Glial cell line-derived neurotrophic factor (GDNF)-dependent activation of the tyrosine kinase receptor RET is necessary for kidney and enteric neuron development, and mutations in RET are associated with human diseases. Activation of RET by GDNF has been shown to require an accessory component, GDNFR-␣ (RETL1). We report the isolation and characterization of rat and human cDNAs for a novel cell-surface associated accessory protein, RETL2, that shares 49% identity with RETL1. Both RETL1 and RETL2 can mediate GDNF dependent phosphorylation of RET, but they exhibit different patterns of expression in fetal and adult tissues. The most striking differences in expression observed were in the adult central and peripheral nervous systems. In addition, the mechanisms by which the two accessory proteins facilitate the activation of RET by GDNF are quite distinct. In vitro binding experiments with soluble forms of RET, RETL1 and RETL2 demonstrate that while RETL1 binds GDNF tightly to form a membrane-associated complex which can then interact with RET, RETL2 only forms a high affinity complex with GDNF in the presence of RET. This strong RET dependence of the binding of RETL2 to GDNF was confirmed by FACS analysis on RETL1 and RETL2 expressing cells. Together with the recent discovery of a GDNF related protein, neurturin, these data raise the possibility that RETL1 and RETL2 have distinctive roles during development and in the nervous system of the adult. RETL1 and RETL2 represent new candidate susceptibility genes and͞or modifier loci for RETassociated diseases.

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.

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Anti-Müllerian hormone (AMH), a TGF-beta family member, determines whether an individual develops a uterus and Fallopian tubes. Mutations in the AMH gene lead to persistent Müllerian duct syndrome in males. The wild-type human AMH protein is synthesized as a disulfide-linked dimer of two identical 70-kDa polypeptides, which undergoes proteolytic processing to generate a 110-kDa N-terminal dimer and a bioactive 25-kDa TGF-beta-like C-terminal dimer. We have studied the biosynthesis and secretion of wild-type AMH and of seven persistent Müllerian duct syndrome proteins, containing mutations in either the N- or C-terminal domain. Mutant proteins lacking the C-terminal domain are secreted more rapidly than full-length AMH, whereas single amino acid changes in both domains can have profound effects on protein stability and folding. The addition of a cysteine in an N-terminal domain mutant, R194C, prevents proper folding, whereas the elimination of the cysteine involved in forming the interchain disulfide bond, in a C-terminal domain mutant, C525Y, leads to a truncation at the C terminus. A molecular model of the AMH C-terminal domain provides insights into how some mutations could affect biosynthesis and function.

Mullerian inhibiting substance (MIS)/anti-Mullerian hormone is a differentiation factor that causes regression of the Mullerian duct in the developing male fetus and an apparent sex reversal of the fetal ovary when inappropriately exposed to it. The purified product is a 140-kilodalton glycoprotein composed of two identical subunits. We show that a C-terminal fragment of MIS, which shares homology with transforming growth factor-beta, causes regression of the Mullerian duct and inhibits the biosynthesis of aromatase in the fetal ovary. However, both activities are enhanced dramatically by addition of the N-terminal portion of MIS. Under conditions where potentiation occurs, the N- and C-terminal domains of MIS reassociate. These results indicate that the N-terminus of MIS, unlike that of the other members of the transforming growth factor-beta family, plays a role in maintaining the biological activity of the C-terminus.