Dynamic localization of G proteins in Dictyostelium discoideum

Abstract

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.

Heterotrimeric G-protein shuttling via Gip1 extends the dynamic range of eukaryotic chemotaxis

“…As discussed below, our analyses suggest Gip1 functions as a regulator of G-protein localization between the cytosol and the plasma membrane. Previous studies have revealed that G proteins undergo translocation to the membrane upon receptor stimulation (17). We found that Gip1 regulates G-protein translocation by its sequestering function (Figs.…”

“…We next investigated the subcellular localization of G proteins, given that they are modified upon cAMP stimulation (17). Gα2 and Gγ conjugated with the fluorescent dye TMR (Gα2-TMR and TMR-Gγ, respectively) via a Halo tag were used for localization analysis.…”

“…Thus, chemical modifications of chemoattractant receptors are also important in eukaryotic chemotaxis as a mechanism to extend the chemotactic range. In addition to the receptor modifications, G proteins are phosphorylated and recruited from the cytosol to the plasma membrane upon receptor stimulation in Dictyostelium cells (16,17), although the relevance of these actions on wide-range sensing and adaptation is unknown.Here we report that a novel regulator of G proteins, G proteininteracting protein 1 (Gip1), is essential for the wide-range chemotaxis in Dictyostelium cells. Gip1 regulates G-protein localization between the cytosol and plasma membrane upon receptor activation, which targets cytosolic G proteins to the membrane in a biased manner along chemoattractant gradients at higher chemotactic ranges.…”

“…Thus, chemical modifications of chemoattractant receptors are also important in eukaryotic chemotaxis as a mechanism to extend the chemotactic range. In addition to the receptor modifications, G proteins are phosphorylated and recruited from the cytosol to the plasma membrane upon receptor stimulation in Dictyostelium cells (16,17), although the relevance of these actions on wide-range sensing and adaptation is unknown.…”

Ras activation and symmetry breaking duringDictyosteliumchemotaxis

“…In Dictyostelium, a shallow gradient of cAMP induces the activation of cAMP receptors and the associated heterotrimeric G-protein Ga2bc in a manner that is approximately proportional to the steepness of the gradient (Elzie et al, 2009;Jin et al, 2000;Xiao et al, 1997). In contrast, activation of Ras is much stronger in the front than in the rear of chemotaxing cells (Charest et al, 2010;Kortholt et al, 2011;Sasaki et al, 2004;Sasaki and Firtel, 2006;Zhang et al, 2008).…”