Identification of a receptor for human müllerian inhibiting substance.
Müllerian inhibiting substance (MIS), a Sertoli cell-derived glycoprotein and member of the transforming growth factor-beta supergene family, plays a key down-stream role in mammalian sex determination. Identification of a receptor for MIS has now been achieved in a MIS-responsive human vulvar carcinoma cell line, A431, using fluorescein isothiocyanate labeling of recombinant human MIS (FITC-rhMIS) and RRAs with iodinated 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 coincubated with molar excesses of unlabeled rhMIS in A431 cells eliminates cell surface and cytosolic fluorescent uptake. Double label experiments with FITC-rhMIS and tetramethyl rhodamine isothiocyanate epidermal growth factor establish separate binding of each ligand, displaceable, respectively, by large molar excesses of unlabeled rhMIS or epidermal growth factor. RRAs reveal a single, high affinity (Kd, 5.8 nM), saturable, low abundance binding species for carboxy-terminal rhMIS. Solubilized supernatants of A431 whole cells cross-linked with 125I-carboxy-terminal rhMIS identify a band with a mol wt of 88,000 on electrophoresis and autoradiography. This identification of a MIS receptor in A431 cells now permits the design of affinity purification protocols using rhMIS, followed by direct protein microsequencing.
Catlin
E A
EA
Neonatology Unit, Massachusetts General Hospital, Boston 02114.
Ezzell
R M
RM
Donahoe
P K
PK
Gustafson
M L
ML
Son
E V
EV
MacLaughlin
D T
DT
D054304:Anti-Mullerian Hormone; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003432:Cross-Linking Reagents; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006023:Glycoproteins; D006131:Growth Inhibitors / Q000378:metabolism; D006801:Humans; D011869:Radioligand Assay; D018000:Receptors, Peptide / Q000378:metabolism*; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; D013696:Temperature; D013735:Testicular Hormones / Q000378:metabolism; D013997:Time Factors; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D006023:Glycoproteins; D006131:Growth Inhibitors; D018000:Receptors, Peptide; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; D013735:Testicular Hormones; C084621:anti-Mullerian hormone receptor; D054304:Anti-Mullerian Hormone; D016650:Fluorescein-5-isothiocyanate