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First published online 7 July 2009
doi: 10.1242/jcs.046300

Journal of Cell Science 122, 2597-2603 (2009)
Published by The Company of Biologists 2009
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Short Report

Dynamic localization of G proteins in Dictyostelium discoideum

Carrie A. Elzie, Jennifer Colby, Morgan A. Sammons and Chris Janetopoulos*

Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA

* Author for correspondence (e-mail: c.janetopoulos{at}vanderbilt.edu)

Accepted 14 May 2009

Summary

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, {alpha}, β and {gamma}. In Dictyostelium discoideum, cAMP binds to the cAMP receptor cAR1, which is coupled to the heterotrimer containing the G{alpha}2 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 {alpha}2- 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 {alpha}2 subunit, while simultaneously promoting β{gamma}-subunit dissociation.

Key words: FRET, G proteins, Chemotaxis


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G-protein dynamics in Dictyostelium

JCS 2009 122: 1505. [Full Text]  





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