scholarly journals Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone receptor-1 and Gqα /G11α induced by agonist stimulation

1999 ◽  
Vol 340 (2) ◽  
pp. 529-538 ◽  
Author(s):  
Tomas DRMOTA ◽  
Jiri NOVOTNY ◽  
Gwyn W. GOULD ◽  
Petr SVOBODA ◽  
Graeme MILLIGAN
Keyword(s):  
Plasma Membrane ◽  
G Proteins ◽  
Hormone Receptor ◽  
Fluorescent Protein ◽  
Thyrotropin Releasing Hormone ◽  
Hek293 Cells ◽  
Low Density ◽  
Rapid Separation ◽  
Releasing Hormone ◽  
Green Fluorescent

The rat thyrotropin-releasing hormone receptor-1 (TRHR-1) was modified by the addition of green fluorescent protein (GFP) and expressed stably in HEK293 cells. Extensive overlap of plasma membrane distribution of autofluorescent TRHR-1-GFP with that of the phosphoinositidase C-linked G-proteins Gqα/G11α, identified by indirect immunofluorescence, was monitored concurrently. Addition of thyrotropin-releasing hormone resulted in rapid separation of TRHR-1-GFP and Gqα/G11α signals as the receptor was internalized. This situation persisted for more than an hour. At longer time periods a fraction of the cellular Gqα/G11α was also internalized, although much of the Gqα/G11α immunoreactivity remained associated with the plasma membrane. Parallel experiments, in which the cellular distribution of TRHR-1-GFP and Gqα/G11α immunoreactivity were monitored in sucrose-gradient fractions following cell disruption, also demonstrated a rapid, agonist-induced movement of TRHR-1-GFP away from the plasma membrane to low-density vesicular fractions. At later time points, a fraction of the cellular Gqα/G11α immunoreactivity was also redistributed to overlapping, but non-identical, low-density-vesicle-containing fractions. Pretreatment of the cells with cytochalasin D or nocodazole prevented agonist-induced redistribution of G-protein but not TRHR-1-GFP, further indicating resolution of the mechanics of these two processes. The combination of a GFP-modified receptor and immunostaining of the G-proteins activated by that receptor allows, for the first time, concurrent analysis of the varying dynamics and bases of internalization and redistribution of two elements of the same signal-transduction cascade.

Protein alterations induced by long-term agonist treatment of HEK293 cells expressing thyrotropin-releasing hormone receptor and G11α protein

2009 ◽  
Vol 109 (1) ◽  
pp. 255-264 ◽  
Author(s):  
Zdenka Drastichova ◽  
Lenka Bourova ◽  
Lucie Hejnova ◽  
Petr Jedelsky ◽  
Petr Svoboda ◽  
...  
Keyword(s):  
Hormone Receptor ◽  
Thyrotropin Releasing Hormone ◽  
Hek293 Cells ◽  
Releasing Hormone ◽  
Protein Alterations

Stimulation of the neurokinin 3 receptor activates protein kinase Cε and protein kinase D in enteric neurons

2008 ◽  
Vol 294 (5) ◽  
pp. G1245-G1256 ◽  
Author(s):  
D. P. Poole ◽  
S. Amadesi ◽  
E. Rozengurt ◽  
M. Thacker ◽  
N. W. Bunnett ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Fluorescent Protein ◽  
Enteric Neurons ◽  
Hek293 Cells ◽  
Protein Kinase D ◽  
Downstream Target ◽  
Neurokinin 3 Receptor ◽  
Green Fluorescent ◽  
Stimulation Of

Tachykinins, acting through NK3 receptors (NK3R), contribute to excitatory transmission to intrinsic primary afferent neurons (IPANs) of the small intestine. Although this transmission is dependent on protein kinase C (PKC), its maintenance could depend on protein kinase D (PKD), a downstream target of PKC. Here we show that PKD1/2-immunoreactivity occurred exclusively in IPANs of the guinea pig ileum, demonstrated by double staining with the IPAN marker NeuN. PKCε was also colocalized with PKD1/2 in IPANs. PKCε and PKD1/2 trafficking was studied in enteric neurons within whole mounts of the ileal wall. In untreated preparations, PKCε and PKD1/2 were cytosolic and no signal for activated (phosphorylated) PKD was detected. The NK3R agonist senktide evoked a transient translocation of PKCε and PKD1/2 from the cytosol to the plasma membrane and induced PKD1/2 phosphorylation at the plasma membrane. PKCε translocation was maximal at 10 s and returned to the cytosol within 2 min. Phosphorylated-PKD1/2 was detected at the plasma membrane within 15 s and translocated to the cytosol by 2 min, where it remained active up to 30 min after NK3R stimulation. PKD1/2 activation was reduced by a PKCε inhibitor and prevented by NK3R inhibition. NK3R-mediated PKCε and PKD activation was confirmed in HEK293 cells transiently expressing NK3R and green fluorescent protein-tagged PKCε, PKD1, PKD2, or PKD3. Senktide caused membrane translocation and activation of kinases within 30 s. After 15 min, phosphorylated PKD had returned to the cytosol. PKD activation was confirmed through Western blotting. Thus stimulation of NK3R activates PKCε and PKD in sequence, and sequential activation of these kinases may account for rapid and prolonged modulation of IPAN function.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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