scholarly journals The Intracellular Trafficking of the G Protein-coupled Receptor TPβ Depends on a Direct Interaction with Rab11

2005 ◽  
Vol 280 (43) ◽  
pp. 36195-36205 ◽  
Author(s):  
Emilie Hamelin ◽  
Caroline Thériault ◽  
Geneviève Laroche ◽  
Jean-Luc Parent
Keyword(s):  
Cell Surface ◽  
Intracellular Trafficking ◽  
Direct Interaction ◽  
Hek293 Cells ◽  
Intracellular Loop ◽  
Recycling Endosome ◽  
Surface Receptors ◽  
The Subject ◽  
Intracellular Domains ◽  
G Protein Coupled

Intracellular trafficking pathways of cell surface receptors following their internalization are the subject of intense research efforts. However, the mechanisms by which they recycle back to the cell surface are still poorly defined. We have recently demonstrated that the small Rab11 GTPase protein is a determinant factor in controlling the recycling to the cell surface of the β-isoform of the thromboxane A2 receptor (TPβ) following its internalization. Here, we demonstrate with co-immunoprecipitation studies in HEK293 cells that there is a Rab11-TPβ association occurring in the absence of agonist, which is not modulated by stimulation of TPβ. We show with purified TPβ intracellular domains fused to GST and HIS-Rab11 proteins that Rab11 interacts directly with the first intracellular loop and the C-tail of TPβ. Amino acids 335–344 of the TPβ C-tail were determined to be essential for the interaction of Rab11 with this receptor domain. This identified sequence appears to be important in directing the intracellular trafficking of the receptor from the Rab5-positive intracellular compartment to the perinuclear recycling endosome. Interestingly, our data indicate that TPβ interacts with the GDP-bound form, and not the GTP-bound form, of Rab11 which is necessary for recycling of the receptor back to the cell surface. To our knowledge, this is the first demonstration of a direct interaction between Rab11 and a transmembrane receptor.

Rab11 regulates the recycling of the β2-adrenergic receptor through a direct interaction

2009 ◽  
Vol 418 (1) ◽  
pp. 163-172 ◽  
Author(s):  
Audrey Parent ◽  
Emilie Hamelin ◽  
Pascale Germain ◽  
Jean-Luc Parent
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Glutathione Transferase ◽  
Direct Interaction ◽  
Small Gtpase ◽  
Cell Surface Receptors ◽  
Wild Type ◽  
Surface Receptors ◽  
Early Endosomes ◽  
Intracellular Domains

The β2ARs (β2-adrenergic receptors) undergo ligand-induced internalization into early endosomes, but then are rapidly and efficiently recycled back to the plasma membrane, restoring the numbers of functional cell-surface receptors. Gathering evidence suggests that, during prolonged exposure to agonist, some β2ARs also utilize a slow recycling pathway through the perinuclear recycling endosomal compartment regulated by the small GTPase Rab11. In the present study, we demonstrate by co-immunoprecipitation studies that there is a β2AR–Rab11 association in HEK-293 cells (human embryonic kidney cells). We show using purified His6-tagged Rab11 protein and β2AR intracellular domains fused to GST (glutathione transferase) that Rab11 interacts directly with the C-terminal tail of β2AR, but not with the other intracellular domains of the receptor. Pull-down and immunoprecipitation assays revealed that the β2AR interacts preferentially with the GDP-bound form of Rab11. Arg333 and Lys348 in the C-terminal tail of the β2AR were identified as crucial determinants for Rab11 binding. A β2AR construct with these two residues mutated to alanine, β2AR RK/AA (R333A/K348A), was generated. Analysis of cell-surface receptors by ELISA revealed that the recycling of β2AR RK/AA was drastically reduced when compared with wild-type β2AR after agonist washout, following prolonged receptor stimulation. Confocal microscopy demonstrated that the β2AR RK/AA mutant failed to co-localize with Rab11 and recycle to the plasma membrane, in contrast with the wild-type receptor. To our knowledge, the present study is the first report of a direct interaction between the β2AR and a Rab GTPase, which is required for the accurate intracellular trafficking of the receptor.

scholarly journals GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

2016 ◽  
Vol 36 (7) ◽  
pp. 1152-1163 ◽  
Author(s):  
Maoxiang Zhang ◽  
Jason E. Davis ◽  
Chunman Li ◽  
Jie Gao ◽  
Wei Huang ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Molecular Mechanisms ◽  
Specific Interaction ◽  
Adaptor Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Intracellular Loop ◽  
Interaction Sites ◽  
G Protein Coupled

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α2-adrenergic receptors (α2-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at thetrans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.

RAB14 and RAB5 Gtpases Regulate Human Erythropoiesis, Potentially Via Opposing Roles in Endosomal Recycling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 937-937
Author(s):  
MinJung Kim ◽  
Tami J. Kingsbury ◽  
Wen-Chih Cheng ◽  
Yee Sun Tan ◽  
Brittany M. Taylor ◽  
...  
Keyword(s):  
Cell Surface ◽  
Conflicts Of Interest ◽  
Degradation Pathway ◽  
Rab Gtpases ◽  
Recycling Endosome ◽  
Hematopoietic Stem ◽  
Surface Receptors ◽  
Protein Levels ◽  
Endosomal Recycling ◽  
Model Cell

Abstract Previously, we reported that the erythroid-expressed miRs, miR-144 and miR-451, target the RAB14 GTPase during human erythropoiesis in the human TF1 erythropoietic model cell line and in primary CD34+ hematopoietic stem-progenitor cells. In response to erythropoietin, endogenous RAB14 levels decreased during erythropoiesis, and RAB14 knockdown increased the numbers of erythroid (CD34- CD71hi CD235ahi) cells, increased b-hemoglobin expression, and decreased ETO2 expression (Kim, BJH, 2015). Taken together, our findings revealed that RAB14 functions as a physiologic inhibitor of human erythropoiesis. RAB GTPases comprise a >60 member subfamily of the large RAS oncogene family, and multiple RAB GTPases play important roles in vesicle trafficking, signal transduction and receptor recycling. RAB14 is a component of the intermediate compartment of the endosomal recycling pathway, between the RAB4/RAB5-dependent early endosome and the RAB11-dependent recycling endosome (Linford, Dev. Cell, 2012). The RAB GTPases are responsible for directing cargo proteins to the recycling vs. degradation pathway during endosome maturation. To test the role of another endosomal RAB GTPases in human erythropoiesis, we examined the consequences of knocking down RAB5 protein levels. In contrast to our findings with RAB14, knockdown of RAB5C decreased the numbers of erythroid cells generated during erythropoiesis, indicating that RAB5C enhances physiologic erythropoiesis. Consistent with this observation, mRNA and protein levels for all three RAB5 isoforms (i.e. RAB5A, B, C) increased during erythropoiesis. The opposing erythropoietic effects of RAB14 and RAB5 GTPases may be due to their contrary roles in endosomal recycling, in that internalization of cell surface receptors is dependent on RAB5 GTPase, whereas RAB14 GTPase is involved in recycling of receptors back to the cell surface. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

2013 ◽  
Vol 24 (11) ◽  
pp. 1649-1660 ◽  
Author(s):  
Susumu Hara ◽  
Shigeki Arawaka ◽  
Hiroyasu Sato ◽  
Youhei Machiya ◽  
Can Cui ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Dopamine Transporter ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Receptor Kinase ◽  
Wild Type ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

scholarly journals Endothelial Cell Inflammation and Barriers Are Regulated by the Rab26-Mediated Balance between β2-AR and TLR4 in Pulmonary Microvessel Endothelial Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Huaping Chen ◽  
Ming Yuan ◽  
Chunji Huang ◽  
Zhi Xu ◽  
Mingchun Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Membrane ◽  
Cell Surface ◽  
Cell Types ◽  
Tlr4 Expression ◽  
Barrier Dysfunction ◽  
Surface Receptors ◽  
G Protein Coupled

Rab26 GTPase modulates the trafficking of cell surface receptors, such as G protein-coupled receptors including α2-adrenergic receptors in some cell types. However, the effect of Rab26 on β2-adrenergic receptor (β2-AR) trafficking or/and Toll-like receptor 4 (TLR4) expression in human pulmonary microvascular endothelial cells (HPMECs) is still unclear. Here, we investigated the role of Rab26 in regulating the expression of β2-ARs and TLR4 in HPMECs and the effect of these receptors’ imbalance on endothelial cell barrier function. The results showed that there was unbalance expression in these receptors, where β2-AR expression was remarkably reduced, and TLR4 was increased on the cell membrane after lipopolysaccharide (LPS) treatment. Furthermore, we found that Rab26 overexpression not only upregulated β2-ARs but also downregulated TLR4 expression on the cell membrane. Subsequently, the TLR4-related inflammatory response was greatly attenuated, and the hyperpermeability of HPMECs also was partially relived. Taken together, these data suggest that basal Rab26 maintains the balance between β2-ARs and TLR4 on the cell surface, and it might be a potential therapeutic target for diseases involving endothelial barrier dysfunction.

scholarly journals Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

2014 ◽  
Vol 15 (11) ◽  
pp. 19700-19728 ◽  
Author(s):  
Fabio Cattaneo ◽  
Germano Guerra ◽  
Melania Parisi ◽  
Marta De Marinis ◽  
Domenico Tafuri ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
G Protein Coupled

scholarly journals High Throughput Fluorescence Polarization: A Homogeneous Alternative to Radioligand Binding for Cell Surface Receptors

2000 ◽  
Vol 5 (2) ◽  
pp. 63-69 ◽  
Author(s):  
Michael Allen ◽  
Julian Reeves ◽  
Geoffrey Mellor
Keyword(s):  
Cell Surface ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
Radioligand Binding ◽  
Low Cost ◽  
G Protein Coupled Receptors ◽  
Cell Surface Receptors ◽  
Binding Assays ◽  
Surface Receptors ◽  
G Protein Coupled

High throughput fluorescence polarization (FP) assays are described that offer a nonradioactive, homogeneous, and low-cost alternative to radioligand binding assays for cell surface receptors (G protein-coupled receptors and ligand-gated ion channels). FP assays were shown to work across a range of both peptide (vasopressin V1a and δ-opioid) and nonpeptide (β-adrenoceptor, 5-hydroxytryptamine3) receptors. Structure-activity relationships were investigated at β1-receptors and were found to be consistent with radioligand binding assays. FP was shown to tolerate up to 5% DMSO with no loss in sensitivity or signal window. From a random set of 1,280 compounds, 1.9% were found to significantly interfere with FP measurement. If fluorescent or quenching compounds were eliminated (3% of all compounds), less than 0.4% of compounds were found to interfere with FP measurement. Assays could be run in 384-well plates with little loss of signal window or sensitivity compared to 96-well plate assays. New advances in FP measurement have therefore enabled FP to offer a high throughput alternative to radioligand binding for cell surface receptors.

scholarly journals Internalization of UT-A1 urea transporter is dynamin dependent and mediated by both caveolae- and clathrin-coated pit pathways

2010 ◽  
Vol 299 (6) ◽  
pp. F1389-F1395 ◽  
Author(s):  
Haidong Huang ◽  
Xiuyan Feng ◽  
Jieqiu Zhuang ◽  
Otto Fröhlich ◽  
Janet D. Klein ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lipid Raft ◽  
Surface Expression ◽  
Membrane Lipid ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Protein Accumulation ◽  
Intracellular Loop ◽  
Cell Plasma ◽  
Cell Surface Biotinylation

Dynamin is a large GTPase involved in several distinct modes of cell endocytosis. In this study, we examined the possible role of dynamin in UT-A1 internalization. The direct relationship of UT-A1 and dynamin was identified by coimmunoprecipitation. UT-A1 has cytosolic NH2 and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH2 and COOH termini. In cell surface biotinylation experiments, coexpression of dynamin and UT-A1 in HEK293 cells resulted in a decrease of UT-A1 cell surface expression. Conversely, cells expressing dynamin mutant K44A, which is deficient in GTP binding, showed an increased accumulation of UT-A1 protein on the cell surface. Cell plasma membrane lipid raft fractionation experiments revealed that blocking endocytosis with dynamin K44A causes UT-A1 protein accumulation in both the lipid raft and nonlipid raft pools, suggesting that both caveolae- and clathrin-mediated mechanisms may be involved in the internalization of UT-A1. This was further supported by 1) small interfering RNA to knock down either caveolin-1 or μ2 reduced UT-A1 internalization in HEK293 cells and 2) inhibition of either the caveolae pathway by methyl-β-cyclodextrin or the clathrin pathway by concanavalin A caused UT-A1 cell membrane accumulation. Functionally, overexpression of dynamin, caveolin, or μ2 decreased UT-A1 urea transport activity and decreased UT-A1 cell surface expression. We conclude that UT-A1 endocytosis is dynamin-dependent and mediated by both caveolae- and clathrin-coated pit pathways.

Abstract 200: Novel Interaction of Spinophilin with Alpha1a-Adrenergic Receptor and its Genetic Variant in Cardiovascular Cells

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Ekaterina Babaeva ◽  
Irina Gradinaru ◽  
Debra A Schwinn ◽  
Anush Oganesian
Keyword(s):  
Genetic Variant ◽  
Direct Interaction ◽  
Cell Types ◽  
Intracellular Loop ◽  
Preliminary Results ◽  
Protein Levels ◽  
Cardiovascular Cells ◽  
Novel Target ◽  
G Protein Coupled

Activation of α 1 -Adrenergic Receptors (α 1 ARs), members of the G protein-coupled receptor (GPCR) superfamily, in response to stimulation of the sympathetic nervous system by catecholamines plays a major role in regulating cardiovascular (CV) function. Among three α 1 AR subtypes (α 1a ,α 1b ,α 1d ), α 1a ARs predominate in human resistant vessels and in heart. Recently, we discovered that naturally occurring human α 1a AR-G247R (247R) genetic variant, identified in the 3 rd intracellular loop (3iL) of the receptor in highly hypertensive patient, triggers constitutive hyperproliferation in CV cells (cardiomyoblasts, smooth muscle cells (SMC) and fibroblasts), which may lead to myocardial fibrosis and remodeling. In fibroblasts and cardiomyoblasts 247R triggered hyperproliferation is due to constitutive active coupling to Gq-independent βarrestin1/MMP/EGFR/ERK dependent pathway, while in SMC it is Gq- and MMP/EGFR/ERK-dependent. Here we report that α 1a AR-WT (WT) and 247R differentially interact with ubiquitous multi-domain scaffold protein spinophilin (SPL) that binds to 3iL of several GPCRs competing with arrestin thereby prolonging their signaling. The role of SPL in CV regulation is poorly studied. We hypothesized that SPL mediates constitutive signaling of 247R and examined whether SPL directly interacts with α 1a AR-WT or 247R. Our preliminary results reveal a direct interaction of SPL with WT and 247R: the SPL-WT interaction appears to be stronger as determined by co-immunoprecipitation. Different domains of SPL differentially interact with WT or 247R. SPL 1-480aa fragment interacts stronger with WT indicating interaction with 3iL, while SPL 480-817 fragment interacts stronger with 247R. Our preliminary results also demonstrate that 247R expression in all three cell types elevates endogenous SPL protein levels. Importantly, inhibition of SPL expression with specific siRNA reduces 247R-triggered hyperproliferation in SMC and cardiomyoblasts to near normal levels, while SPL knockdown has no effect in WT cells. Thus, we identified SPL as a novel protein involved in interacting and signaling of α 1a AR and its genetic variant in CV cells and that SPL could be considered as a potentially novel target in α 1a AR-mediated cardiovascular disorders.

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