Summary/Abstract: Heterotrimeric G proteins are the major intracellular enzymes that receive signals from plasma membrane G protein coupled receptors (GPCRs) and transduce them to a broad array of intracellular effectors. Our lab has contributed much to the understanding of the function of a pair of G protein regulators, Ric-8A and Ric-8B. We identified Ric-8A and Ric-8B as G protein subunit binding proteins and showed that they possess the ability to accelerate the rate at which G subunits release GDP and bind GTP. This activity is similar to GPCR action towards G protein heterotrimers. GPCRs stimulate G protein GDP/GTP exchange for the purpose of activating the G proteins to transmit signals. We have yet to find conclusive evidence that Ric-8 proteins facilitate G GDP/GTP exchange for signaling purposes. We did find that deletion of the Ric-8A or Ric-8B genes in mice caused embryonic lethality. Culture of Ric-8A or Ric-8B knockout embryonic stem cells that were attained from viable embryos prior to death demonstrated that the cells had dramatically reduced levels of G proteins. This prompted our investigation into the role that Ric-8 proteins have in regulating G protein abundance. We found that Ric-8 proteins are molecular chaperones that facilitate protein folding of newly made G subunits. When G proteins are made in cells lacking Ric-8 proteins, they are misfolded and rapidly degraded. We reconcile the in vitro GDP/GTP exchange stimulatory activity of Ric-8 with the folding function by proposing that Ric-8 proteins bind the intermediate of the in vitro exchange reaction in cells, newly-synthesized, nucleotide-free G proteins to facilitate first time GTP binding. Until now, all evidence indicated that Ric-8 proteins acted constitutively to fold G proteins. The premise of this new application is based on our recent data that show that Ric-8 activities are subject to dramatic regulation by post-translational phosphorylation. We will investigate a new link between mitogenic oncogene stimulus that leads to Ric-8 deregulation (dephosphorylation) and possible remodeling of cellular G protein levels. We have made important strides in our long-time collaborative efforts to investigate the structural basis by which Ric-8 proteins regulate G proteins. The work in this application will define the way that phosphorylation regulates Ric-8 function.