9218823 Gundersen Recent reports describing in vivo phosphorylation of the guanine nucleotide-binding regulatory protein (G protein) alpha subunits, G alpha-i in hepatocytes, G alpha-z in platelets, and G alpha-2 in Dictyostelium suggest that alpha subunit phosphorylation is an important mechanism of G protein function. The significance of alpha subunit phosphorylation however remains unknown. Dictyostelium offers a powerful system for analyzing this question through the use of molecular biology and biochemical methodologies. The goal of this research is to examine the role of G alpha-2 phosphorylation in Dictyostelium. The site(s) of phosphorylation, which occurs on serine in G alpha-2. will be identified using peptide mapping, alpha subunit chimeral, and site-directed mutagenesis. The function of G alpha-2 phosphorylation will be determined by transforming G alpha-2 sequences that can not be phosphorylated into a cell line lacking the G alpha-2 gene. Chemotaxis and differentiation in the transformants will be compared to wild-type. The G alpha-2 effectors adenylyl cyclase, guanylyl cyclase and phospholipase C will be assayed to identify changes resulting from the loss of G alpha-2 phosphorylation. Phosphorylated and nonphosphorylated G alpha-2 will be purified to compare GTP binding affinity and GTPase. Purified G alpha-2 will also be used to produce a polyclonal antiserum. The kinase that phosphorylates G alpha-2 will be characterized by developing an in vitro assay and purified using standard chromatographic approaches. %%% Guanine nucleotide-binding regulatory proteins (G proteins) play an essential role in the signal transducing pathways of numerous extracellular stimuli which include hormones, neurotransmitters, growth factors, odorants, and light. Recent research has identified a number of stimulus-response pathways in which G proteins play a role, and a number of types of G protein subunits have been identified. Thus, the system for signal transduction in cells is very complex. In fact, most stimuli probably do not activate a single biochemical response pathway, but instead activate a network of response pathways. A key question is, how to cells coordinate and regulate these diverse networks of responses to come out with an appropriate response to an environmental or internal stimulus? G proteins play a key role in signal transduction pathways and are likely to be important sites for modulation. The goal of this research is to understand the mechanisms that modify G proteins by covalent attachment of phosphate groups (phosphorylation) under some physiological conditions. The system to be used is the slime mold, Dictyostelium. This relatively simple organism offers important experimental advantages for these studies. The results of this research should provide important new insights into the molecular mechanisms that regulate the responses of cells to environmental and internal signals. ***
