polyclonal anti-AMH antibody and 1 h with an FITC-conjugated goat antibody raised against IgG. Then cells were fixed and examined under microscope. All mutant receptors (green) bind AMH, except Arg54Cys.

Figure 3.

Expression at the cell surface and AMH binding of mutant receptors. (A) Cell-surface expression of mutant AMHRII in COS cells. Two days after the transfection, COS cells were biotinylated, immunoprecipitated with an anti-HA antibody, resolved onto a 4–20% SDS–PAGE, transferred to a nitrocellulose membrane and probed with horseradish peroxydase-conjugated streptavidin as described in Materials and Methods. All mutant receptors were expressed at the cell-surface membrane. (B) AMH binding. Three days after transfection with mutant receptors, COS cells were successively incubated for 4 h with 3.5 nm human recombinant AMH, 2 h with 3 µg/ml of a rabbit polyclonal anti-AMH antibody and 1 h with an FITC-conjugated goat antibody raised against IgG. Then cells were fixed and examined under microscope. All mutant receptors (green) bind AMH, except Arg54Cys.

Bioactivity of mutant receptors

To investigate whether these receptors mediate the effect of AMH upon target genes, we transfected them into P19 cells which have all the components needed for AMH signal transduction except its type II receptor and tested the ability of AMH to stimulate an AMH reporter gene. Gouédard et al. (3) have previously shown that P19 cells are responsive to AMH only after the transfection of a normal AMHRII gene. Here, we co-transfected P19 cells with a Smad1-Gal4 fusion protein (i.e. the N-terminus of Smad1 linked to the DNA-binding domain of the Gal4 transcription factor), a luciferase reporter gene under the control of a Gal4-dependent promoter and mutant receptors. After AMH treatment, the Smad1-Gal4 fusion protein is phosphorylated and translocated to the nucleus, where it activates the Gal4-luc reporter gene (4). Results are shown in Figure 4. None of the mutant receptors was able to mediate AMH activation of Smad1, except Gly142Val. However, the stimulation of the reporter gene for cells transfected with Gly142Val (48.15 ± 3.30%, n = 4) was significantly lower (P < 0.05) than for cells transfected with the WT receptor (73.78 ± 5.60%, n = 3).

Figure 4.

Ability of mutant AMHRII to mediate AMH activation of Smad1. P19 cells were co-transfected with the different AMHRII cDNAs, Gal4-Smad1, Gal4-luc and pRLTK vectors and treated with 10 µg/ml AMH for 24 h. Lysates were assayed for firefly and Renilla luciferase activity. Each experiment was done in triplicate, and the results were expressed as a percentage of stimulation of the reporter gene in the presence of AMH. AMH stimulated Gal4-Smad1 reporter gene when WT AMHRII was transfected in P19 cells (P < 0.001). Only Gly142Val was able to mediate this AMH effect (P < 0.001), although with a lower efficiency (P < 0.05) than WT AMHRII.

Figure 4.

Ability of mutant AMHRII to mediate AMH activation of Smad1. P19 cells were co-transfected with the different AMHRII cDNAs, Gal4-Smad1, Gal4-luc and pRLTK vectors and treated with 10 µg/ml AMH for 24 h. Lysates were assayed for firefly and Renilla luciferase activity. Each experiment was done in triplicate, and the results were expressed as a percentage of stimulation of the reporter gene in the presence of AMH. AMH stimulated Gal4-Smad1 reporter gene when WT AMHRII was transfected in P19 cells (P < 0.001). Only Gly142Val was able to mediate this AMH effect (P < 0.001), although with a lower efficiency (P < 0.05) than WT AMHRII.

Receptors truncated upstream of the transmembrane domain are not stable

In order to investigate the nature of the trafficking defect of the three truncated receptors (Arg80Stop, Arg97Stop and Ile145Stop), we first compared mRNA levels of the mutant receptors 24, 48 and 72 h after transfection (Fig. 5A). The level of mRNA is highest 48 h after transfection and is similar for the three mutant receptors and the WT receptor, indicating that the low expression of these mutant receptors in cell lysates is not due to a lower expression of their mRNAs. Pulse chase experiments were then performed to test the stability of mutant receptors (Fig. 5B). In contrast to WT receptor, the three mutant receptors are barely detectable after a 30 min chase and undetectable at longer chase times. No labeled protein is found in the culture medium, providing further evidence that the truncated receptors are not secreted despite the lack of a transmembrane domain. Confocal microscopy was used to localize truncated receptors inside the cells (Fig. 5C). The number of cells that are stained is much lower for Ile145Stop than for WT, confirming that it is unstable. There is also a difference in their staining patterns, suggesting that they are in different subcellular compartments. Finally, COS cells were treated with a proteasome inhibitor, clasto-lactacystin, to determine whether mutant receptors were degraded by a proteasome-mediated process. As expected, there was no effect of this treatment on the level of WT receptor, but the amount of mutant receptors detected in cell lysates was higher in the presence of clasto-lactacystin (Fig. 5D), indicating that they are degraded by the proteasome complex.

Figure 5.

Instability of receptors truncated upstream of the transmembrane domain. (A) Mutant AMHRII mRNA expression. Total RNAs were extracted 24, 48 and 72 h after the transfection of COS cells. Northern blot analysis was performed on 5 µg of total RNA using an AMHRII probe. Blots were quantified by phosphorimaging and normalized with a ribosomal probe. The kinetics mRNA expression was comparable for mutant and WT receptors. (B) Stability of Arg80Stop, Arg97Stop, Ile145Stop and WT receptors in COS cells. Three days after transfection, cells were metabolically labeled with [35S]Met/Cys for 30 min and then cultured for various period of time in cold medium. After precipitation of cell lysates and media with an anti-HA antibody, immunoprecipitates were resolved onto 4–20% SDS–PAGE and analyzed by autoradiography. Exposure was for 4 days for WT AMHRII and 10 days for mutant receptors. Mutant receptors remain visible in cell lysates only 1 h after the beginning of the pulse, whereas WT AMHRII is stable for 6 h. No receptors were detectable in cell medium. (C) Localization of mutant receptors in COS cells by confocal microscopy. Three days after transfection, COS cells were co-labeled with anti-HA antibody and FITC anti-mouse IgG for the detection of AMHRII (green) and rhodamine 6G for labeling the ER (red). The number of labeled cells is much lower and the staining is less homogeneous for Ile145Stop than for WT. (D) Degradation of mutant AMHRII by the proteasome. Two days after transfection, cells were treated for 24 h with 10 µm of clasto-lactacystin. Cell lysates were resolved onto 4–20% SDS–PAGE and analyzed by western blotting with an anti-HA antibody. The amount of truncated receptors was enhanced when cells were treated with clasto-lactacystin.

Figure 5.

Instability of receptors truncated upstream of the transmembrane domain. (A) Mutant AMHRII mRNA expression. Total RNAs were extracted 24, 48 and 72 h after the transfection of COS cells. Northern blot analysis was performed on 5 µg of total RNA using an AMHRII probe. Blots were quantified by phosphorimaging and normalized with a ribosomal probe. The kinetics mRNA expression was comparable for mutant and WT receptors. (B) Stability of Arg80Stop, Arg97Stop, Ile145Stop and WT receptors in COS cells. Three days after transfection, cells were metabolically labeled with [35S]Met/Cys for 30 min and then cultured for various period of time in cold medium. After precipitation of cell lysates and media with an anti-HA antibody, immunoprecipitates were resolved onto 4–20% SDS–PAGE and analyzed by autoradiography. Exposure was for 4 days for WT AMHRII and 10 days for mutant receptors. Mutant receptors remain visible in cell lysates only 1 h after the beginning of the pulse, whereas WT AMHRII is stable for 6 h. No receptors were detectable in cell medium. (C) Localization of mutant receptors in COS cells by confocal microscopy. Three days after transfection, COS cells were co-labeled with anti-HA antibody and FITC anti-mouse IgG for the detection of AMHRII (green) and rhodamine 6G for labeling the ER (red). The number of labeled cells is much lower and the staining is less homogeneous for Ile145Stop than for WT. (D) Degradation of mutant AMHRII by the proteasome. Two days after transfection, cells were treated for 24 h with 10 µm of clasto-lactacystin. Cell lysates were resolved onto 4–20% SDS–PAGE and analyzed by western blotting with an anti-HA antibody. The amount of truncated receptors was enhanced when cells were treated with clasto-lactacystin.

The truncated receptors are not secreted because of a defective signal peptide

The lack of secretion of the truncated receptors may be due to a folding problem within the ER or caused by a defect in translocating to the ER, a process that requires a functional signal sequence. Although a comparison of the human AMHRII signal sequence to the TGF-β type II receptor (TβRII) signal sequence (Fig. 6A) does not reveal any obvious defect, we considered it a possibility, since previous attempts to make a soluble receptor Fc fusion protein, in which the ECD of AMHRII (human or rabbit) was fused to the Fc region of human IgG1, had also failed. As shown in Figure 6B, an AMHRII-ECD-Fc fusion protein with the endogenous signal sequence failed to be secreted, although it was detectable in the cell lysate. However, when we substituted the signal sequence of TβRII for the signal sequence of AMHRII, the fusion protein was detected in the culture medium (Fig. 6B). These results strongly suggest that the truncated AMH receptors are not secreted into the ER because the AMHRII signal peptide is not functional. The implications these findings have for the translocation of WT AMHRII into the ER are considered in Discussion.

Figure 6.

Lack of secretion of receptors truncated upstream of the transmembrane domain is due to AMHRII signal peptide. (A) Comparison of human AMHRII and TβRII signal peptides. (B) Secretion in the culture medium of an AMHRII-ECD-Fc fusion protein with the signal sequence of TβRII. Two days after transfection of cDNAs coding for the ECD of AMHRII fused to the Fc region of human IgG1 with either the endogenous signal sequence of AMHRII (AMHRII-ECD-Fc) or the signal sequence of TβRII (TβRII-AMHRII-ECD-Fc), COS cells lysates and culture media were analyzed by western blotting with an anti-Fc antibody. Only TβRII-AMHRII-ECD-Fc was detected in the culture medium.

Figure 6.

Lack of secretion of receptors truncated upstream of the transmembrane domain is due to AMHRII signal peptide. (A) Comparison of human AMHRII and TβRII signal peptides. (B) Secretion in the culture medium of an AMHRII-ECD-Fc fusion protein with the signal sequence of TβRII. Two days after transfection of cDNAs coding for the ECD of AMHRII fused to the Fc region of human IgG1 with either the endogenous signal sequence of AMHRII (AMHRII-ECD-Fc) or the signal sequence of TβRII (TβRII-AMHRII-ECD-Fc), COS cells lysates and culture media were analyzed by western blotting with an anti-Fc antibody. Only TβRII-AMHRII-ECD-Fc was detected in the culture medium.

Molecular models of the AMHRII extracellular and intracellular domains

In order to investigate the effect of the AMHRII mutations at the molecular level, three-dimensional models of the extracellular and intracellular domains of AMHRII were built by homology modeling. The model of the AMHRII ECD was constructed using crystal structures of three type II receptors of TGF-β family members as templates: activin type II receptor (ActRII), TβRII and bone morphogenetic proteins type II receptor (BMPRII) (Fig. 7A). The low-sequence identity (<20%) between the AMHRII ECD and these templates makes the homology-modeling process somewhat challenging. In particular, the region between the β1 and β2 strands and the region between the β5 and β6 strands are not well conserved. In the first region (from Glu28 to Leu49), AMHRII presents a longer sequence than any of the other receptors, and the templates display different structural features (simple helix for ActRII, double helix for BMPRII and β-sheet for TβRII). For AMHRII, secondary structure prediction programs suggest a small β-strand between Gly40 and Leu43, but our attempt to generate a stable model with such a structural feature was unsuccessful. The second region between the β5 and β6 strands shows a longer sequence for AMHRII than for both ActRII and BMPRII, but shorter than for TβRII. Secondary structure prediction programs predict a coil for this region in AMHRII. Overall, ActRII is the best template based on sequence identity, but an insert within the M-loop of ActRII makes it a poor template for this region. BMPRII, on the other hand, has no insert and is better conserved in this region. Therefore, there is no clear best template for the modeling of AMHRII and we decided to generate our models on the basis of the three templates mentioned earlier.

Figure 7.

Sequence alignments used for homology modeling of the extracellular and intracellular domains of AMHRII. (A) Sequence alignment of the ECD of AMHRII with ActRII, BMPRII and TβRII. (B) Sequence alignment of the intracellular domain of AMHRII with ActRIIB.

Figure 7.

Sequence alignments used for homology modeling of the extracellular and intracellular domains of AMHRII. (A) Sequence alignment of the ECD of AMHRII with ActRII, BMPRII and TβRII. (B) Sequence alignment of the intracellular domain of AMHRII with ActRIIB.

In modeling the ECD of AMHRII, a large number of structures were generated in order to explore a vast conformational space and to obtain a reliable model. The quality of the models was judged both in energetic and structural terms, and the model with the best consensus between structure and energy was identified and subjected to minimization. Our best model of the AMHRII ECD reproduces the general three-finger toxin fold of type II receptors (Fig. 8A) and displays five disulfide bridges between Cys24 and Cys61, Cys55 and Cys79, Cys60 and Cys87, Cys111 and Cys116, and between Cys92 and Cys109. Four of these disulfide bridges are conserved in the other three receptors [C1–C3, C2–C4, C6–C7 and C9–C10 (numbering shown on Fig. 7A is from reference (15)]. The C5 cysteine forms a bridge with the C8 cysteine in most other proteins containing a three-finger toxin fold, but AMHRII contains no C8 cysteine. Instead, the C5 cysteine of AMHRII (Cys87) is predicted to form a disulfide bridge with Cys60. TβRII is also missing the C5–C8 disulfide bridge.

Figure 8.

Molecular modeling of AMHRII. The two upper figures show three dimensional models of AMHRII extracellular (A) and intracellular (B) domains. Residues affected by mutations and structural features are labeled. The four lower figures show portions of the intracellular domain affected by specific mutations: (C) Asp491His, (D) Arg406Gln, (E) Arg504Cys, (F) Asp426Gly. Nitrogen atoms are shown in blue and oxygen atoms in red.

Figure 8.

Molecular modeling of AMHRII. The two upper figures show three dimensional models of AMHRII extracellular (A) and intracellular (B) domains. Residues affected by mutations and structural features are labeled. The four lower figures show portions of the intracellular domain affected by specific mutations: (C) Asp491His, (D) Arg406Gln, (E) Arg504Cys, (F) Asp426Gly. Nitrogen atoms are shown in blue and oxygen atoms in red.

The three-dimensional model of the AMHRII kinase domain was built using the recently released structure of the activin type II B receptor (ActRIIB) (16) as template, which shares ∼35% sequence identity with AMHRII (Fig. 7B). The alignment requires a few insertions, the most significant one appearing in the activation loop in the region between residues 360 and 375 of AMHRII. No information is available on the structure of this region of the protein, and the secondary structure prediction program predicts only a large coil, consistent with the rich proline density of this part of the kinase. To better explore the conformational space of this region and look for alternative conformations with respect to the template, a large series of loop optimizations were performed (17).

Our best model of the AMHRII kinase domain presents the general fold of a two-domain kinase, with an N-lobe consisting mainly of a five-stranded β-sheet and a C-lobe, which is mainly α-helical. The activation loop of AMHRII differs from the one in ActRII by the presence of the 3-10 helix at the residues from 367 to 370 (Fig. 8B). Amino acids affected by the six naturally occurring mutations reported in this study are all located outside the ATP-binding site. Moreover, these mutations are mainly located at the solvent accessible part of the C-lobe region and involve the domains IX, X and XI of the kinase. Residues 444–452 (deleted in mutation del6331–6357) constitute part of the αG helix and the loop that precedes it. Arg406 is located at the N-terminal end of the αF helix, and Asp491 lies in a small loop between the αH and αI helices. Arg504 lies in the core of the αI helix, and Asp426 lies in a loop after the αF helix. The potential effects of the six mutations on the structure and function of AMHRII are discussed in what follows.

DISCUSSION

A number of diseases have now been found to be caused by mutations of type II receptors of the TGF-β family. TβRII mutations are associated with various cancers (18,19), Marfan syndrome (20,21) and Loeys–Dietz syndrome (20,22), whereas BMPRII mutations have been found in patients with familial primary pulmonary hypertension (PPH) (23–27). AMHRII mutations are responsible for approximately half of the cases of PMDS. In this article, we have studied 10 natural mutations of AMHRII, in order to understand how these mutations affect its structure, biosynthesis or function. Two of these mutations are located in the ECD, which is involved in binding AMH, six affect the cytoplasmic domain, which contains the serine/threonine kinase and is responsible for downstream signaling, and two mutations result in the deletion of both the intracellular and transmembrane domains. We have also generated models of both the extracellular and intracellular domains. In what follows, we discuss these mutations in the context of our biochemical analyses and these molecular models.

Two of the mutant receptors, Arg54Cys and Gly142Val, have amino acid substitutions in the ECD. Arg54 is located at the end of the β2 strand just prior to the M-loop (Fig. 8A). The disulfide bridge between Cys55 and Cys79 is a key component in defining the conformation of this part of the protein and is conserved in all proteins with a three-finger toxin fold structure. In addition, our model predicts that Cys60 forms a disulfide bridge with Cys87, a bridge not observed in the other type II receptors. Substitution of cysteine for Arg54 in the Arg54Cys mutant may perturb the formation of these disulfide bonds and cause the formation of alternative bridges. Changes in the disulfide bridges could lead to an altered conformation of the entire ECD or more locally of the M-loop, consistent with the observation that the Arg54Cys mutant receptor cannot bind AMH (Fig. 3B). Such an alteration of the M-loop conformation could subsequently disrupt an interaction with AMH, since the M-loop of ActRIIB is involved in direct interactions with the hormone (28–30). However, the M-loop of AMHRII is shorter than the M-loop of ActRIIB, and at present, there is no direct evidence that it can contribute to ligand binding.

No template is available for modeling the membrane proximal region of the ECD, where the Gly142Val mutation is located. By similarity with other receptors, residue 142 lies in the last section of the ECD immediately adjacent to the membrane. It is possible that the larger side chain of valine creates some steric hindrance and disturbs the contacts between these entities. Consequently, the effectiveness of this mutant receptor in transducing an AMH signal might be reduced, consistent with the results of Figure 4, showing a reduced activation of the reporter gene by this mutant receptor compared with the WT one.

All six receptors with mutations in the cytoplasmic domain were expressed on the cell membrane and could bind AMH, but were unable to activate a Smad1-dependent reporter gene. Two of the mutants contain substantial deletions. Mutant del1692 lacks the entire kinase domain and therefore is incapable of transducing an AMH signal. However, like similar receptors of the TGF-β family, it is dominant negative in vitro (14). Mutant del6331–6357 is missing residues 444–452, which are located at the top of the C-lobe in the X region of the kinase domain. This deletion should lead to a substantial conformational change in the AMHRII fold, since residues 444–452 constitute part of the αG helix and the loop that precedes it (Fig. 8B). These residues are also located immediately upstream of insert 2, a region in which TβRII is crucial for catalytic activity (31).

Two other mutations, Arg406Gln and Asp491His, affect residues that are structurally close together and are part of polar patches at the surface of the C-lobe region, which are conserved in all type II receptors. Our model of the kinase domain shows that Asp491 lies in a small loop between the αH and αI helices. Its side chain points out to the solvent and it does not interact with any neighboring residues, which include three other polar residues: Asp489, Glu493 and Arg495 (Fig. 8C). These four residues are all conserved in the other type II receptors. In TβRII, mutations of the analogous residues for Glu493 (Glu526Gln) (32) or Arg495 (Arg528His) (33) lead to loss of kinase activity. This is also true for mutations of BMPRII at the analogous residues for Asp489 (Asp485Gly) (25) and Arg495 (Arg491Trp,Gln) (27). The substitution of histidine for aspartic acid in the Asp491His AMHRII mutation may decrease the polarity of this exposed region. This polar patch also contains residue Thr497, predicted to be phosphorylated by the NetPhos 2.0 program (34). Although phosphorylation of this site has not been confirmed experimentally, Thr497 is conserved across species and in other type II receptors (it is a serine in the activin type II receptors). Finally, Asp491 immediately precedes a proline which, when mutated to a leucine in TβRII, can no longer phosphorylate the type I TGF-β receptor (35).

Arg406 is located at the N-terminus of the αF helix, where one or two consecutive polar residues are present in all type I and type II receptors (Fig. 8D). In our model, the guanidium group of Arg406 forms hydrogen bonds with the backbone oxygen of His316 and the side chain of the Glu318 on the αE helix. Substitution of glutamine for arginine in the Arg406Gln mutant may destabilize this interaction between the αE and αF helices. Because of the proximity of this region to the activation loop of the kinase, this could affect substrate recognition or kinase activity. However, one interesting aspect of this mutation has to be mentioned here. Whereas human AMHRII presents an arginine at this residue, rat AMHRII and mouse AMHRII have a glutamine at the same position. This suggests that there might be compensatory effects near residue 406 of human AMHRII that allow it to accommodate arginine but not glutamine. One possibility is residue 310, which is a glutamine in human and a glutamic acid in rat and mouse and is close to His316. However, our model indicates that, in human, residue 310 is too far away to interact with Arg406. Another possible explanation could be the size of the residues in the E–F interface. In human AMHRII, two residues close to Arg406, Met403 and Ile410, are respectively a threonine and valine in rat and mouse. The larger amino acids found in the human should increase the distance between the two helices. Consequently, an arginine may be required in the human at position 406 in order to form hydrogen bonds with His316 and Glu318 and stabilize the E–F interface. Consistent with the sensitivity of this region of the kinase domain to small alterations, mutation of the analogous residue for Ile410 in TβRII, Val447, to an alanine leads to the loss of kinase activity (19).

Arg504 lies on the αI helix and is conserved in all type II receptors. Our homology model indicates that the side chain of this residue is mainly pointing to the solvent accessible face of the helix and that the guanidinium nitrogens interact with two negatively charged residues, Glu482 and Asp486 (Fig. 8E). These residues are located on the αH helix and, interestingly, are part of the conserved polar region which includes Asp491 (discussed earlier). A similar salt bridge is found in ActRIIB between residues Arg477 and Glu459 (16). These interactions should contribute to stabilizing the αH–αI interhelical contacts and may be destabilized by the Arg504Cys mutation. The importance of Arg504 for signaling is consistent with the loss of kinase activity observed in the TβRII mutation Arg537Cys (21); Arg537 of TβRII is analogous to Arg504 of AMHRII.

In our model, Asp426 lies in the FG loop, a region with apparent high flexibility. Asp426 is also part of a large polar patch, which includes residues Arg428, Asp430, Ser431, Ser432 and Thr469 (Fig. 8F). Within this patch, both Ser431 and Thr469 are predicted as possible sites of phosphorylation by the NetPhos 2.0 program (34). Whereas Ser431 is not conserved across species or in other type II receptors, Thr469 is conserved across species, but not in other type II receptors. It is possible that mutation of Asp426 to glycine could affect phosphorylation of Thr469, however further experimentation will be required to confirm that this site is phosphorylated. Disease-associated mutations have been found in TβRII (Arg460Cys, His) (36) and BMPRII (Cys420Arg) (25) at residues that correspond to Arg423 and Cys424 of AMHRII.

Two mutations, Arg80Stop and Arg97Stop, were particularly interesting because they truncated AMHRII upstream of the transmembrane domain. Neither of these naturally truncated receptors nor an artificial truncated receptor corresponding to the entire ECD of AMHRII (Ile145Stop) was secreted into the culture medium, the typical fate of a type I membrane protein after its transmembrane domain has been removed. Secretion of soluble receptors occurs as a natural process in a number of cases where alternative splicing leads to the deletion of the transmembrane domain or as an artificial process in many cases as a result of protein engineering (37,38). Our results indicate that the AMHRII signal sequence is defective in its ability to transit to the ER, since the replacement of the endogenous AMHRII signal sequence with that of TβRII allowed secretion of a soluble AMHRII ECD fusion protein. That secretion can be rescued using a different signal sequence appears to rule out an alternative hypothesis where the truncation of AMHRII leads to a misfolded protein which cannot be translocated to the ER. The actual defect in the AMHRII signal sequence is not clear since the means scores predicted by the SignalP 3.0 program (39) are normal. However, there is a sharp decrease in the S-score around position 10, not observed with either TβRII or ActRII. Another factor may be the two proline residues present in the cleavage site. Park et al. (40) have shown that misfolded proteins retained in the cytoplasm because of a missing signal sequence are degraded via the proteosome, although the recognition and delivery mechanism that is employed is different than the one used for the elimination of misfolded secretory proteins in the ER. This is consistent with our finding that the Arg80Stop, Arg97Stop and Ile145Stop mutant receptors are degraded via the proteosome (Fig. 5D).

Three types of membrane proteins have been identified (11). Type I membrane proteins are initially targeted to the ER by an N-terminal cleavable signal peptide and then anchored in the membrane by a segment of approximately 20 hydrophobic residues that stops further translocation (i.e. a transmembrane domain). In type II membrane proteins, a signal anchor sequence (approximately 18–25 apolar amino acids) is responsible for both the insertion and anchoring of the protein in the membrane, with its C-terminal end on the outside of the cell. Type III membrane proteins are similar to type II proteins, except that the signal anchor sequence directs the N-terminal end of the protein to the outside of the cell (11). By virtue of possessing a signal peptide and by comparison with the other TGF-β family type I and II receptors, AMHRII has been classified as a type I membrane protein. However, our results indicate that AMHRII does not possess a functional signal peptide, suggesting that its transmembrane domain functions as a signal anchor, which is responsible for both the insertion and the orientation of the receptor in the membrane. In this regard, it is worth noting that the transmembrane domain of AMHRII is a little longer than that of TβRII (27 versus 23 amino acids). Thus, it appears that AMHRII has evolved from being a type I membrane protein to being a type III membrane protein.

Although AMH is considered an atypical member of the TGF-β family, exerting its effects exclusively in the reproductive organs, its signaling pathway appeared initially to conform to the rules. Indeed, cloning of the AMH type II receptor was made possible by its homologies to cloned receptors of the same family (2) but none of these yielded a satisfactory template for molecular modeling of the ECD, forcing us to generate a large number of structures to obtain a model capable of reliably analyzing structure/function relationships of natural AMHRII mutations. Our present results indicate that AMHRII also strays from the beaten path by virtue of its inefficient signal sequence, whose function is probably taken up by the transmembrane domain.

MATERIALS AND METHODS

Cells and transfection assays

COS-7 and mouse embryonal carcinoma P19 were cultured at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) with Glutamax-1, 4.5 g/l d-glucose and 4 mg/l pyridoxine–HCl (Invitrogen) containing 10% fetal calf serum (Invitrogen), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen). Twenty-four hours prior to transfection, COS-7 and P19 cells were seeded in six-well plates at, respectively, 105 cells/ml and 0.5 × 105 cells/ml. For pulse-chase and biotinylation experiments, COS cells were seeded at 0.25 × 105 cells/ml in 100 mm plates. Transient transfection was performed with the Lipofectamine Plus kit (Invitrogen).

Constructs

The AMHRII cDNA was cloned into the pALTER vector and used as a template for site-directed mutagenesis using the Altered Sites Mutagenesis System kit (Promega). Mutagenic primers designed to create some of the AMHRII mutations shown in Figure 1 are indicated in Table 1. The mutations del1692 and Arg406Gln were reproduced as described (14). The stop codon of the mutant receptor cDNAs was transformed into a HindIII restriction site by site-directed mutagenesis in order to subclone the inserts into the mammalian expression vector pCDM8 (Invitrogen), upstream of the influenza virus HA epitope sequence as described in Faure et al. (13). To ensure that required changes were present, plasmid constructs were checked by enzymatic digestion and DNA sequencing.

Table 1.

Mutagenic primers for AMHRII cDNA changes. Bold letters indicate mutated nucleotides and the arrows the location of the deletions.

Mutagenic primers Sequences (5′–3′) 
Arg54Cys AGA GCT ATC TGC TGC CTC T 
Arg80Stop GCC TGG CTC ATC ACT AAG CTT GCA TCC TTG CAT TTC 
Arg97Stop GCC AGG GCT GGG GTG AAG CTT GGG ACT TGG GTC ACA 
Gly142Val GCT GCC CCA GTT GAG TCC AT 
Ile145Stop CAG CAC CAG TGC CAT AAG CTT GGA CTC ACC TGG GGC 
Lys230Arg CGG TGG GAA GGC CCG GAT GGC AAC CAG 
del1692 CTC AGG CAG ClCC TGC AGC TC 
Arg406Gln ATC AGC TCG TTG GAG GGC C 
Asp426Gly GCT GCC CAG GTT TGA GGC CT 
del6331–6357 CCA CCC TTC CAA CTG GCC TAT GAG GCA GAA l CTA TGG GCC TTG GCA GTG CAG GAG AGG AGG 
Asp491His TTG GGA TGC ACA CCC AGA AG 
Arg504Gly TGT ACA GCA GTG CCT GGC TGC 
Mutagenic primers Sequences (5′–3′) 
Arg54Cys AGA GCT ATC TGC TGC CTC T 
Arg80Stop GCC TGG CTC ATC ACT AAG CTT GCA TCC TTG CAT TTC 
Arg97Stop GCC AGG GCT GGG GTG AAG CTT GGG ACT TGG GTC ACA 
Gly142Val GCT GCC CCA GTT GAG TCC AT 
Ile145Stop CAG CAC CAG TGC CAT AAG CTT GGA CTC ACC TGG GGC 
Lys230Arg CGG TGG GAA GGC CCG GAT GGC AAC CAG 
del1692 CTC AGG CAG ClCC TGC AGC TC 
Arg406Gln ATC AGC TCG TTG GAG GGC C 
Asp426Gly GCT GCC CAG GTT TGA GGC CT 
del6331–6357 CCA CCC TTC CAA CTG GCC TAT GAG GCA GAA l CTA TGG GCC TTG GCA GTG CAG GAG AGG AGG 
Asp491His TTG GGA TGC ACA CCC AGA AG 
Arg504Gly TGT ACA GCA GTG CCT GGC TGC 

A cDNA encoding the signal sequence of human TβRII (residues 1–23 of Swiss Prot Accession P37173) fused to the mature ECD of human AMHRII (residues 18–145 of Swiss Prot Accession Q16671) was generated by polymerase chain reaction. This cDNA and one encoding the signal sequence and ECD of human AMHRII (residues 1–145 of Swiss Prot Accession Q16671) were then ligated to a cDNA encoding the hinge, CH2 and CH3 domains of human IgG1 (residues 104-330 of Swiss Prot Accession P01857), and cloned into the expression vector pNE001.

In vitro translation

The mutant AMHRII cDNAs in pCDM8 (20 ng/ml) were incubated for 90 min in a transcription–translation-coupled rabbit reticulocyte lysate system (Promega) in the presence of T7 RNA polymerase and 20 µCi l-[35S]-methionine (1000 Ci/mmol; GE Healthcare), following the manufacturer's protocol. Reaction mixtures (5 µl) were resolved by SDS–PAGE on 4–20% gradient gels (Bio-Rad Laboratories) and analyzed by autoradiography.

RNA extraction and northern blot analysis

The kinetics of mutant AMHRII RNA synthesis was studied by northern blotting. COS cells were cultured for 24, 48 or 72 h after transfection without changing the medium. Total RNAs were extracted with the RNA Plus extraction kit (Qiagen). Five micrograms were separated by electrophoresis through a 1.2% agarose/formaldehyde gel, blotted onto a Hybond-N membrane (GE Healthcare) and hybridized with a 32P-labeled AMHRII cDNA probe. A rabbit ribosomal probe was used as an internal control, and a 0.24–9.5 kb RNA ladder (Invitrogen) was used as a size marker. Blots were scanned on a PhosphorImager (Molecular Dynamics) and semi-quantified with ImageQuant software (Molecular Dynamics).

Confocal microscopy analysis

Transfected COS cells were seeded in four-dish glass slide Lab-Tec chambers at 0.5 × 105 cells. After 2 days, cells were washed, fixed and permeabilized by incubation with methanol/acetone (v/v) for 2 min at room temperature. After blocking the cells with PBS containing 10% goat serum (Invitrogen) at room temperature for 1 h at 4°C, they were incubated overnight with an anti-HA mAb (Roche Diagnostics) in PBS containing 10% goat serum, then washed with PBS three times and incubated with an FITC-labeled anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 2 h at 4°C. After two washes, cells were labeled with 0.5 µm Rhodamine 6G (Molecular Probes) for 30 min at 4°C. Coverslips were mounted in Vectashield (Vector Laboratories) and cells were examined with a Zeiss LSM-510 confocal scanning laser microscope (Jena, Germany) equipped with a 25 mW argon laser and a 1 mW helium–neon laser, using a plan apochromat ×63 objective (oil immersion). Green fluorescence was observed with a 505–550 nm band-pass emission filter under 488 nm laser illumination, and red fluorescence was observed with a 560 long-pass emission filter under 543 nm laser illumination. Sections were collected every 0.4 µm along the Z-axis. Projections of three medium images for FITC and Rhodamine 6G were done for each sample.

Western blot analysis

COS cells were transfected with WT or mutant AMHRII cDNAs and incubated for various times in the appropriate medium as described for RNA studies. For clasto-lactacystin studies, cells were treated for 24 h with 10 µm of clasto-lactacystin β-lactone (Calbiochem) 2 days after transfection. After washing, cells were lysed as described previously (13). Protein concentration of the supernatant was determined using the BCA assay (Pierce Chemical). Proteins were subjected to SDS–PAGE (Bio-Rad Laboratories) and transferred onto a Protran BA85 nitrocellulose membrane (Schleicher & Schuell). Detection of HA-tagged receptors was performed as described (3) using a mAb against HA (Roche Diagnostics). Blots were then probed with a peroxidase-labeled anti-mouse or anti-human antibody at 1:5000 (Jackson Laboratories). Bands were visualized with the ECL detection reagent (GE Healthcare).

Metabolic labeling and immunoprecipitation

COS cells transfected with mutant AMHRII cDNAs were incubated in methionine and cysteine-free DMEM (ICN Biomedicals) containing 100 µg/ml [35S] methionine and cysteine (ICN Biomedicals) for 30 min at 37°C and then cultured for different periods of time in the same medium containing cold methionine and cysteine. COS cell lysates and media were immunoprecipitated overnight at 4°C with 5 µg/ml of anti-HA mAb (Roche Diagnostics) and protein-A Sepharose beads (GE Healthcare). Immunoprecipitates were washed five times with 50 mm Tris–HCl, 150 mm NaCl, 0.5% Triton X-100. Proteins were eluted from the pellets by boiling in Laemmli buffer, 10 mm DTT, subjected to SDS–PAGE electrophoresis and visualized by autoradiography.

Cell-surface biotinylation

Cell-surface biotinylation of COS cells was performed as described previously (13). Briefly, cells were incubated with sulfosuccinimidyl-6-(biotinamido) hexanoate (Sulfo-NHS-LC-biotin, Pierce Chemical) for 2 h at 4°C, lysed and immunoprecipitated with 5 µg/ml of anti-HA antibody. After electrophoresis of the complexes and electroblotting on a nitrocellulose membrane, they were detected with horseradish peroxidase-conjugated streptavidin and chemiluminescence.

AMH binding

COS cells were seeded at 105 cells/ml in four-well Permanox Lab-Tek chambers and transfected 24 h later with the mutant receptors. Three days later, cells were incubated for 4 h at 37°C with 3.5 nm human recombinant AMH in DMEM/1% FCS medium. After rinsing with DMEM, cells were incubated for 2 h at 37°C with 3 µg/ml of a rabbit polyclonal anti-AMH antibody. After rinsing with PBS, cells were then incubated for 1 h with an FITC-conjugated goat antibody raised against IgG. After rinsing with PBS, cells were fixed for 5 min in methanol/acetone (v/v). After hydratation, slides were mounted in Vectashield containing propidium iodide (Vector Laboratories), and cells were examined with a Zeiss microscope (Jena, Germany).

Reporter assays

The Gal4-Smad1 reporter system was a generous gift of Dr A. Atfi. P19 cells were co-transfected with 1 µg of Gal4-Smad1, 1 µg of Gal4-luc, 1 µg of the different mutant cDNA expression vectors and 50 ng of pRLTK as a control for transfection efficiency. At the end of the transfection, AMH (10 µg/ml) was added to the culture medium for 24 h. Cells were washed twice with PBS and lysed for 20 min under rocking in 500 µl of passive lysis buffer. Twenty microliters were analyzed for firefly and Renilla luciferase activity according to the manufacturer instructions (Dual Luciferase Kit, Promega) using a Lumat LB95507 luminometer (EG&G Berthold). Results were normalized to Renilla luciferase activity. Each experiment was done in triplicate and the results were expressed as a percentage of the stimulation of the reporter gene in presence of AMH.

Molecular modeling

The search for structural templates for AMHRII structural modeling of the extracellular and intracellular domains was performed with mGenThreader (41) and Psi-blast (42). The resulting best hit structures were downloaded from the Protein Data Bank (PDB) (43). The template proteins used for the homology modeling of the extracellular region are the mouse activin type IIB receptor (PDB code 1bte) (15), the sheep TβRII receptor (PDB code 2hlq) and the human BMPRII receptor (code 1m9z). The modeling of the intracellular region was based on the recently released structure of the human ActRIIB receptor (pdb code 2qlu). The sequence alignments were performed using TCOFFEE and invoking for structural information. Secondary structure prediction was provided by mGen and the program PSI-PRED (41). The resulting alignments were used for building the three-dimensional models using the Modeler V9.2 package (44), and post-optimization of the major loop insert was performed with fast refinement (17). For the ECD, different constraints were applied for checking the different combinations of disulfide bridges. Models presenting the lowest energy and the highest three-dimensional quality, as evaluated by Errat (45), were kept as possible models. Finally, relaxation of the selected models was performed by a full minimization of the structure using the Charmm program with the Charmm22 force field. Molecular dynamics calculations are now underway.

FUNDING

This work was supported by the Association pour la Recherche contre le Cancer (grant no. 3734 to N.d.C.). J.-D.M. thanks the Spanish Ministerio de Ciencia e Innovación for funding through the project no. CTQ2008-06866-C02-01 and consolider-ingenio 2010 no. CSD2007-00006. L.M. thanks the Spanish Ministerio de Ciencia e Innovación for funding through the ‘Ramón y Cajal’ Program and the project no. CTQ2008-02403/BQU.

ACKNOWLEDGEMENTS

We thank Henrik Nielsen at the Center for Biological Sequence Analysis for help with signal sequence analysis and Valérie Nicolas for help with confocal microscopy.

Conflict of Interest statement. None declared.

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Author notes

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.