The long term objective of this proposal is to develop a detailed molecular understanding of signaling through G protein pathways. Many hormones, neurotransmitters, autocrine and paracrine factors function through receptors that use heterotrimeric G proteins as transducers. Both G protein alpha-subunits as well as the betagamma subunits regulate effectors. Among the best studied of the effectors is adenylyl cyclase (AC2). In the previous term, we had identified a region of AC2 involved in receiving signals from Gbetagamma subunits. This region contains a functionally important motif that also plays a role in Gbetagamma regulation of Ca2+ and K+ channels. We have also identified the region 85-145 of Gbeta subunits as being important for interactions with effectors such as AC2 and AC1, as well as phospholipase C-beta2. We have resolved this region into a signal transfer region (STR) and general binding domains (GBD). We have also identified a region (aa 660-682) of AC6 involved in low-affinity interaction with Galphas. A Ser in this region, when phosphorylated by protein kinase A results in blockade of AC6 stimulation by high concentrations of Galphas. Research in the current term build on these findings. We will study signal transfer from Gbetagamma subunits to effectors such as adenylyl cyclase and phospholipase C-beta2. We will analyze in detail STRs and GBDs by site directed mutagenesis and by peptide mimetics. We will evaluate the role of the C-terminal region of Ggamma and the importance of the prenyl group at the C-terminus in effector interactions and signal transfer to the effector. We will also analyze the regions of adenylyl cyclases involved in receiving signals from Galphas and Galphai. Additionally, we will study how protein kinase A regulates signaling by these Galpha subunits in an isoform specific manner. In a first step towards developing definitive structural information about the full length adenylyl cyclase, we propose to set up a purification procedure to produce mg quantities of the AC6 that would be suitable for crystallization. It is anticipated that these studies will lead to the identification of key intracellular sites that are potential targets for therapeutic agents that have their effects by modulation of G protein signaling pathways.
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