The Saccharomyces cerevisiae mating pheromone response pathway is arguably the best understood multi-tiered, MAPK signaling cascade in any eukaryote. However, coordinating the changes in gene expression and cell morphology necessary for mating involves an elaborate network of interlocking events rather than a simple linear pathway. In addition, feedback mechanisms exist to modulate the efficiency and duration of these signaling events at essentially every step. Moreover, this signaling pathway must evoke an appropriate response upon the correct stimulus, yet avoid adventitious activation. Also, it is now appreciated that many components required for pheromone response are also utilized for a different developmental outcome, termed filamentous/invasive growth, in response to nutrient limitation. How different extracellular signals impinge on the same MAPK cascade, yet are deciphered differently, is not understood fully in any organism. Yeast continues, therefore, to provide opportunities to examine basic aspects of the organization, specificity, fidelity, and regulation of MAPK signaling pathways, including how the same components can be coupled to different upstream inputs and downstream responses in the same cell type. Specific aims include: (1) Characterization of Ste5 scaffold protein, including crystal structure determination, elucidating the mechanism of its regulated nucleocytoplasmic transport, and genetic and biochemical analysis of the role of its RING-H2 domain in Ste5 oligomerization, in intra- and intermolecular ubiquitinylation, and in interaction with Gbeta/gamma (Ste4-Ste18). (2) Development of a new method for visualization of signaling protein dynamics in real time in live cells based on exploitation of the fluorescent properties of cyanobacterial phycobiliproteins, also applicable to functional genomics. (3) Genetic and biochemical characterization of the adaptor protein, Ste50, and its interaction with the small GTPase, Cdc42, including solution structure determination. (4) Testing a hypothesis, using yeast RGS protein, Sst2, that DEP domains associate with Gbeta/gamma. (5) Exploring the possibility that the requirement for PKA function in filamentation is to sustain MAPK signaling by blocking MAPK phosphatase action. (6) Investigating the role of phosphoinositides in membrane recruitment of Bem1 adaptor protein and in the mechanisms by which pheromone signaling interdicts both bud formation and cytokinesis. (7) Biochemical characterization of repression and promoter-specific discrimination by transcriptional regulators, Dig1 and Dig2, in Ste12-dependent gene expression. Studying yeast MAPK signaling may provide insights for anti-cancer therapy because inappropriate MAPK activation in humans, evoked by well-known oncoproteins (e.g. Ras and Raf), leads to tumor formation.