Sci. STKE, 3 July 2001
GTPases Illuminating Active Forms
Green fluorescent protein (GFP) and its variants that emit light of different wavelengths have been used by Mochizuki et al. to develop a fluorescent resonance energy transfer (FRET) method to detect the activation state of small guanosine triphosphatases (GTPases). A chimeric fusion protein consisting of either the Ras or Rap1 GTPase fused to the yellow emitting form of GFP (YFP) and the Ras-binding domain fused to the cyan-emitting form of GFP (CFP) undergo FRET when the GTPase half of the protein is activated. When cells transfected with either of these reporters were activated with growth factors (epidermal growth factor or nerve growth factor, depending on the cell type), the distribution of active Ras or Rap1 could be monitored by FRET. Ras was activated at the plasma membrane and the active form spread inward, whereas Rap1 was activated at the perinuclear region and activity spread outward. Treatment of cells with an inhibitor of endocytosis showed that activation of Rap1 required the endocytosis of active receptors, whereas Ras did not. The potential applications of these proteins and the use of this method to study questions related to cell signaling are addressed by Bos.
N. Mochizuki, S. Yamashita, K. Kurokawa, Y. Ohba, T. Nagai, A. Miyawaki, M. Matsuda, Spatio-termporal images of growth-factor-induced activation of Ras and Rap1. Nature 411, 1065-1068 (2001). [Online Journal]
J. L. Bos, Glowing switches. Nature 411, 1006-1007 (2001). [Online Journal]
Citation: Illuminating Active Forms. Sci. STKE 2001, tw5 (2001).
Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882