Article
- The EMBO Journal (2008) 27, 727 - 735
- doi:10.1038/emboj.2008.10
Published online: 14 February 2008
Subject Categories:
A novel switch region regulates H-ras membrane orientation and signal output
Daniel Abankwa1, Michael Hanzal-Bayer1, Nicolas Ariotti1, Sarah J Plowman1, Alemayehu A Gorfe2, Robert G Parton1,4, J Andrew McCammon2,3 and John F Hancock1
- Molecular Cell Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia
- Department of Chemistry and Biochemistry, Centre for Theoretical Biological Physics, La Jolla, CA, USA
- Department of Pharmacology, Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA, USA
- Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, Queensland, Australia
Correspondence to:
Daniel Abankwa, Molecular Cell Biology, Institute for Molecular Bioscience, University of Queensland, 306 Carmody Rd, Brisbane, Queensland 4072, Australia. Tel.: +61 733 462 033; Fax: +61 733 462 101; E-mail: d.abankwa@imb.uq.edu.au
John F Hancock, Molecular Cell Biology, Institute for Molecular Bioscience, University of Queensland, 306 Carmody Rd, Brisbane, Queensland 4072, Australia. Tel.: +61 733 462 033; Fax: +61 733 462 101; E-mail: j.hancock@imb.uq.edu.au
Received 10 September 2007; Accepted 11 January 2008
Abstract
The plasma membrane nanoscale distribution of H-ras is regulated by guanine nucleotide binding. To explore the structural basis of H-ras membrane organization, we combined molecular dynamic simulations and medium-throughput FRET measurements on live cells. We extracted a set of FRET values, termed a FRET vector, to describe the lateral segregation and orientation of H-ras with respect to a large set of nanodomain markers. We show that mutation of basic residues in helix 
4 or the hypervariable region (HVR) selectively alter the FRET vectors of GTP- or GDP-loaded H-ras, demonstrating a critical role for these residues in stabilizing GTP- or GDP-H-ras interactions with the plasma membrane. By a similar analysis, we find that the 
2–3 loop and helix 5 are involved in a novel conformational switch that operates through helix 4 and the HVR to reorient the H-ras G-domain with respect to the plasma membrane. Perturbation of these switch elements enhances MAPK activation by stabilizing GTP-H-ras in a more productive signalling conformation. The results illustrate how the plasma membrane spatially constrains signalling conformations by acting as a semi-neutral interaction partner.
Keywords:
- FRET,
- microdomain,
- nanocluster,
- plasma membrane,
- Ras
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