IDENTIFICATION OF A RECEPTOR FOR HUMAN MULLERIAN-INHIBITING SUBSTANCE

Mullerian inhibiting substance (MIS), a Sertoli cell-derived glycoprot ein and member of the transforming growth factor-beta supergene family , plays a key down-stream role in mammalian sex determination. Identif ication of a receptor for MIS has now been achieved in a MIS-responsiv e human vulvar carcinoma cell line, A431, using fluorescein isothiocya nate labeling of recombinant human MIS (FITC-rhMIS) and RRAs with iodi nated carboxy-terminal rhMIS. Confocal fluorescence microscopy of A431 cells incubated on ice with 30-nm concentrations of covalently bound FITC-rhMIS reveals specific punctate cell surface fluorescent signal. Cytosolic fluorescent signal is seen after incubation at 37 C for 1 h as well as occasional apparent perinuclear accumulation. FITC-rhMIS co incubated with molar excesses of unlabeled rhMIS in A431 cells elimina tes cell surface and cytosolic fluorescent uptake. Double label experi ments with FITC-rhMIS and tetramethyl rhodamine isothiocyanate epiderm al growth factor establish separate binding of each ligand, displaceab le, respectively, by large molar excesses of unlabeled rhMIS or epider mal growth factor. RRAs reveal a single, high affinity (K(d), 5.8 nm), saturable, low abundance binding species for carboxy-terminal rhMIS. Solubilized supernatants of A431 whole cells cross-linked with I-125-c arboxy-terminal rhMIS identify a band with a mol wt of 88,000 on elect rophoresis and autoradiography. This identification of a MIS receptor in A431 cells now permits the design of affinity purification protocol s using rhMIS, followed by direct protein microsequencing.