Cells sense and respond to their environment when receptors on their surface are activated by stimuli such as light, hormones or chemical substances. Many cell surface receptors function by interacting with proteins known as G-proteins, and thus are called G-protein-coupled receptors (GPCRs). G-proteins, in turn, activate or inhibit various "effector" proteins such as enzymes or ion channels, leading to changes in cellular processes such as gene expression, cell division and cell migration. While much is known about GPCRs and G-protein-signaling, the molecular arrangement of these molecules before and during signaling is not fully understood. In particular, it is not known if GPCRs and G-proteins are bound to each other before the receptors are activated, or if these molecules interact with each other only after receptor activation. In addition, inactive G-proteins are made up of 2 functional components (G alpha and G beta/gamma) bound together as a Galpha-beta-gamma complex. It is thought that GPCRs activate G-proteins by promoting the dissociation of this complex into separate G alphaand G beta/gamma subunits, but this idea has not been thoroughly examined in living cells. This project will use a newly developed imaging technique to address these questions in intact cells. Thus this project will provide new information concerning the fundamental mechanisms of signaling molecules that serve critical functions in organisms ranging from plants to yeast to human beings. In addition, this will provide a valuable new tool for the study the study of protein-protein interactions in living cells. This technique is simple to implement, requires equipment that is already widely available, and builds on an extensive literature devoted to the study of binding interactions with fluorescence recovery after photobleaching (FRAP). Several graduate and undergraduate students will be involved in the project where they will learn concepts and techniques in the fields of cell biology and biophysics.
