Mullerian Inhibiting Substance (MIS), as described by Jost over 40 years ago, is a fetal testicular inhibitor responsible in the male embryo for regression of the Mullerian ducts, the anlagen of the uterus, Fallopian tube, and vagina. We speculated that the regression caused by MIS in fetal tissue could be recapitulated in Mullerian derived tumors such as cervical, endometrial, Fallopian tube, and ovarian carcinomas. To pursue this hypothesis we first purified bovine MIS and then cloned the bovine and human MIS genes. We scaled up produCtion of recombinant human MIS from Chinese hamster ovary (CHO) cells transfected with the MIS gene. After purification to homogeneity, we then found that holo-MIS must be cleaved to be activated and that the small C-terminal portion of the molecule is largely responsible for the biological activity, although the larger N- terminal domain may contribute to this activity. To characterize the receptor for Mullerian Inhibiting Substance, we have cloned and sequenced four full length cDNAs encoding novel serine/threonine kinase transmembrane proteins (R1-R4), one of which, R1, cross-links labelled MIS and localizes by in situ hybridization to MIS target tissues, the fetal Mullerian duct and the developing oocyte. Overexpression of R1 in MIS sensitive A431 cells resulted functionally in enhanced inhibition of EGF receptor autophosphorylation. MISR1 is thus a candidate Component for the MIS receptor. We will prove MIS binding to endogenous R1 by co-precipitation with specific antibodies to R1 and correlate MIS specific functional activity with cross-linking. """"""""Protein Interaction Trap"""""""" strategy will be used to screen libraries constructed in yeast to uncover transmembrane proteins that interact with R1C. Thus we hope to discover MIS specific type II receptor. The ultimate test of MISR1 function can be assessed by using homologous recombination to generate a null mutation. We have constructed a targeting vector containing an MISR1 null mutation, for transfection into embryonic stem cells. From these we will generate homozygous R1 deficient genotypes, and assess the resulting phenotypes. Similar approaches can be used to study serine/threonine kinase receptors R2-R4. We have recent evidence that the R4 is a type I TGF-beta receptor. Expression of R4, and not R1, 2, or 3, rescues function in a TGF-beta type I receptor deficient cell line, and binding and subsequent gene expression requires that the type I receptor be kinase competent. We hope to systematically define the remaining orphan ligands which we suspect may be Activin, Inhibin, or bone morphogenesis, then to uncover their receptor component partners. Each of the serine/threonine kinase receptors and their ligands are important in reproductive function. Receptor ligand partners can be used to design target-specific regimens to assist or to control reproductive development and function, and to treat reproductive tumors.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
1R01HD032112-01
Application #
2205057
Study Section
Reproductive Biology Study Section (REB)
Project Start
1994-08-01
Project End
1999-05-31
Budget Start
1994-08-01
Budget End
1995-05-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
Fujino, Akihiro; Arango, Nelson A; Zhan, Yong et al. (2009) Cell migration and activated PI3K/AKT-directed elongation in the developing rat Müllerian duct. Dev Biol 325:351-62
Szotek, Paul P; Chang, Henry L; Zhang, LiHua et al. (2007) Adult mouse myometrial label-retaining cells divide in response to gonadotropin stimulation. Stem Cells 25:1317-25
Gupta, Vandana; Yeo, Giminna; Kawakubo, Hirofumi et al. (2007) Mullerian-inhibiting substance induces Gro-beta expression in breast cancer cells through a nuclear factor-kappaB-dependent and Smad1-dependent mechanism. Cancer Res 67:2747-56
Zhan, Yong; Fujino, Akihiro; MacLaughlin, David T et al. (2006) Mullerian inhibiting substance regulates its receptor/SMAD signaling and causes mesenchymal transition of the coelomic epithelial cells early in Mullerian duct regression. Development 133:2359-69
Tran, Trinh T; Segev, Dorry L; Gupta, Vandana et al. (2006) Mullerian inhibiting substance regulates androgen-induced gene expression and growth in prostate cancer cells through a nuclear factor-kappaB-dependent Smad-independent mechanism. Mol Endocrinol 20:2382-91
Arango, Nelson A; Huang, Tiffany T; Fujino, Akihiro et al. (2006) Expression analysis and evolutionary conservation of the mouse germ cell-specific D6Mm5e gene. Dev Dyn 235:2613-9
Szotek, Paul P; Pieretti-Vanmarcke, Rafael; Masiakos, Peter T et al. (2006) Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. Proc Natl Acad Sci U S A 103:11154-9
Gupta, V; Carey, J L; Kawakubo, H et al. (2005) Mullerian inhibiting substance suppresses tumor growth in the C3(1)T antigen transgenic mouse mammary carcinoma model. Proc Natl Acad Sci U S A 102:3219-24
Arango, Nelson A; Szotek, Paul P; Manganaro, Thomas F et al. (2005) Conditional deletion of beta-catenin in the mesenchyme of the developing mouse uterus results in a switch to adipogenesis in the myometrium. Dev Biol 288:276-83
Houk, Christopher P; Pearson, Elliot J; Martinelle, Nina et al. (2004) Feedback inhibition of steroidogenic acute regulatory protein expression in vitro and in vivo by androgens. Endocrinology 145:1269-75

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