Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 107, Issue 3, Pages 1130-1135Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0903907107
Keywords
FRET; nanocluster; signal transduction; small G protein
Categories
Funding
- National Institute of General Medical Sciences [R01GM066717]
- Swiss National Science Foundation [PA00A-111446]
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The structural elements encoding functional diversity among Ras GTPases are poorly defined. The orientation of the G domain of H-ras with respect to the plane of the plasma membrane is recognized by the Ras binding domain of C-Raf, coupling orientation to MAPK activation. We now show that two other proteins, phosphoinositide-3-kinase-alpha and the structurally unrelated galectin-1, also recognize G-domain orientation. These results rationalize the role of galectin-1 in generating active GTP-H-ras signaling nanoclusters. However, molecular dynamics simulations of K-ras membrane insertion and fluorescence lifetime imaging microscopy (FLIM)-Forster resonance energy transfer (FRET) imaging of the effector interactions of N-Ras, K-Ras, and M-ras suggest that there are two hyperactive, signaling-competent orientations of the Ras G domain. Mutational and functional analyses establish a clear relationship between effector binding and the amphilicities of helix alpha 4 and the C-terminal hypervariable region, thus confirming that these structural elements critically tune the orientation of the Ras G domain. Finally, we show that G-domain orientation and nanoclustering synergize to generate Ras isoform specificity with respect to effector interactions.
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