G protein-mediated signaling underlies the action of many growth factors, hormones, and neurotransmitters. Although initially conceived as linear and unidirectional, many G protein-related cell signaling pathways now are known to converge (and diverge) at many levels. For example, members of the large family of RGS proteins exhibit multiple biochemical activities in addition to their signature capacity to promote G-alpha-mediated GTP hydrolysis. This program project (PPG) applies a multidisciplinary approach to gain mechanistic and structural insight into important examples of multifunctional proteins in G protein signaling. We have assembled investigators with expertise in cancer biology (Der), in signaling in yeast (Dohlman), and in the molecular pharmacology/biochemistry (Harden), molecular biology/bioinformatics (Siderovski), and structure (Sondek) of G protein signaling. A Protein Core will play a central role in the PPG by facilitating high-throughput cloning, expression, purification, and biophysical characterization of proteins. The mechanism(s) of R7-family RGS proteins in """"""""upstream"""""""" regulation of receptor/G protein interaction and cross-talk among heterotrimeric G proteins will be determined in Project I through studies with mammalian and C. elegans proteins. The multifunctional nature of the newly identified PLC-epsilon isozyme will be delineated in Project II through studies identifying interactors for the N-terminal RasGEF and C-terminal Ras-association domains, the role of PLC-epsilon in Ras-promoted cell transformation, and the domain(s) in PLC-epsilon that interacts with G-alpha-subunits. Project III will focus on the yeast protein Sst2, which was the first RGS protein discovered, and which contains an N-terminal DEP domain of unknown function also found in the mammalian R7-family RGS proteins studied in Projects I and IV. Yeast two-hybrid screens have identified proteins that bind the Sst2 N-terminal domain, and which activate the stress response signal. The domains of Sst2 responsible for G protein effector activity will be defined, as will the mechanism(s) of stress response signaling. Project IV will establish the structural basis for several functional interactions mediating cross-talk in G protein signaling by solving the structures of the beta5-R7 dimer and the GoLoco domain of R12-family RGS proteins, which has been shown to inhibit GDP release by G-alpha-i-subunits. This PPG will provide major new mechanistic insights into the molecular complexities that function across G protein signaling pathways and should illuminate new drug targets within fundamental processes that underlie diseases as diverse as cancer, heart disease, and mental disorders.
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