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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036927-13
Application #
2684832
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1982-08-01
Project End
1999-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
13
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Yu, Olivia M; Benitez, Jorge A; Plouffe, Steven W et al. (2018) YAP and MRTF-A, transcriptional co-activators of RhoA-mediated gene expression, are critical for glioblastoma tumorigenicity. Oncogene 37:5492-5507
Dusaban, Stephanie S; Chun, Jerold; Rosen, Hugh et al. (2017) Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes. J Neuroinflammation 14:111
Yung, Bryan S; Brand, Cameron S; Xiang, Sunny Y et al. (2017) Selective coupling of the S1P3 receptor subtype to S1P-mediated RhoA activation and cardioprotection. J Mol Cell Cardiol 103:1-10
Yu, Olivia M; Miyamoto, Shigeki; Brown, Joan Heller (2016) Myocardin-Related Transcription Factor A and Yes-Associated Protein Exert Dual Control in G Protein-Coupled Receptor- and RhoA-Mediated Transcriptional Regulation and Cell Proliferation. Mol Cell Biol 36:39-49
Yu, Olivia M; Brown, Joan Heller (2015) G Protein-Coupled Receptor and RhoA-Stimulated Transcriptional Responses: Links to Inflammation, Differentiation, and Cell Proliferation. Mol Pharmacol 88:171-80
Dusaban, Stephanie S; Kunkel, Maya T; Smrcka, Alan V et al. (2015) Thrombin promotes sustained signaling and inflammatory gene expression through the CDC25 and Ras-associating domains of phospholipase C?. J Biol Chem 290:26776-83
Dusaban, Stephanie S; Brown, Joan Heller (2015) PLC? mediated sustained signaling pathways. Adv Biol Regul 57:17-23
Sayyah, Jacqueline; Bartakova, Alena; Nogal, Nekeisha et al. (2014) The Ras-related protein, Rap1A, mediates thrombin-stimulated, integrin-dependent glioblastoma cell proliferation and tumor growth. J Biol Chem 289:17689-98
Zhao, Xia; Ding, Eric Y; Yu, Olivia M et al. (2014) Induction of the matricellular protein CCN1 through RhoA and MRTF-A contributes to ischemic cardioprotection. J Mol Cell Cardiol 75:152-61
Dusaban, Stephanie S; Purcell, Nicole H; Rockenstein, Edward et al. (2013) Phospholipase C epsilon links G protein-coupled receptor activation to inflammatory astrocytic responses. Proc Natl Acad Sci U S A 110:3609-14

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