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Dynamic localization of G proteins in Dictyostelium discoideum
Carrie A. Elzie, Jennifer Colby, Morgan A. Sammons, Chris Janetopoulos


Extracellular stimuli exert their effects on eukaryotic cells via serpentine G-protein-coupled receptors and mediate a vast number of physiological responses. Activated receptors stimulate heterotrimeric G-proteins, consisting of three subunits, α, β and γ. In Dictyostelium discoideum, cAMP binds to the cAMP receptor cAR1, which is coupled to the heterotrimer containing the Gα2 subunit. These studies provide in vivo evidence as to how receptors influence the localization of the G-protein complex prior to and after ligand binding. Previous work has shown that the state of the heterotrimer could be monitored by changes in fluorescence (or Förster) resonance energy transfer (FRET) between the α2- and β-subunits of D. discoideum. We now report the kinetics of G-protein activation as a loss of FRET prior to and after cAMP addition by using total internal reflection fluorescence microscopy (TIRFM). We also performed photobleaching experiments to measure G-protein recovery times. Our data show that inactive and active G-proteins cycle between the cytosol and plasma membrane. These data suggest that cAR1 activation slows the membrane dissociation (`off') rate of the α2 subunit, while simultaneously promoting βγ-subunit dissociation.


  • Supplementary material available online at

  • We thank the Devreotes lab for providing the cAR1 and cAR3 nulls. This research was supported in part by the Vanderbilt Institute for Integrative Biosystems Research and Education. Support for J.C. was provided by the Systems Biology Bioengineering Undergraduate Research Experience. Program support for C.A.E. was provided by the Vanderbilt Biomedical Research and Education Training Office, an Institutional Research and Academic Career Developmental Award from the NIGMS/NIH 5K12 GM068543-04, and NIHGM080370 to C.J. Deposited in PMC for release after 12 months.

  • Accepted May 14, 2009.
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