The overall goal of our research is to understand how the large number of G protein alpha and betagamma isoforms lead to specificity in cell signaling, especially how the multiple isoforms of the betagamma dimer selectively regulate signaling. The goal of this project period is to understand the interaction between two major signaling pathways in the cell membrane, one utilized by Gi linked receptors to activate hematopoietic cells and another used by Gs linked receptors to inhibit activation of these cells. In neutrophils and macrophages, activation of Gi coupled receptors release the bg dimer activating superoxide production, cell shape changes and cell migration. Cellular targets for bg dimers include phosphatidylinositol 3-kinase and the Rac GEF, PRex1. Activation of Ptdlns 3-kinase generates PIP3 in the plasma membrane and Rac is a central mediator of superoxide generation and migration. As P-Rex1 activation is modulated by PIP3 and the G protein betagamma subunit, the synergistic actions of the betagamma dimer on PI 3-kinase and P-Rex1 combine to activate neutrophils and macrophages. Receptors that raise cyclic AMP levels inhibit the activation of hematopoietic cells. Thus, phosphorylation of important regulatory sites in hematopoietic cells is central to the inhibitory response. We discovered that P-Rex1 can be phosphorylated in vitro by the cyclic AMP-dependent protein kinase (PKA). We propose to explore the regulation of the Rac specific GEF, P-Rex1, by the signals generated following activation of Gi and Gs coupled receptors. The work will be performed via the following Specific Aims.
Aim 1 : To examine the ability of pure G protein a and bg subunits to regulate P-Rex1 in synthetic lipid vesicles containing PIP3 and Rac to determine which G protein subunits modify P-Rex1 activity. We will also determine how P-Rex1 responds to activation of G protein coupled receptors in HEK-293 cells, macrophages and HL60 cells.
Aim 2 : We will phosphorylate pure, recombinant P-Rex1 with PKA and examine the effect of phosphorylation on P-Rex1 activity in the presence of PIP3 and G protein betagamma subunits. We will also determine the signals leading to phosphorylation of P-Rex1 in cells and what amino acid residues are phosphorylated by activation of PKA in cells.
Aim 3 : We will use small, inhibitory RNA's delivered to HEK-293 cells, mouse macrophages or HL-60 cells by stable infection with plasmids or retroviruses to determine which isoforms of G proteins regulate P-Rex1 activity in a cellular context. RNA expression will be verified by QPCR and protein expression will be examined by western blotting with selective antibodies. Parameters examined will include: receptor - alpha:betagamma interaction, adenylyl cyclase (cAMP), PLC-beta (Ca2+), Ptdlns 3-kinase (PIP3), P-Rex1 (Rac activation) and cell migration.
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