Richard L. Cate

Processing of Anti-Müllerian Hormone Regulates Receptor Activation by a Mechanism Distinct from TGF-β

TGF-β family ligands are translated as prepropeptide precursors and are processed into mature C-terminal dimers that signal by assembling a serine/threonine kinase receptor complex containing type I and II components. Many TGF-β ligands are secreted in a latent form that cannot bind their receptor, due to the pro-region remaining associated with the mature ligand in a noncovalent complex after proteolytic cleavage. Here we show that anti-Müllerian hormone (AMH), a TGF-β family ligand involved in reproductive development, must be cleaved to bind its type II receptor (AMHRII), but dissociation of the pro-region from the mature C-terminal dimer is not required for this initial interaction. We provide direct evidence for this interaction by showing that the noncovalent complex binds to a soluble form of AMHRII in an ELISA format and to AMHRII immobilized on Sepharose. Binding of the noncovalent complex to Sepharose-coupled AMHRII induces dissociation of the pro-region from the mature C-terminal dimer, whereas no dissociation occurs after binding to immobilized AMH antibodies. The pro-region cannot be detected after binding of the AMH noncovalent complex to AMHRII expressed on COS cells, indicating that pro-region dissociation may occur as a natural consequence of receptor engagement on cells. Moreover, the mature C-terminal dimer is more active than the noncovalent complex in stimulating Sma- and Mad-related protein activation, suggesting that pro-region dissociation contributes to the assembly of the active receptor complex. AMH thus exemplifies a new mechanism for receptor engagement in which interaction with the type II receptor promotes pro-region dissociation to generate mature ligand.

Natural Mutations of the anti-Müllerian Hormone type II Receptor Found in Persistent Müllerian Duct Syndrome affect Ligand Binding, Signal Transduction and Cellular Transport

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.

Anti-Müllerian Hormone Recruits BMPR-IA in Immature Granulosa Cells

Anti-Müllerian hormone (AMH) is a member of the TGF-β superfamily secreted by the gonads of both sexes. This hormone is primarily known for its role in the regression of the Müllerian ducts in male fetuses. In females, AMH is expressed in granulosa cells of developing follicles. Like other members of the TGF-β superfamily, AMH transduces its signal through two transmembrane serine/threonine kinase receptors including a well characterized type II receptor, AMHR-II. The complete signalling pathway of AMH involving Smads proteins and the type I receptor is well known in the Müllerian duct and in Sertoli and Leydig cells but not in granulosa cells. In addition, few AMH target genes have been identified in these cells. Finally, while several co-receptors have been reported for members of the TGF-β superfamily, none have been described for AMH. Here, we have shown that none of the Bone Morphogenetic Proteins (BMPs) co-receptors, Repulsive guidance molecules (RGMs), were essential for AMH signalling. We also demonstrated that the main Smad proteins used by AMH in granulosa cells were Smad 1 and Smad 5. Like for the other AMH target cells, the most important type I receptor for AMH in these cells was BMPR-IA. Finally, we have identified a new AMH target gene, Id3, which could be involved in the effects of AMH on the differentiation of granulosa cells and its other target cells.

Anti-Müllerian Hormone Deficiency and Resistance

Anti-Mullerian hormone causes the regression of the Mullerian ducts in the male fetus. AMH is expressed at high levels by testicular Sertoli cells from early fetal life through puberty. The human AMH gene maps to chromosome 19. AMHR2 gene, encoding the AMH type 2 receptor, maps to chromosome 12. Mutations in either gene are responsible for the Persistent Mullerian Duct Syndrome (PMDS), characterized by the presence of the uterus and fallopian tubes in an otherwise normally virilized male. AMH is also a useful serum biomarker of testicular function in pediatric patients.

[29] – Detection of DNA in Southern Blots with Chemiluminescence

Publisher Summary The analysis of DNA by membrane hybridization techniques is important in the characterization of cloned genes, investigation of genetic diseases, detection of pathogens, forensic determinations, as well as many other areas of biology. The analysis of DNA by chemiluminescence is discussed in this chapter. Chemiluminescent organic reactions can be classified in several ways. The majority of these reactions require a critical step that involves oxidation of a substrate with molecular oxygen or its synthetic equivalent. The oxidation of luminol is a classic example of a chemiluminescent reaction in which the key oxidative step involves hydrogen peroxide and aminophthalhydrazide in the presence of suitable catalysts. The mechanism leading to AMPPD luminescence in the presence of alkaline phosphatase involves two steps. In the first step, dephosphorylation by alkaline phosphatase occurs, generating a moderately stable anion, AMP-D. The second step involves a further breakdown of AMP-D to adamantanone and the charge-transfer excited state of methyl m-oxybenzoate anion, which emits light. Chemiluminescence detection with AMPPD can be performed using various experimental formats, such as solution, bead, and membrane hybridizations.