Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Abstract

Heterotrimeric G proteins are localized to the plasma membrane where they transduce extracellular signals to intracellular effectors. G proteins also act at intracellular locations, and can translocate between cellular compartments. For example, G␣ s can leave the plasma membrane and move to the cell interior after activation. However, the mechanism of G␣ s translocation and its intracellular destination are not known. Here we use bioluminescence resonance energy transfer (BRET) to show that after activation, G␣ s rapidly associates with the endoplasmic reticulum, mitochondria, and endosomes, consistent with indiscriminate sampling of intracellular membranes from the cytosol rather than transport via a specific vesicular pathway. The primary source of G␣ s for endosomal compartments is constitutive endocytosis rather than activity-dependent internalization. Recycling of G␣ s to the plasma membrane is complete 25 min after stimulation is discontinued. We also show that an acylation-deacylation cycle is important for the steady-state localization of G␣ s at the plasma membrane, but our results do not support a role for deacylation in activity-dependent G␣ s internalization.Heterotrimeric G proteins are best known for their role in transducing signals from the extracellular environment across the plasma membrane (1). However, it is becoming increasingly apparent that these signaling molecules have important functions in other cellular locations (2). Therefore, it is important to know how G proteins are distributed among various intracellular compartments, and how these proteins traffic between the plasma membrane, the cytosol, cytoskeletal elements, and intracellular organelles both in their resting state and when they are activated.The most well studied example of activity-dependent G protein trafficking occurs in the retina, where intense illumination promotes the dissociation of transducin from rod outer segment disks and the subsequent translocation of both G␣ t and G␤␥ into the rod inner segment (3). Activity-dependent translocation of G␣ and G␤␥ subunits occurs in other cells as well (4, 5), most notably for G␣ s , the subunit responsible for stimulation of adenylate cyclase. Stimulation of a cognate receptor or inhibition of GTPase activity promotes activation and translocation of G␣ s from the plasma membrane to the cell interior (6 -8).One important yet poorly understood aspect of G␣ s internalization is the intracellular destination of this G protein. Cell fractionation studies have shown that a large amount of constitutively active G␣ s is found in the soluble fraction, suggesting that active G␣ s may simply become soluble in the cytosol (8, 9). However, only a small amount of G␣ s enters the soluble fraction after receptor-mediated activation, consistent with vesicle-mediated internalization and continued association with membranes. Imaging studies have shown that internalized G␣ s can appear to be diffuse in the cytosol (8, 9), but association with intracellular vesicles has also been observed (10 -12)...

Components of the Gs signaling cascade exhibit distinct changes in mobility and membrane domain localization upon β2‐adrenergic receptor activation

“…However, Gα s , Gγ 2 and AC5 were also not excluded from CCSs and, therefore, it is likely that some of these molecules could be inadvertently internalized together with β 2 AR‐arrestin complexes, and thus participate in signaling in the endosomal compartment. This result is consistent with a previous study that showed the absence of agonist‐stimulated G s accumulation in endosomes, and implies that the stoichiometry of β 2 AR cascade components is likely to differ in plasma membrane and intracellular compartments …”

“…This result is consistent with a previous study that showed the absence of agonist-stimulated G s accumulation in endosomes, and implies that the stoichiometry of β 2 AR cascade components is likely to differ in plasma membrane and intracellular compartments. 35 Diffusion coefficients observed in our single-molecule experiments for receptors and G proteins in unstimulated cells are generally consistent with previous reports, including Valentine and Haggie 36 and Calebiro et al 37 who also studied β 2 AR. The slightly higher β 2 AR diffusion rates that we observed can be attributed to differences in temperature and possibly cell type (as shown in Valentine and Haggie).…”

DHHC5 Mediates β-Adrenergic Signaling in Cardiomyocytes by Targeting Gα Proteins

“…One possible explanation is that [ 3 H]palmitate loading only labels a small fraction of the total G α pool, which cannot be detected using high-performance liquid chromatography. Additionally, another recent study in HEK cells observed redistribution of G α proteins to internal membranes after ISO stimulation, and the authors suggested that such events are independent of the acylation-deacylation cycles ( 59 ). Recent advances in palmitoylation detection methods allow us to better study dynamic palmitoylation of endogenous G α proteins with endogenous lipids and their functional outcomes in specific cell types.…”

Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells

“…A plasmid encoding the S1 subunit of pertussis toxin was kindly provided by Stephen R. Ikeda (NIAAA, National Institutes of Health, Rockville, MD). Plasmids encoding cerulean–TM–venus, venus–kras, venus–rab5, venus–giantin, and Gβγ–venus have been described previously and were used in this study under conditions that are essentially the same as described previously ( 11 , 22 – 25 ). Several different GPCR–luciferase constructs were made by appending either Rluc8 or Nluc directly to the receptor C terminus either by QuikChange PCR or by subcloning into pRluc8-N1 or pNluc-N1 vectors.…”