When activated by their transmembrane receptors, heterotrimeric guanine nucleotide-binding proteins (G proteins) dissociate into α and βγ subunits to act on plasma membrane-associated targets. Thus, G proteins are typically thought to reside in association with the cytoplasmic surface of the plasma membrane. Two articles from the same group described research using various fluorescence imaging methods in live cells--including fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and photoactivation of a fluorescent protein--to directly examine G protein localization. Chisari et al. focused on G proteins in the basal (unstimulated) state; they found that fluorescently tagged αo, β1, and various γ subunits shuttled between the plasma membrane and the internal membranes of CHO cells, with a t1/2 of less than a minute. G protein movement was not blocked by pharmacological inhibitors of vesicular trafficking and was still rapid at 10oC, which, together with the rapidity of the movement, suggested that it depended on diffusion rather than vesicular trafficking. Treatment with 2-bromopalmitate, however, which inhibits palmitoylation, inhibited shuttling. Fluorescence resonance energy transfer (FRET) analysis indicated that G proteins were resident as heterotrimers in internal membranes as well as in the plasma membrane.
Saini et al., investigated the movement of βγ when β1 was complexed with different fluorescently labeled γ subunits after receptor-mediated activation in CHO cells stably expressing M2 muscarinic receptors. Treatment with the muscarinic agonist carbachol led to the movement of βγ containing 6 of 12 γ subunit types from the plasma membrane to internal membranes, whereas treatment with antagonist or agonist withdrawal led to translocation back to the plasma membrane. Intriguingly, βγ subunits were targeted to distinct compartments, depending on specific γ subunit identity. γ1, γ5, γ9, and γ11 translocated to one compartment, identified as the Golgi, whereas γ13 translocated to another, identified as the endoplasmic reticulum. The rates of translocation differed as well, falling into either a "rapid" (t1/2 ~ 6 to 13 s)or a "slow" (t1/2 ~ 39 to 85 s)group. The ability of specific subunits to translocate, and the speed with which they did so, was closely related to their structure, and mutation of C-terminal residues enabled nontranslocating subunits to do so. Translocation occurred in various cell types after activation by various receptors. Like the shuttling of heterotrimers in the basal state, translocation was inhibited by 2-bromopalmitate and was likely diffusion mediated. Thus, the authors conclude that both likely involve the same process and speculate that the ability of only certain γ subunits to translocate and their targeting to distinct compartments add additional layers of complexity to regulation of G protein signaling.
M. Chisari, D. K. Saini, V. Kalyanaraman, N. Gautam, Shuttling of G protein subunits between the plasma membrane and intracellular membranes. J. Biol. Chem. 282, 24092-24098 (2007). [Abstract] [Full Text]
D. K. Saini, V. Kalyanaraman, M. Chisari, N. Gautam, A family of G protein βγ subunits translocate reversibly from the plasma membrane to endomembranes on receptor activation. J. Biol. Chem. 282, 24099-24108 (2007). [Abstract] [Full Text]