Our long-term goals are to study transmembrane signaling via heterotrimeric G-protein in development. The structure of G-proteins have been solved, as have many features of their roles in signaling. Unsolved, however, is the role of specific G-proteins in mediating development. In the current award period, we provide compelling evidence that G-proteins play obligate roles in development, demonstrated in Xenopus, zebrafish and mammalian cells. Using rat P19 embryonal carcinoma stem cells, we have reported that Galpha13 signaling via MEKK, MEK, and c-Jun N- terminal kinase is obligate for morphogen-induced formation of primitive endoderm. For the next award period, we propose to study downstream signaling between Galpha13 and MEKKs, more specifically three important targets: TEC kinases, small molecular weight G-proteins (smgs), and GEFs. Dominant negative and constitutively activated versions of key molecules will be expressed to test the central hypotheses of this signal paradigm. We now show that morphogen induces activation of canonical beta-catenin pathway to Lef/Tcf-sensitive genes. The beta-catenin pathway activated by morphogen can be activated independently by use of novel Frizzled homolog, chimeric receptors with the beta2-adrenergic receptor. Activation/translocation of the novel protein Dishevelled (Dvl) are critical to beta-catenin stabilization and, the presence of DEP domain in Dvl may provide another possible linkage to smgs (RhoA) and thereby to JNK. Dvl mutants lacking specific protein-binding motifs, like the DEP domain, will be expressed to define the role of each in morphogen action. Dvl participates in a complex composed of beta-catenin, APC, GSK-3, PP2-A, with Axin. Axin has been shown to act as a scaffold for the complex and recently to possess an MEKK-binding site implicated in signaling. We propose to test the exciting hypothesis that Axin acts as integrator of signals emanating from both Galpha13 and beta- catenin, mediating activation of JNK and beta-catenin- regulated Lef/Tcf-sensitive genes. Each of these pathways are implicated in developmental defects and cancer in humans.
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