The specific research in this proposal is directed toward two members of the FIGS protein family throught to be important in proper neuronal functioning: RGS12 and RGS14. Discovery of putative Galphai/o- binding (""""""""GoLoco"""""""") motifs has led us to the hypothesis that RGS12 and RGS14 coordinate heterotrimeric G-protein signaling pathways by modifying the GDP/GTP cycle or serving as scaffolds for Galpha subunits. We will determine the selectivity and structural determinants of RGS12 and RGS14 GoLoco regions by a combination of yeast-two hybrid, in vitro coprecipitation, surface plasmon resonance binding assays, and co-immunoprecipitation studies. We will also determine the effects of GoLoco association on the Galpha GDP/GTP-cycle through in vitro measurements of nucletide binding and hydrolysis. Finally, using x-ray crystallography, we will ascertain the atomic resolution structure of a Galpha/GoLoco complex to understand, at the molecular level, the specificity and activity demonstrated by GoLoco motifs. Our long term objectives are to define the molecular mechanisms governing RGS protein activity to provide future therapeutic targets for disease states and clinical disorders associated with aberrant Gprotein coupled receptor signaling. The initial objectives outlined in this proposal are to define the molecular basis of the binding specificity, structural determinants, and biochemical activities of isolated GoLoco regions of RGS12 and RGS14 in order to better understand the role that RGS12 and 14 play in regulating signal transduction in neurons.
| Webb, Christina K; McCudden, Christopher R; Willard, Francis S et al. (2005) D2 dopamine receptor activation of potassium channels is selectively decoupled by Galpha-specific GoLoco motif peptides. J Neurochem 92:1408-18 |
| McCudden, C R; Hains, M D; Kimple, R J et al. (2005) G-protein signaling: back to the future. Cell Mol Life Sci 62:551-77 |
| Willard, Francis S; Kimple, Randall J; Siderovski, David P (2004) Return of the GDI: the GoLoco motif in cell division. Annu Rev Biochem 73:925-51 |
| Kimple, Randall J; Willard, Francis S; Hains, Melinda D et al. (2004) Guanine nucleotide dissociation inhibitor activity of the triple GoLoco motif protein G18: alanine-to-aspartate mutation restores function to an inactive second GoLoco motif. Biochem J 378:801-8 |
| Willard, Francis S; Kimple, Randall J; Kimple, Adam J et al. (2004) Fluorescence-based assays for RGS box function. Methods Enzymol 389:56-71 |
| Kimple, Randall J; Willard, Francis S; Siderovski, David P (2004) Purification and in vitro functional analyses of RGS12 and RGS14 GoLoco motif peptides. Methods Enzymol 390:416-36 |
| Martin-McCaffrey, Luke; Willard, Francis S; Oliveira-dos-Santos, Antonio J et al. (2004) RGS14 is a mitotic spindle protein essential from the first division of the mammalian zygote. Dev Cell 7:763-9 |
| Kimple, Randall J; Jones, Miller B; Shutes, Adam et al. (2003) Established and emerging fluorescence-based assays for G-protein function: heterotrimeric G-protein alpha subunits and regulator of G-protein signaling (RGS) proteins. Comb Chem High Throughput Screen 6:399-407 |
| Rojas, Rafael J; Kimple, Randall J; Rossman, Kent L et al. (2003) Established and emerging fluorescence-based assays for G-protein function: Ras-superfamily GTPases. Comb Chem High Throughput Screen 6:409-18 |
| Kimple, Randall J; Willard, Francis S; Siderovski, David P (2002) The GoLoco motif: heralding a new tango between G protein signaling and cell division. Mol Interv 2:88-100 |
