G protein-coupled receptors represent one of the most important pharmacological targets for the treatment of cardiovascular and pulmonary diseases. Receptors of this class are well known to transmit signals to intracellular effector enzymes, including members of the mitogen activated protein (MAP) kinase family. Using yeast as a model system, we have recently found that a G protein alpha subunit Gpa1 can also activate the phosphatidylinositol 3-kinase Vps34. There are several novel features of this finding. First, activated Gpa1 binds directly to Vps34 at the endosome rather than at the plasma membrane, where G proteins and effectors are typically found. This interaction leads to elevated production of the phospholipid second messenger phosphatidylinositol 3-phosphate (PI3P). Second, PI3P binds directly to the MAP kinase Fus3 and apparently promotes its recruitment to the endosome. Third, activated Gpa1 binds directly to Fus3 and may further contribute to its recruitment and activation at the endosome. Fourth, unactivated Gpa 1 binds directly to Vps15, a protein kinase required for Vps34 activity. Our goal is to determine the functional consequences of these unprecedented interactions, and to eventually extend these findings to homologous proteins in mammalian cells. To achieve our objective, three specific aims are proposed:
Aim 1. Determine the function of MAP kinase binding to PI3P at the endosome. Our hypothesis is that PI3P binding leads to the recruitment and activation of Fus3 at the endosome. We further hypothesize that selective recruitment of Fus3 but not other MAP kinases contributes to signal specificity. Here we will determine how PI3P at the endosome modulates Fus3 activity and substrate-specificity.
Aim 2. Determine the function of MAP kinase binding to the G protein. Our hypothesis is that Gpa1 recruits and activates Fus3 at the endosome, possibly in conjunction with PI3P. Here we will determine how Gpa1, PI3P and other Fus3-binding proteins cooperate to modulate Fus3 activity and substrate specificity.
Aim 3. Determine the function of Vps15 protein kinase activity at the endosome. Vps15 kinase activity is required for Vps34 PI3-kinase function. Our hypothesis is that unactivated Gpa1 inhibits Vps15 kinase activity, and in this manner attenuates Vps34 signaling. Here we will determine how Gpa1 modulates the function of Vps15 and identify protein substrates for Vps15 kinase activity. These experiments will reveal the mechanism by which Gpa1 and Vps 15 cooperate to regulate Vps34-catalyzed PI3P production. The underlying premise of this work is that new drug therapies will come from the discovery of new drug targets. Consequently, a fuller understanding of the different ways that cells transmit their signals, particularly those that act in new and unexpected locations within the cell, could eventually lead to fundamentally new therapies for human disease. Delineating such new pathways is the principal objective of this proposal. ? ? ?
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