Rho guanine-nucleotide exchange factors (RhoGEFs) comprise a large family of intracellular signaling proteins that couple diverse inputs to the activation of the small GTPase Rho and ultimately to dynamic cell architecture changes brought about by Rho's role in modifying a cell's actin cytoskeleton. Cellular processes under the control of Rho GTPases include smooth muscle cell contractility, cell adhesion, cell migration, cell proliferation, neurite extension and retraction, gene expression and cell division. A sub-family of three RhoGEFs, termed regulator of G protein signaling domain-containing RhoGEFs (RGS-RhoGEFs), is specifically activated by heterotrimeric G proteins at the plasma membrane. The RGS-RhoGEFs thus mediate signaling from several important G protein-coupled receptors (GPCR) to activation of Rho and changes in a cell's actin cytoskeleton. The research proposed in this application will focus on one RGS-RhoGEF, termed leukemia-associated RhoGEF (LARG). Critical physiological pathways mediated by LARG include contraction of vascular smooth muscle cells in response to vasoconstrictors angiotensin II and endothelin, and genetic deletion of LARG prevents salt-induced hypertension in the mouse. Thus, LARG has potential as a therapeutic target in the treatment of cardiovascular disease. Work in this laboratory has recently uncovered a novel and unexpected role for LARG in cell division. In cultured cells, LARG is localized at specific mitotic structures, including centrosomes and mitotic spindles in early mitosis and the cytokinesis cleavage furrow and midbody in late mitosis/cytokinesis. Moreover, LARG undergoes mitotic-dependent phosphorylation, and depletion of LARG causes a strong late cytokinesis defect. This application will focus on defining and understanding this new role for LARG. To address this question, the major objectives of this proposal are 1) Define the role of LARG in mitosis;2) Characterize mitotic-dependent phosphorylation of LARG;and 3) Investigate mitotic localization of LARG and the role LARG in recruiting critical proteins to the midbody. These objectives will be pursued by a variety of experimental approaches, including cultured cells, time-lapse microscopy, immunofluorescence microscopy, pharmacological inhibitors, mutational analysis, and biochemical assays.

Public Health Relevance

Rho guanine-nucleotide exchange factors (RhoGEFs) are key intracellular signaling proteins, connecting activated cell-surface receptors to dynamic changes in a cell's interior cytoskeleton. The RhoGEFs mediate a number of physiological responses that involve changes in a cell's shape, including smooth muscle cell contractility, cell migration, and developmental processes, and represent potential and novel therapeutic targets in disease states such as hypertension and cancer. The research in this application will provide new knowledge about the functions of RhoGEFs and thus better define how the RhoGEFs can be therapeutically inhibited in disease without affecting their critical normal functions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062884-11
Application #
8308638
Study Section
Special Emphasis Panel (ZRG1-VH-B (02))
Program Officer
Dunsmore, Sarah
Project Start
2001-04-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
11
Fiscal Year
2012
Total Cost
$317,913
Indirect Cost
$112,808
Name
Thomas Jefferson University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Martz, Matthew K; Grabocka, Elda; Beeharry, Neil et al. (2013) Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission. Mol Biol Cell 24:2785-94
Bhattacharyya, Raja; Banerjee, Jayashree; Khalili, Kamel et al. (2009) Differences in Galpha12- and Galpha13-mediated plasma membrane recruitment of p115-RhoGEF. Cell Signal 21:996-1006
Banerjee, Jayashree; Fischer, Christopher C; Wedegaertner, Philip B (2009) The amino acid motif L/IIxxFE defines a novel actin-binding sequence in PDZ-RhoGEF. Biochemistry 48:8032-43
Grabocka, Elda; Wedegaertner, Philip B (2007) Disruption of oligomerization induces nucleocytoplasmic shuttling of leukemia-associated rho Guanine-nucleotide exchange factor. Mol Pharmacol 72:993-1002
Escriba, Pablo V; Wedegaertner, Philip B; Goni, Felix M et al. (2007) Lipid-protein interactions in GPCR-associated signaling. Biochim Biophys Acta 1768:836-52
Grabocka, Elda; Wedegaertner, Philip B (2005) Functional consequences of G alpha 13 mutations that disrupt interaction with p115RhoGEF. Oncogene 24:2155-65
Banerjee, Jayashree; Wedegaertner, Philip B (2004) Identification of a novel sequence in PDZ-RhoGEF that mediates interaction with the actin cytoskeleton. Mol Biol Cell 15:1760-75
Thiyagarajan, Manimekalai M; Stracquatanio, RoseAnn P; Pronin, Alexey N et al. (2004) A predicted amphipathic helix mediates plasma membrane localization of GRK5. J Biol Chem 279:17989-95
Day, Peter W; Tesmer, John J G; Sterne-Marr, Rachel et al. (2004) Characterization of the GRK2 binding site of Galphaq. J Biol Chem 279:53643-52
Bhattacharyya, Raja; Wedegaertner, Philip B (2003) Characterization of G alpha 13-dependent plasma membrane recruitment of p115RhoGEF. Biochem J 371:709-20

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