There is now substantial evidence that ligands for certain G- protein coupled receptors can stimulate pathways that lead to growth and gene expression. The objective of the proposed research is to understand how this occurs and what distinguishes G-protein coupled receptors that lead to these responses from those that do not. This questions will be addressed by comparing the M3 muscarinic cholinergic (mAChR) and thrombin receptor in 1321N1 astroglial cells. Both receptors activate phospholipase C, phospholipase D and protein kinase C and induce immediate early genes. However, only thrombin activates Ras, induces transcriptional activation of AP-1 sensitive genes, and is mitogenic. To examine the divergent effects of these two G-protein coupled receptors in 1321N1 cells, we propose the following: (1) to identify the G-proteins that interact with and are activated by mAChR and thrombin receptors, using co-immunoprecipitation of G- proteins and receptors with receptor antibodies and analyzing agonist stimulated binding of labelled guanine nucleotides to specific G-proteins using G-protein antibodies. (2) to determine which G protein subunits mediate the genetic and mitogenic responses to thrombin by examining AP-1 mediated increases in gene expression and BrdU incorporation into DNA following microinjection of antibodies to G-proteins and microinjection ore transient expression of activated G-protein subunit expression constructs. (3) to examine the kinetics of PKC, Raf-1 kinase, and MAP kinase activation in response to thrombin and mAChR stimulation, analyze these responses in relationship to the time established for commitment to thrombin-induced mitogenic and genetic responses, and express or microinject dominant interfering mutants and antibodies to delineate involvement of these kinases in thrombin-induced mitogenesis and gene expression. (4) to define the pathways through which the thrombin receptor activates Ras, examining the ability of thrombin to increase guanine nucleotide releasing factor (GNRF) activity using purified GTP-labelled Ras protein, and testing antibodies and proteins corresponding to SH2, SH3 and PH domains of known GNRFs and adaptor molecules to determine whether similar proteins participate in thrombin-receptor mediated activation of Ras-dependent responses. These experiments should lead to a better understanding of the potential for hormones that activate G-protein receptors to serve as physiological regulators of cell growth and gene expression.
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