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Vol. 17, Issue 5, 2177-2189, May 2006
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Department of Pharmacology, Wayne State University, Detroit, MI 48201
Submitted October 3, 2005;
Revised February 1, 2006;
Accepted February 7, 2006
Monitoring Editor: J. Silvio Gutkind
The Ras-GRF1 exchange factor has regulated guanine nucleotide exchange factor (GEF) activity for H-Ras and Rac1 through separate domains. Both H-Ras and Rac1 activation have been linked to synaptic plasticity and thus could contribute to the function of Ras-GRF1 in neuronal signal transduction pathways that underlie learning and memory. We defined the effects of Ras-GRF1 and truncation mutants that include only one of its GEF activities on the morphology of PC12 phaeochromocytoma cells. Ras-GRF1 required coexpression of H-Ras to induce morphological effects. Ras-GRF1 plus H-Ras induced a novel, expanded morphology in PC12 cells, which was characterized by a 10-fold increase in soma size and by neurite extension. A truncation mutant of Ras-GRF1 that included the Ras GEF domain, GRF
N, plus H-Ras produced neurite extensions, but did not expand the soma. This neurite extension was blocked by inhibition of MAP kinase activation, but was independent of dominant-negative Rac1 or RhoA. A truncation mutant of Ras-GRF1 that included the Rac GEF domains, GRFC, produced the expanded phenotype in cotransfections with H-Ras. Cell expansion was inhibited by wortmannin or dominant-negative forms of Rac1 or Akt. GRFC binds H-Ras.GTP in both pulldown assays from bacterial lysates and by coimmunoprecipitation from HEK293 cells. These results suggest that coordinated activation of H-Ras and Rac1 by Ras-GRF1 may be a significant controller of neuronal cell size.
Abbreviations used: CRIB, Cdc42/Rac1-binding domain; DH, Dbl homology domain; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GRFC, Ras-GRF1 residues 1-631; GRFN, Ras-GRF1 residues 632-1262; GST, glutathione S-transferase; HA, hemagglutinin-1 epitope; LPA, lysophosphatidic acid; NGF, nerve growth factor; PH, plekstrin homology domain; PI3K, phosphatidylinositol 3-kinase; Raf.RBD, Ras-binding domain of Raf; TBST, TBS with Tween-20.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Raymond R. Mattingly (r.mattingly{at}wayne.edu).
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