A subset of G-protein coupled receptors, including those for thrombin and the lysophospholipids S1P and LPA, have a remarkable capacity to promote DNA synthesis and actin cytoskeletal rearrangement in mammalian cells. These receptors couple not only to Gq to stimulate phospholipase C (PLC) but also to G12/13 to activate Rho. The coordinate regulation of these two signaling pathways elicits a unique cellular response profile which suggests a role for these receptors and their ligands in cellular responses to injury. Whether Gq and G12 signaling are mediated through one or different subtypes of the receptor, and how these responses are coordinated, is an important question central to the aims of this proposal.
In Aim #1, the capacity for Gq versus G12/13 to regulate PLC, and the contribution of these pathways to the activation of phosphoinositide (PI) hydrolysis by GPCR agonists will be explored. The hypothesis to be tested is that there is more than a single pertussis toxin-insensitive pathway for regulating PLC and that some receptor subtypes have the potential to induce PI hydrolysis through a G12/13 mediated activation of PLC epsilon. Proposed studies will examine PLC activation by endogenous PAR1 thrombin and LPA receptors in 1321N1 cells, and use cells from S1P and LPA receptor knockout mice to ask if subtypes of these receptors stimulate PLC via different mechanisms.
Aim #2 examines the role of specific Rho family proteins and of altered PIP2 levels in mediating GPCR effects on the cytoskeleton. The hypothesis to be tested is that distinct patterns of Rho small G-protein activation are associated with the cytoskeletal responses to GPCR agonists and that there is a relationship between the ability to regulate cellular PIP2 levels and the activation or localization of the small G-proteins. Proposed experiments will examine the kinetics of activation of various Rho family proteins, their relationship to cell rounding, invasiveness and migration, and the role of the PIP2 in regulation of these responses.
Aim #3 examines the role of the lysophospholipid receptors in astroglial and vascular smooth muscle cell migration and proliferation and in pathophysiological responses in vivo. The hypothesis to be tested is that LPA and SIP receptors regulate vascular smooth muscle cell (VSMC) and astroglial cell proliferation and migration through specific receptor-mediated signaling pathways and that these ligands play a critical role in responses to cell injury. Proposed experiments will utilize interventions that induce vascular or brain injury to test the pathophysiological role of these receptors in mediating astrogliosis and intimal thickening. The basis for the observed changes will be related to information regarding LPA and S1P signaling pathways regulating migration and proliferation in astrocytes and VSMC from WT and LPA or S1P receptor knockout mice.
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