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 Ga-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 biologylbioinformatics (Siderovski), and structure (Sondek) of G protein signaling. A protein core will play a central role in the PPG by facilitating highthroughput 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-e 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-e in Ras-promoted cell transformation, and the domain(s) in PLC-e that interacts with Ga- subunits. Project III will focus on new signaling roles of the yeast Ga-subunit Gpa1, new effector roles of the yeast RGS protein Sst2, and the role of Scp160 in transmitting both downstream signals acting as a Gpa1 activated RNA binding protein and upstream signals as a modulator of Gpa1, potentially as a novel RGS protein. The delineation of novel signaling activities in yeast will guide complementary studies in mammalian systems. Project IV will establish the structural basis for several functional interactions mediating cross-talk in G protein signaling by solving the structures of the ?5-R7 dimer and the GoLoco domain of R12-family RGS proteins, which has been shown to inhibit GDP release by Gai-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 ? ? Many cell stimulators act via cell surface receptors, G proteins, and effector enzymes. RGS proteins inactivate the signal by accelerating G protein GTPase activity. In yeast the beta/gamma subunits (Ste4/Ste18) have long been regarded as the signal-transmitting component of the G protein. We identified recently a positive signaling role for the G protein alpha subunit (Gpa1). Our goal is to identify effectors of the Gpa1-initiated signaling pathway(s). Our hypothesis is that the Gpa1 effectors include the RGS protein Sst2 and a novel RNA binding protein Scp160, since deletion of either blocks Gpa1 signaling. Our approach will be to determine how Sst2 and Scp160 transmit the downstream signal, and establish whether Scp160 accelerates Gpa1 GTPase activity, in the manner of Sst2. There are three specific aims of the proposal:
Aim 1 : Determine the mechanism of Sst2 effector activity. Two-hybrid screening revealed a number of proteins that bind selectively to Sst2 or Gpa1. We will confirm each interaction, identify additional binding partners; and through deletion and overexpression of each gene we will establish their role in Gpa1-initiated signaling in vivo Aim 2: Determine the mechanism of Scp160 effector activity. Scp160 was previously shown to bind mRNA. We will determine if Scp160 recruits selected mRNAs to the site of active signaling by sequencing RNAs bound to Scp160, and determine if these RNAs or their encoded protein translocate to the mating projection upon signal activation. We will determine if Scp160 transmits a signal through these RNA-encoded proteins, through deletion and overexpression of each gene.
Aim 3 : Determine the mechanism of Scp160 desensitization. Deletion of the SCP160 gene results in a pheromone-supersensitive phenotype, resembling that of the sst2 deletion mutant. We will map the Gpa1-binding domain of Scp160, and determine its ability to accelerate Gpa1 GTPase activity. ? ? ?
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