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.
Showing the most recent 10 out of 29 publications
