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Autosomal Recessive Segregation of a Truncating Mutation of Anti-Müllerian Type II Receptor in a Family Affected by the Persistent Müllerian Duct Syndrome Contrasts with Its Dominant Negative Activity in Vitro

Unité de Recherches sur l’Endocrinologie du Développement, INSERM, U-493, Département de Biologie, Ecole Normale Supérieure (L.M.-Z., L.G., C.B., M.D., S.I., J.-Y.P., N.J., N.d.C.), 92120 Montrouge, France; INSERM, U-10, Hôpital Bichat (L.L.), 75018 Paris, France; and Department of Pediatrics, Addenbrookes’s Hospital (I.A.H.), Cambridge, United Kingdom CB2 2QQ

Address all correspondence and requests for reprints to: Dr. Nathalie di Clemente, Unité de Recherches sur l’Endocrinologie du Développement, INSERM, U-493, Département de Biologie, Ecole Normale Supérieure, 92120 Montrouge, France. E-mail: clemente{at}wotan.ens.fr

Abstract

Anti-Müllerian hormone belongs to the TGFß family whose members exert their effects by signaling through two related serine/threonine kinase receptors. Mutations of the anti-Müllerian hormone type II receptor occur naturally, causing the persistent Müllerian duct syndrome. In a family with two members with persistent Müllerian duct syndrome and one normal sibling, we detected two novel mutations of the anti-Müllerian hormone type II receptor gene. One, transmitted by the mother to her three sons, is a deletion of a single base leading to a stop codon, causing receptor truncation after the transmembrane domain. The other, a missense mutation in the substrate-binding site of the kinase domain, is transmitted by the father to the two sons affected by persistent Müllerian duct syndrome, indicating a recessive autosomal transmission as in other cases of persistent Müllerian duct syndrome. Truncating mutations in receptors of the TGFß family exert dominant negative activity, which was seen only when each of the mutant anti-Müllerian hormone receptors was overexpressed in an anti-Müllerian hormone-responsive cell line. We conclude that assessment of dominant activity in vitro, which usually involves overexpression of mutant genes, does not necessarily produce information applicable to clinical conditions, in which mutant and endogenous genes are expressed on a one to one basis.

ANTI-MÜLLERIAN HORMONE (AMH), also called Müllerian inhibiting substance, is a member of the TGFß superfamily that is synthesized by somatic cells of the gonads. In male fetuses, AMH is responsible for the early regression of Müllerian ducts, the anlagen of uterus and Fallopian tubes in females (1). AMH also down-regulates cell maturation and transcription of various steroidogenic proteins in gonads of both sexes (2, 3, 4) and in gonadal cell lines (5).

Members of the TGFß family exert their effects by signaling through two related receptors formed by a ligand-binding extracellular domain, a single transmembrane region, and an intracellular domain with serine/threonine kinase activity. Mutations that truncate these receptors immediately after the transmembrane domain or that destroy their kinase activity are classical dominant negative inhibitors of cellular biological functions when overexpressed at the cell surface membrane and thus are in a position to interfere with the function of the endogenous receptor (6, 7, 8).

Autosomal dominant transmission has been demonstrated in two disorders linked to germline mutations of receptors of the TGFß family: hereditary hemorrhagic telangiectasia type 2 due to mutations of the orphan receptor activin receptor-like kinase 1 (ALK1) (9) and familial primary pulmonary hypertension associated with mutations of the bone morphogenetic protein receptor type II (BMPR-II) (10). The lack of appropriate biological tests does not allow discrimination between haploinsufficiency or dominant negative activity of the mutations.

AMH is the only other member of the TGFß family in which mutations of signaling molecules occur naturally in the germline. A rare form of male pseudohermaphroditism, the persistent Müllerian duct syndrome (PMDS), which is characterized by the persistence of Müllerian derivatives in otherwise normally virilized males (11), may be due to spontaneous mutations of either the AMH or the AMH type II receptor (AMHR-II) gene (12) and is transmitted as a recessive autosomal trait (13), although X-linked inheritance has been suspected in two kindreds affected by PMDS of unknown molecular etiology (14, 15). We now describe two novel mutations of AMHR-II, detected in a family (K family) affected by PMDS. One truncates the receptor immediately after the transmembrane domain, and the other is a missense mutation of the substrate-binding site of the kinase domain. Both would be expected to exhibit dominant negative activity, as suggested by experimental mutations engineered in receptors of other TGFß family members. However, in the K family, clinical symptoms were restricted to individuals exhibiting two abnormal alleles. To understand this discrepancy, the effects of both mutated receptors on the normal endogenous receptor were studied in vitro at various levels of expression.

Materials and Methods

Single stranded conformation polymorphism (SSCP)-PCR of the AMH type II receptor gene

DNA was extracted from peripheral blood lymphocytes. PCR amplification of exons and intron-exon boundaries of AMHR-II gene was performed on patient DNA using 11 pairs of oligonucleotide primers as previously described (16). The PCR amplification products were purified by electrophoresis through a 1.5% low melting agarose gel (SeaPlaque GTG, FMC Bioproducts, Rockland, ME), recovered by centrifugation through SpinX (Costar, Cambridge, MA) and submitted to a second round of PCR amplification in the presence of [³²P]deoxy-CTP for 20 cycles. These radiolabeled reaction products were analyzed by SSCP-PCR after digestion with appropriate restriction enzymes as previously described (16).

Cloning, sequencing, and restriction analysis

Gene PCR products exhibiting abnormal migration by SSCP-PCR were sequenced using the Thermo Sequenase radiolabeling terminator cycle sequencing kit (Amersham Pharmacia Biotech, Arlington, IL). When the mutation altering the AlwNI restriction site was detected, its presence in other family members was investigated by restriction analysis of 200 ng PCR-amplified DNA digested for 4 h with 1 IU AlwNI restriction enzyme (New England Biolabs, Inc., Beverly, MA) according to the manufacturer’s instructions.

For patients heterozygous for the mutation altering the AlwNI site, the two AMHR-II alleles were amplified in toto (7 kbp) by the Expand Long PCR kit (Roche Molecular Biochemicals, Indianapolis, IN), isolated by cloning in the PGEM-T vector, and tested by AlwNI restriction analysis. Clones yielding normal results were sequenced in toto by the dideoxynucleotide termination with BigDye terminators premix (PE Applied Biosystems, Foster City, CA) using an ABI Prism 377 automatic sequencer (PE Applied Biosystems).

Site-directed mutagenesis

The 1.8-kbp AMHR-II cDNA (17) subcloned into pALTER vector in the SP6-T7 orientation at the EcoRI site was used as a template for creating the influenza virus hemagglutinin epitope (HA) and the FLAG-tagged two mutant receptors found on the AMHR-II gene. The Altered Sites Mutagenesis System kit (Promega Corp., Madison, WI) was used according to the manufacturer’s instructions.

The first mutation, a deletion of an adenosine at cDNA position 674, was reproduced by the reverse 20-mer mutagenic primer 5'-CTCAGGCAGC CCTGCAGCTC-3', and the stop codon generated nine codons downstream by this mutation was replaced by the HindIII restriction site (underlined) using the reverse mutagenic primer 5'-CCTCCTTCCCGGAAAGCTTCCTGGGAGAAACAC-3'.

The second mutation, which converted an arginine at position 406 into a glutamine, was reproduced with the reverse primer 5'-ATCAGCTCGTTGGAGGGCC-3', and replacement of the natural stop codon by an HindIII site was performed with the reverse primer 5'-AACTGCATATAAGCTTCACAGGAGAAAGG-3'.The replacement of the signal peptide cleavage site by an EcoRI site was performed with the reverse primer 5'-CCTGTTTGGGGAATTCTCCACAGCTG-3'.

Depending upon the constructs, the mutated AMHR-II cDNAs were excised from the mutagenesis vector pALTER either by HindIII and inserted immediately upstream of the first HA codon of the mammalian expression vector pCDM8/HA (18) or by EcoRI and inserted downstream of the last FLAG codon of the mammalian expression vector pFLAG-CMV-1 vector (Eastman Kodak Co., New Haven, CT). The mutations and the junctions of the mutant cDNAs with the different epitopes were checked by enzymatic digestion and DNA sequencing.

Cell lines and transfections

COS-6 cells were cultured in DMEM (Life Technologies, Inc., Cergy-Pontoise, France) containing 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Eurobio, les Ulis, France). They were seeded at 3 x 10⁵ cells on poly-D-lysine-coated, one-chambered Lab-Tek microscope slides (Nunc, Naperville, IL) for binding experiments and on six-well, 35-mm plates (Techno Plastic Products AG, Trasadingen, Switzerland) for cell surface biotinylation and interaction experiments. The cells were transiently transfected the following day with wild-type or mutant AMHR-II cDNAs using the diethylaminoethyl-dextran/chloroquine method as previously described (19).

SMAT-1 cells derived from mouse immature Sertoli cells by targeted oncogenesis (20) were grown in DMEM (Life Technologies, Inc.) supplemented with 10% FCS, 1 x amino acid mix (Eurobio), 100 U/ml penicillin, and 100 µg/ml streptomycin (Eurobio). Cells were plated at 3 x 10⁵ cells/well in six-well, 35-mm plates and transiently transfected with a total of 2 µg cDNA using the Lipofectamine Plus reagent package (Life Technologies, Inc.) according to the manufacturer’s instructions.

Cell surface biotinylation

After incubation for 2 h at 4 C with sulfosuccinimidyl 6-(biotinamido) hexanoate (Pierce Chemical Co., Rockford, IL; 1 mg/ml), a membrane-impermeable biotin reagent labeling only cell surface proteins, transfected COS cells were lysed and immunoprecipitated with 5 µg/ml anti-HA monoclonal antibody (BabCO, Richmond, CA) as previously described (18). The immunoprecipitates were submitted to an electrophoresis on 4–20% SDS-PAGE followed by electroblotting on a nitrocellulose membrane. The detection of the immune complexes was performed with horseradish peroxidase-conjugated streptavidin (1:1000) and chemiluminescence (ECL kit, Amersham Pharmacia Biotech, Piscataway, NJ).

Binding experiments

Exposure of COS cells, transiently transfected with wild-type or mutant AMHR-II cDNAs, to 1 nmol/liter iodinated AMH for 4 h at 25 C and autoradiography were performed as previously described (19).

Interaction experiments

For interaction experiments between wild-type and mutant AMHR-II, COS cells were transiently transfected with FLAG-tagged mutant and HA-tagged wild-type AMHR-II cDNAs. Two days later, cell lysates were immunoprecipitated with anti-FLAG M2 affinity gel (Eastman Kodak Co.) according to the manufacturer’s instructions. The immunoprecipitates were then analyzed by Western blotting using an anti-HA antibody (1 µg/ml).

For interaction experiments between wild-type or mutant AMHR-II and type I receptors, COS cells were transfected with FLAG-tagged AMHR-II cDNAs and HA-tagged BMPR-IB or ALK1 cDNAs. Forty-eight hours later, cells were incubated for 45 min in the presence or absence of AMH (357 nmol/liter). Cell lysates were immunoprecipitated with an anti-HA antibody (5 µg/ml) and analyzed by Western blotting with an anti-FLAG antibody (Eastman Kodak Co.; 10 µg/ml).

Northern blotting analysis

Effect of AMH on cytochrome P450 side-chain cleavage enzyme (P450scc) expression was studied by Northern blotting. SMAT-1 cells were transfected with mutated AMHR-II cDNAs and incubated for 72 h in the appropriate medium in the presence or absence of recombinant human AMH (17) at 71.4 nmol/liter. Total RNAs were isolated with the RNA Plus extraction kit (Quantum Biotechnologies, Montreuil, France), and 5 µg were loaded onto an agarose gel. After transfer onto a Hybond-N membrane, the blots were analyzed successively with P450scc and AMHR-II probes as previously described (4). A rabbit ribosomal probe was used as an internal control, and a 0.24- to 9.5-kb RNA ladder (Life Technologies, Inc.) was used as a size marker. Blots were scanned on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and were semiquantified with ImageQuant Software (Molecular Dynamics, Inc.).

Reporter gene assay

The AMHR-II-luc reporter gene was constructed by inserting 5.5 kbp human AMHR-II promoter starting 28 bp upstream of the initiation codon between the XhoI and HindIII sites of pGL2-Basic vector (Promega Corp.), which contains the luciferase gene. SMAT-1 cells were cotransfected with this construct (1 µg) and either the mutant AMHR-II cDNAs (0.5 µg) or empty pCDM8 vector, and treated the same day with AMH (71.4 nmol/liter) for 40 h. Luciferase activity was determined with the luciferase reporter gene assay kit (Roche, Indianapolis, IN) using a Lumat LB95507 luminometer (EG&G Berthold, Bad Wildbad, Germany) and was normalized for protein concentration.

Results

Clinical and genetic data

PMDS was diagnosed in two 46,XY brothers, S1 and S2, who were of normal height and had normal external genitalia, apart from bilateral nonpalpable testes. The youngest brother, S3, was clinically normal. The index case, S1, underwent laparoscopy at 7 yr of age to localize the left testis after a difficult mobilization of an intraabdominal right testis to the right pubic region. A left testis was identified in the pelvis, surrounded by a Fallopian tube. No laparotomy was performed to confirm the presence of a midline uterus. The second brother, S2, also cryptorchid, underwent laparotomy at 4 yr of age. Bilateral gonads were found on the pelvic wall, connected to a lumbar pedicle attached to bilateral Fallopian tubes and vasa deferentia, associated with a midline hypoplastic uterus. In both children, testis size was apparently normal; biopsy showed immature testicular tissue with seminiferous tubules but no Leydig cells. Serum T responded well to hCG (1500 U twice weekly for 3 wk; respectively, 27.0 and 40.0 nmol/liter in S1 and S2). An LH-releasing hormone test was normal, and pubertal maturation was seen to proceed at the expected age at subsequent follow-up visits. The next stage in surgical management will involve removal of intraabdominal testes, insertion of testicular prostheses, and removal of Müllerian structures. The absence of Müllerian derivatives in the youngest sibling was ascertained by lack of clinical abnormalities and a normal pelvic ultrasonogram. The serum AMH concentrations were, respectively, 327, 36, and 380 pmol/liter in S1 (10 yr), S2 (7 yr), and S3 (5 yr). These results are normal for S1 and S3, but unexpectedly low for S2; unfortunately, the assay could not be repeated. The phenotype/genotype correlation of the family is shown in Fig. 1.

View larger version (36K): [in this window] [in a new window]   Figure 1. Detection and transmission of truncation and R406Q AMHR-II mutations in the K family. A, Transmission of the mutations. F, Father; M, mother; S1–S3, siblings 1–3. S1 and S2 are affected by PMDS (*); S3 is clinically normal. B, Detection of the truncation mutation by restriction analysis using AlwNI enzyme. In the absence of this mutation, as in F, three fragments are generated (279, 167, and 21 bp; the latter is not visible on the figure). The mutation destroys an AlwNI restriction site, so that restriction analysis produces only two restriction fragments (279 and 188 bp, respectively). In heterozygotes (M and S1–S3), all three fragments (279, 188, and 167 bp) are visible. C, Detection of R406Q mutation by automatic sequencing of a PCR amplification product of S2 DNA. The C at position 6051 in the wild-type DNA (antisense strand) is replaced by a T in the mutated allele. As the patient is heterozygous for this mutation, the chromatogram shows two superposed peaks, corresponding to T and C.

Two novel mutations of the AMHR-II gene were detected. One, detectable by SSCP-PCR, consists of deletion of an adenosine at position 1692 in exon 5. This mutation leads to a modification of the reading frame 28 amino acids after the transmembrane domain and the formation of a stop codon (TAA) at positions 1834–1837, resulting in a truncated receptor (Fig. 2). It destroys an AlwNI restriction site, allowing quick ascertainment of the pattern of transmission. The mutation was transmitted by the mother to her three sons, only two of whom are affected by PMDS (Fig. 1). In the latter, a second mutation, a transition of a guanosine to an adenosine at position 6051 in exon 9, was detected on the other allele by automatic sequencing of the gene in toto. This mutation leads to the replacement of an arginine by a glutamine at position 406 (R406Q) and thus changes a positively charged amino acid to a neutral one at the end of kinase subdomain VIII. This mutation is carried by the father and was transmitted to his two affected sons (Fig. 1).

View larger version (15K): [in this window] [in a new window]   Figure 2. Schematic representation showing the cytoplasmic domain of the normal and truncated AMHR-II. The nucleotide and corresponding predicted protein sequence beginning 24 amino acids after the transmembrane (TM) domain are indicated. For the normal receptor (top), the AlwNI-cut site is indicated by a vertical dash, and the adenosine 1692, which is deleted in the mutant gene, is underlined. For the truncated receptor (bottom), the site of the deletion is indicated by an arrowhead, and the amino acids resulting from the frameshift are shaded.

The two mutations were reproduced by site-directed mutagenesis of the normal AMHR-II cDNA, and the resulting mutated cDNAs were tagged with HA. The ability of these mutant receptors to reach the cell surface was tested after transfection of their cDNAs into COS cells and biotinylation of cell surface components as previously described (18). One wide band corresponding to the mature form of each receptor was detected: a 80-kDa band for wild-type and R406Q mutant receptors and a 30-kDa protein for the truncated receptor (Fig. 3). These results showed that both mutated receptors are expressed at the cell surface, a prerequisite for AMH binding. To confirm this, COS cells were transfected with wild-type or mutant cDNAs and incubated with iodinated AMH. Autoradiography showed that both mutated receptors bound AMH normally (Fig. 4).

View larger version (60K): [in this window] [in a new window]   Figure 3. Expression of wild-type and mutant AMHR-II proteins at the cell surface. COS cells expressing normal or mutant receptors were biotinylated, immunoprecipitated with an anti-HA antibody, subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probed with horseradish peroxidase-conjugated streptavidin as described in Materials and Methods. The 30-kDa truncated receptor and the 80-kDa wild-type and R406 mutant receptors appear as wide bands. A thin 80-kDa band is an artifact always detected with the anti-HA antibody when the film is overexposed. The results shown are representative of three independent experiments.

View larger version (113K): [in this window] [in a new window]   Figure 4. Binding capacity of wild-type and mutant AMHR-II. COS cells were transfected with wild-type and mutant AMHR-II cDNAs, incubated 3 d later with 1 nmol/liter iodinated AMH for 4 h at 25 C, and processed for autoradiography. Exposure was for 5 d. Both the truncated and R406Q receptors bind AMH, like wild-type (WT) AMHR-II. A negative COS cell is indicated by an arrow. The results shown are representative of four independent experiments.

An important criterion for dominant negative activity of type II receptors is their ability to sequester the endogenous type II receptor. To investigate the formation of wild-type and mutant receptor heterodimers, COS cells were cotransfected with cDNAs encoding mutant receptors tagged with the FLAG epitope and with the cDNA encoding the wild-type receptor tagged with the HA epitope. After immunoprecipitation of the complexes with an anti-FLAG antibody, the wild-type AMHR-II was revealed by Western blotting with an anti-HA antibody under denaturing conditions. Both mutated receptors were able to interact with the wild-type AMHR-II and with each other (Fig. 5A). The same results were obtained after cotransfection of the mutant receptor cDNAs tagged with the HA epitope and the wild-type AMHR-II tagged with the FLAG epitope (results not shown).

View larger version (37K): [in this window] [in a new window]   Figure 5. Interaction between AMH type II and type I receptors. A, COS cells were transiently cotransfected with FLAG-tagged mutant AMHR-II cDNAs and HA-tagged wild-type AMHR-II. After immunoprecipitation with an anti-FLAG affinity gel, complexes were loaded on a 4–20% gel and revealed by Western blotting with an anti-HA antibody. Lane 1, Mock-transfected cells; lane 2, cotransfection with two wild-type AMHR-II cDNAs, respectively, tagged with HA and FLAG epitopes; lane 3, cotransfection with wild-type and truncated AMHR-II cDNAs; lane 4, cotransfection with wild-type and R406Q AMHR-II cDNAs; lane 5, cotransfection with R406Q (HA) and truncated (FLAG) AMHR-II cDNAs. B, COS cells were transiently cotransfected with FLAG-tagged wild-type or mutant AMHR-II cDNAs and HA-tagged BMPR-IB or ALK1. Forty-eight hours later, cells were treated with AMH (357 nmol/liter) when indicated in the figure. After immunoprecipitation with an anti-HA antibody, complexes were loaded on a 4–20% gel and revealed by Western blotting with an anti-FLAG antibody. Lane 1, Mock-transfected cells; lanes 2 and 3, cotransfection with wild-type AMHR-II and BMPR-IB cDNAs; lanes 4 and 5, cotransfection with truncated AMHR-II and BMPR-IB cDNAs; lanes 6 and 7, cotransfection with R406Q AMHR-II and BMPR-IB cDNAs; lane 8, cotransfection with wild-type HA- and FLAG-AMHR-II cDNAs; lane 9, cotransfection with wild-type AMHR-II and ALK1 cDNAs.

Effects of mutant receptors on AMH repression of P450scc mRNAs

The ability of the mutated receptors to specifically block the function of the endogenous receptor and thus behave as a dominant negative mutant in vitro was assessed on two AMH biological effects and by two different methods. Both tests were performed in an AMH-responsive cell line of Sertoli cell origin, SMAT-1 (20), which expresses all of the AMH transduction machinery (21). The first bioassay was based upon the capacity of AMH to decrease the RNA level of testicular P450scc (4) and took advantage of P450scc expression by SMAT-1 cells, a characteristic of immature Sertoli cells (22). In SMAT-1 cells, 3 d of treatment with AMH down-regulated basal P450scc expression by 49.8 ± 2.2% (P < 0.001; n = 8; not shown), an effect comparable to that obtained either in testicular tissue of transgenic mice overexpressing AMH or in AMH-treated purified Leydig cells (4, 23). The ability of the mutated receptors to block this AMH effect was tested by Northern blotting 3 d after transient transfection of their cDNA. The amount of cDNA used was that recommended by the manufacturer, but due to the variability of the transfection efficiency, the expression of mutated receptors varied from one experiment to another. Thus, to relate the inhibitory effect of the truncated receptor to its expression level, the same filters were then hybridized with an AMHR-II probe, which can detect both the endogenous (1.8 kb) and the truncated (0.9 kb) AMHR-II mRNAs (Fig. 6A). The effect of truncated receptor on P450scc mRNA down-regulation by AMH was expressed as a percentage of that achieved with mock-transfected cells, taken as 100%. Figure 6B shows that the effect of AMH is significantly decreased by the truncated receptor only when the ratio of mutant over endogenous receptor mRNA is greater than 3. Transfection of the R406Q receptor cDNA decreased the AMH effect by 30.4 ± 6.5% (P < 0.05; n = 4), but no comparison with the ratio of abnormal vs. endogenous receptor mRNA was possible because they could not be distinguished by Northern blotting (Fig. 6A).

View larger version (21K): [in this window] [in a new window]   Figure 6. Effect of AMH on expression of P450scc in SMAT-1 cell line transfected with truncated AMHR-II cDNA. SMAT-1 cells transiently transfected with either the empty vector (mock-transfected cells) or the truncated receptor were cultured for 72 h in the presence or absence of 71.4 nmol/liter AMH. Northern blot analysis was performed on 5 µg total RNA, using P450scc and AMHR-II probes successively. Blots were quantified by phosphorimaging and normalized with a ribosomal probe. A, The AMHR-II probe allows detection of the endogenous (1.8 kb) and truncated (0.9 kb) receptor mRNA. The R406Q receptor mRNA could not be distinguished from the endogenous one. B, The effect of truncated receptor on P450scc mRNA down-regulation by AMH was expressed as a percentage of that achieved using mock-transfected cells, which was taken as 100%. A significant inhibition of the effect of AMH on P450scc mRNA was observed only when the ratio (R) of the transfected truncated receptor mRNA over the endogenous receptor was greater than 3, as shown by t test. *, P < 0.05. The data shown are the average of three independent experiments ± SEM.

Because regulation of steroidogenesis is multifactorial and obviously not AMH specific, we next asked whether AMH, like other peptide hormones (24), might regulate the transcription of its own receptor. Preliminary results obtained by Northern blotting (not shown) had demonstrated that AMHR-II mRNAs are repressed after 24–72 h of treatment with AMH in SMAT-1 cells. An AMHR-II reporter gene (AMHR-II-luc) was constructed with 5.5 kbp of the human AMHR-II promoter driving the expression of the luciferase gene. Treatment with AMH for 40 h induced a 49.8 ± 2.7% (P < 0.001; n = 4) decrease in AMHR-II-luc activity in SMAT-1 cells (not shown). Cotransfection of either the truncated or the R406Q receptor cDNAs blocked the effect of AMH by, respectively, 90.5 ± 8.2% (P < 0.01; n = 4) and 66.7 ± 11.1% (P < 0.01; n = 4; Fig. 7). Taken together, these results demonstrate that both mutated receptors can, when overexpressed, act as dominant negative mutants to block the functional response to AMH.

View larger version (19K): [in this window] [in a new window]   Figure 7. Effect of AMH on AMHR-II-luc reporter gene transcription. SMAT-1 cells were cotransfected with the AMHR-II-luc reporter gene and either empty vector (mock-transfected cells) or mutant receptor constructs. Cells were incubated for 40 h with AMH (71.4 nmol/liter), and luciferase activity was determined. The effect of mutated receptors on the inhibition of AMHR-II-luc transcription by AMH was expressed as a percentage of that achieved using mock-transfected cells, which was taken as 100%. **, P < 0.01. The data shown are the average of four independent experiments; each performed in triplicate ± SEM.

PMDS is a rare form of male pseudohermaphroditism characterized by the lack of regression of Müllerian ducts in male individuals. Two anatomical forms have been described. The association of unilateral cryptorchidism and contralateral hernia characterizes the most common form. More rarely, PMDS takes the form of bilateral cryptorchidism, the uterus is fixed in the pelvis, and both testes are embedded in the broad ligament. Regression of Müllerian ducts normally occurs in male human fetuses at 8 wk of fetal development under the influence of AMH. The hormone binds to a receptor, AMHR-II, expressed in the mesenchyme of Müllerian ducts, initiating a transduction cascade (25) that leads to the disappearance of Müllerian ducts at 10 wk of fetal age. Mutations of both the AMH and AMHR-II genes block Müllerian regression. Our laboratory has currently studied 69 families with PMDS, 31 associated with AMH and 27 with AMHR-II mutations (12). All displayed recessive autosomal transmission, 2 mutated alleles being required for expression of the PMDS phenotype. Such was also the case for the K family. Persistence of Müllerian derivatives was seen only when both AMHR-II alleles were abnormal. This contrasts with the dominant transmission of other spontaneous mutations of receptors of the TGFß family (9, 10).

Recessive autosomal transmission is the rule when the amount of protein synthesized by a single allele is sufficient to ensure normal physiological function. If this is not the case, individuals heterozygous for the mutation will also exhibit the phenotype for the disease; in other words, the transmission will be autosomal dominant, a condition known as haploinsufficiency. The deficiency mechanism assumes that the quantity of functional protein within the cell is a limiting step for hormone action and that the normal allele is not up-regulated to compensate for the nonfunctional one. Dominant transmission may also be due to the interference of the mutated allele with the function of the normal allele, a condition known as dominant negative activity. Dominant negative activity implies that the mutated allele sequesters ligand or cofactors, forms nonfunctional dimers, or competes with the wild-type allele for binding to a common target sequence (26). Generalized resistance to thyroid hormone is a good example. This condition, usually due to mutations in the ligand-binding domain of TRß, clinically segregates in an autosomal dominant manner and inhibits the function of the normal allele in ligand binding studies (27). Interestingly, complete deletion of virtually all of the coding region of the human TRß gene segregates in an autosomal recessive manner (28), because a receptor that is not expressed at all obviously cannot block the activity of a normal allele.

Members of the TGFß family, which includes AMH, signal through two membrane-bound serine/threonine kinase receptors, called receptors I and II. Type II, the primary receptor, binds the ligand on its own, causing recruitment and phosphorylation of the type I receptor, which, in turn, activates pathway-specific Smad transcription factors (29).

Dominant negative versions have been engineered by truncation of the activin (30), bone morphogenetic proteins (31), or TGFß (32) type II receptors or by the introduction of point mutations that inactivate the kinase domain (6, 32).

The truncation mutation detected in the K family, due to a 1-base deletion in exon 5, leads to a stop codon that interrupts the receptor protein between the transmembrane and kinase domains. This region was that chosen to engineer truncated versions of receptors for various members of the TGFß family. It ensures anchorage of the abnormal receptor in the cytoplasmic membrane, a prerequisite for dominant negative activity, which was indeed always observed, as referenced above.

The R406Q mutation changes a positively charged to a neutral residue at the end of kinase subdomain VIII. Kinase subdomain VIII plays a major role in the recognition of peptide substrates, providing a hydrophobic pocket to accommodate hydrophobic residues of substrates, as judged by comparison with PKA, which has been crystallized and shows sequence homology to the serine/threonine kinase domain of the TGFß type II receptor (33, 34). A point mutation of the TGFß type II receptor gene that prevents substrate recognition displays dominant negative activity (35).

Thus, mutations of receptors of the TGFß family similar to those observed in the K family are dominant, whereas in the K family, individuals with only one mutated allele (sibling 3 for the truncation mutation and the father for the R406Q one) are phenotypically normal. Could differences in cell trafficking explain this discrepancy? To exert dominant negative activity, i.e. compete with the normal receptor in the signaling complex, probably formed by two molecules of both the type II and the type I receptor (36), the mutated receptors must be expressed at the cell surface, bind the ligand, and interact with normal receptors. Nevertheless, the signaling complex is nonfunctional because the kinase domain, required to transphosphorylate the type I receptor, is either absent (7, 37) or inactivated by mutations in key amino acids (6, 32).

Cell trafficking and protein binding capacity are normal in mutated receptors of the K family. Both are normally expressed at the cell surface, bind iodinated AMH, and interact with the wild-type AMH type II receptor. They also form a complex with the recently identified candidate AMH type I receptor, BMPR-IB (21), but the AMHR-II/BMPR-IB interaction is not specific, as could be expected in COS cells (38).

We believe that the discrepancy between the recessive mode of transmission in the K family and the dominant or dominant negative activity of similar mutations reported in the literature can only be explained by differences in the level of expression of the mutated gene. Probably in human heterozygous individuals, the normal and mutated genes are expressed on a one to one basis. It is therefore not appropriate to compare this condition to those in which the abnormal gene is overexpressed, as it is in all of the experiments using transfected cells or transgenic animals. Indeed, Fig. 6 clearly shows that the truncated AMHR-II acquires dominant negative activity only when its mRNA is overexpressed relative to that of endogenous AMHR-II. In experiments using a reporter gene, the mutated genes exhibit dominant negative activity forthwith, but this technique is much more sensitive than Northern blotting (39).

In conclusion, we were able to reconcile an autosomal recessive segregation of clinical features with the dominant negative biological activity characteristic of kinase-deficient type II receptors of the TGFß family. Assessment of dominant activity in vitro, which usually involves overexpression of mutant genes, does not necessarily produce information applicable to clinical conditions, in which mutant and endogenous genes are expressed on a one to one basis.

Acknowledgments

We are grateful to Drs. Rodolfo Rey and Danièle Carré-Eusèbe for critical reading of the manuscript, and to Dr. Joan Massagué for the generous gift of BMPR-IB and ALK1 constructs.

Footnotes

This work was supported by fellowships from la Ligue Contre le Cancer et la Fondation pour la Recherche Médicale (to L.G.), Contract BMH4 from the Biomed2 Program of the European Community.

Abbreviations: ALK1, Activin receptor-like kinase 1; AMH, anti-Müllerian hormone; AMHR-II, anti-Müllerian hormone type II receptor; BMPR-IB, bone morphogenetic type I receptor; HA, influenza virus hemagglutinin epitope; P450scc, cytochrome P450 side-chain cleavage enzyme; PMDS, persistent Müllerian duct syndrome; P450scc, cytochrome P450 side-chain cleavage; SSCP, single strand conformation polymorphism.

Received December 1, 2000.

Accepted May 11, 2001.

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