Regulation of Golgi structure and secretion by receptor-induced G protein βγ complex translocation (original) (raw)
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Regulation of Golgi structure and secretion by receptor-induced G protein complex translocation
Proceedings of The National Academy of Sciences, 2010
We show that receptor induced G protein βγ subunit translocation from the plasma membrane to the Golgi allows a receptor to initiate fragmentation and regulate secretion. A lung epithelial cell line, A549, was shown to contain an endogenous translocating G protein γ subunit and exhibit receptor-induced Golgi fragmentation. Receptor-induced Golgi fragmentation was inhibited by a shRNA specific to the endogenous translocating γ subunit. A kinase defective protein kinase D and a phospholipase C β inhibitor blocked receptor-induced Golgi fragmentation, suggesting a role for this process in secretion. Consistent with βγ translocation dependence, fragmentation induced by receptor activation was inhibited by a dominant negative nontranslocating γ3. Insulin secretion was shown to be induced by muscarinic receptor activation in a pancreatic β cell line, NIT-1. Induction of insulin secretion was also inhibited by the dominant negative γ3 subunit consistent with the Golgi fragmentation induced by βγ complex translocation playing a role in secretion.
The Journal of biological chemistry, 2007
The present model of G protein activation by G protein-coupled receptors exclusively localizes their activation and function to the plasma membrane (PM). Observation of the spatiotemporal response of G protein subunits in a living cell to receptor activation showed that 6 of the 12 members of the G protein ␥ subunit family translocate specifically from the PM to endomembranes. The ␥ subunits translocate as ␥ complexes, whereas the ␣ subunit is retained on the PM. Depending on the ␥ subunit, translocation occurs predominantly to the Golgi complex or the endoplasmic reticulum. The rate of translocation also varies with the ␥ subunit type. Different ␥ subunits, thus, confer distinct spatiotemporal properties to translocation. A striking relationship exists between the amino acid sequences of various ␥ subunits and their translocation properties. ␥ subunits with similar translocation properties are more closely related to each other. Consistent with this relationship, introducing residues conserved in translocating subunits into a nontranslocating subunit results in a gain of function. Inhibitors of vesicle-mediated trafficking and palmitoylation suggest that translocation is diffusion-mediated and controlled by acylation similar to the shuttling of G protein subunits (Chisari, M., Saini, D. K., Kalyanaraman, V., and Gautam, N. (2007) J. Biol. Chem. 282, 24092-24098). These results suggest that the continual testing of cytosolic surfaces of cell membranes by G protein subunits facilitates an activated cell surface receptor to direct potentially active G protein ␥ subunits to intracellular membranes.
Journal of Biological Chemistry, 2007
The present model of G protein activation by G protein-coupled receptors exclusively localizes their activation and function to the plasma membrane (PM). Observation of the spatiotemporal response of G protein subunits in a living cell to receptor activation showed that 6 of the 12 members of the G protein ␥ subunit family translocate specifically from the PM to endomembranes. The ␥ subunits translocate as ␥ complexes, whereas the ␣ subunit is retained on the PM. Depending on the ␥ subunit, translocation occurs predominantly to the Golgi complex or the endoplasmic reticulum. The rate of translocation also varies with the ␥ subunit type. Different ␥ subunits, thus, confer distinct spatiotemporal properties to translocation. A striking relationship exists between the amino acid sequences of various ␥ subunits and their translocation properties. ␥ subunits with similar translocation properties are more closely related to each other. Consistent with this relationship, introducing residues conserved in translocating subunits into a nontranslocating subunit results in a gain of function. Inhibitors of vesicle-mediated trafficking and palmitoylation suggest that translocation is diffusion-mediated and controlled by acylation similar to the shuttling of G protein subunits (Chisari, M., Saini, D. K., Kalyanaraman, V., and Gautam, N. (2007) J. Biol. Chem.
Frontiers in Cell and Developmental Biology, 2022
The biosynthetic transport route that constitutes the secretory pathway plays a fundamental role in the cell, providing to the synthesis and transport of around one third of human proteins and most lipids. Signaling molecules within autoregulatory circuits on the intracellular membranes of the secretory pathway regulate these processes, especially at the level of the Golgi complex. Indeed, cancer cells can hijack several of these signaling molecules, and therefore also the underlying regulated processes, to bolster their growth or gain more aggressive phenotypes. Here, we review the most important autoregulatory circuits acting on the Golgi, emphasizing the role of specific signaling molecules in cancer. In fact, we propose to draw awareness to highlight the Golgi-localized regulatory systems as potential targets in cancer therapy.
The Journal of Cell Biology
A well-characterized cell-free assay that reconstitutes Golgi transport is shown to require physically fragmented Golgi fractions for maximal activity. A Golgi fraction containing large, highly stacked flattened cisternae associated with coatomer-rich components was inactive in the intra-Golgi transport assay. In contrast, more fragmented hepatic Golgi fractions of lower purity were highly active in this assay. Control experiments ruled out defects in glycosylation, the presence of excess coatomer or inhibitory factors, as well as the lack or consumption of limiting diffusible factors as responsible for the lower activity of intact Golgi fractions. Neither Brefeldin A treatment, preincubation with KCl (that completely removed associated coatomer) or preincubation with imidazole buffers that caused unstacking, activated stacked fractions for transport. Only physical fragmentation promoted recovery of Golgi fractions active for transport in vitro. Rate-zonal centrifugation partially separated smaller transport-active Golgi fragments with a unique v-SNARE pattern, away from the bulk of Golgi-derived elements identified by their morphology and content of Golgi marker enzymes ( N -acetyl glucosaminyl and galactosyl transferase activities). These fragments released during activation likely represent intra-Golgi continuities involved in maintaining the dynamic redistribution of resident enzymes during rapid anterograde transport of secretory cargo through the Golgi in vivo.
G protein subunit dissociation and translocation regulate cellular response to receptor stimulation
PLOS One, 2009
We examined the role of G proteins in modulating the response of living cells to receptor activation. The response of an effector, phospholipase C-b to M3 muscarinic receptor activation was measured using sensors that detect the generation of inositol triphosphate or diacylglycerol. The recently discovered translocation of Gbc from plasma membrane to endomembranes on receptor activation attenuated this response. A FRET based G protein sensor suggested that in contrast to translocating Gbc, non-translocating Gbc subunits do not dissociate from the aq subunit on receptor activation leading to prolonged retention of the heterotrimer state and an accentuated response. M3 receptors with tethered aq induced differential responses to receptor activation in cells with or without an endogenous translocation capable c subunit. G protein heterotrimer dissociation and bc translocation are thus unanticipated modulators of the intensity of a cell's response to an extracellular signal. Citation: Chisari M, Saini DK, Cho J-H, Kalyanaraman V, Gautam N (2009) G Protein Subunit Dissociation and Translocation Regulate Cellular Response to Receptor Stimulation. PLoS ONE 4(11): e7797.
Journal of Cell Biology, 1967
Since in the pancreatic exocrine cell synthesis and intracellular transport of secretory proteins can be uncoupled (1), it is possible to examine separately the metabolic requirements of the latter process. To this intent, guinea pig pancreatic slices were pulse labeled with leucine-SH for 3 man and incubated post-pulse for 37 rain in chase medium containing 5 X I 0--4M cycloheximide and inhibitors of glycolysis, respiration, or oxidative phosphorylation. In each case, the effect on transport was assessed by measuring the amount of labeled secretory proteins found in zymogen granule fractions isolated from the corresponding slices. This assay is actually a measure of the efficiency of transport of secretory proteins from the cisternae of the rough endoplasrnic reticulum (RER) to the condensing vacuoles of the Golgi complex which are recovered in the zymogen granule fraction (16). The results indicate that transport is insensitive to glycolytic inhibitors (fluoride, iodoacetate) but is blocked by respiratory inhibitors (N2, cyanide, Antimycin A) and by inhibitors of oxidative phosphorylation (dinitrophenol, oligomycin). Except for Antimycin A, the effect is reversible. Parallel radioautographic studies and cell fractionation procedures applied to microsomal subfractions have indicated that the energy-dependent step is located between the transitional elements of the RER and the small, smooth-surfaced vesicles at the periphery of the Golgi complex. Radiorespirometric data indicate that the substrates oxidized to support transport are endogenous long-chain fatty acids.
Characterization of a 58 kDa Cis-Golgi protein in pancreatic exocrine cells
Journal of Cell Science, 1992
We have studied the biochemical characteristics and localization of a 58 kDa cis-Golgi marker protein (p58) in rat pancreatic exocrine cells. The protein remained associated with membranes after extraction at alkaline pH and was largely resistant to proteases, added to intact microsomes. By electrophoresis p58 could be resolved into two bands which in two-dimensional gels separated into several charge variants around pI 5.5. This size and charge heterogeneity of p58 did not appear to be due to acylation, glycosylation or phosphorylation. In non-reduced gels p58 migrated as two kinetically related, high relative molecular mass forms, apparently corresponding to disulfide-linked homo-dimers and-hexamers. Immuno-electron microscopy localized p58 to both the fenestrated cis-Golgi cisternae and small Golgi vesicles or buds as well as large, pleiomorphic structures, scattered throughout the cells and associated with distinct smooth ER (endoplasmic reticulum) clusters. These findings correlated with cell fractionation results showing the concentration of p58 in two microsomal subfractions, banding at intermediate densities between the rough ER and trans-Golgi in sucrose gradients. Our results indicate that p58 is a major component of pre-and cis-Golgi elements and could be part of the transport machinery that operates in these membranes. Together with results obtained with other cell types, these observations suggest that the peripheral smooth ER clusters are involved in the early stages of the secretory pathway in the pancreatic acinar cells.
Alteration of Golgi structure in senescent cells and its regulation by a G protein γ subunit
Cellular Signalling, 2011
Cellular senescence is a process wherein proliferating cells undergo permanent cell cycle arrest while remaining viable. Senescence results in enhanced secretion of proteins that promote cancer and inflammation. We report here that the structure of the Golgi complex which regulates secretion is altered in senescent cells. In cells where senescence is achieved by replicative exhaustion or in cells wherein senescence has been induced with BrdU treatment dependent stress, the Golgi complex is dispersed. The expression of a G protein γ subunit, γ11, capable of translocation from the plasma membrane to the Golgi complex on receptor activation increases with senescence. Knockdown of γ11 or overexpression of a dominant negative γ3 subunit inhibits Golgi dispersal induced by senescence. Overall these results suggest that in cellular senescence an upregulated G protein gamma subunit mediates alterations in the structure of the Golgi.