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.

Intracellular signalling

2010 ◽  
pp. 169-176
Author(s):  
R. Andres Floto
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Intracellular Signalling ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Transmission Of Information ◽  
Effective Transmission

This section outlines the general principles of intracellular signalling. Focusing on cell surface receptors, the requirements for effective transmission of information across the plasma membrane are outlined. The principal mechanisms utilized in mammalian signal transduction are described. For each, the pathological consequences of aberrant signalling and means by which pathways can be pharmacologically targeted are described in molecular terms....

scholarly journals Redirecting Retroviral Tropism by Insertion of Short, Nondisruptive Peptide Ligands into Envelope

2002 ◽  
Vol 76 (7) ◽  
pp. 3558-3563 ◽  
Author(s):  
Timothy J. Gollan ◽  
Michael R. Green
Keyword(s):  
Cell Surface ◽  
Leukemia Virus ◽  
Envelope Proteins ◽  
Peptide Ligands ◽  
Viral Envelope ◽  
Moloney Murine Leukemia Virus ◽  
Cell Surface Receptors ◽  
Wild Type ◽  
Surface Receptors

ABSTRACT A potentially powerful approach for in vivo gene delivery is to target retrovirus to specific cells through interactions between cell surface receptors and appropriately modified viral envelope proteins. Previously, relatively large (>100 residues) protein ligands to cell surface receptors have been inserted at or near the N terminus of retroviral envelope proteins. Although viral tropism could be altered, the chimeric envelope proteins lacked full activity, and coexpression of wild-type envelope was required for production of transducing virus. Here we analyze more than 40 derivatives of ecotropic Moloney murine leukemia virus (MLV) envelope, containing insertions of short RGD-containing peptides, which are ligands for integrin receptors. In many cases pseudotyped viruses containing only the chimeric envelope protein could transduce human cells. The precise location, size, and flanking sequences of the ligand affected transduction specificity and efficiency. We conclude that retroviral tropism can be rationally reengineered by insertion of short peptide ligands and without the need to coexpress wild-type envelope.

scholarly journals Rapid Transport of Internalized P-Selectin to Late Endosomes and the Tgn

2000 ◽  
Vol 151 (1) ◽  
pp. 107-116 ◽  
Author(s):  
Kimberly S. Straley ◽  
Samuel A. Green
Keyword(s):  
Cell Surface ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Endosomal Sorting ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Granule Membrane

Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles. We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins. P-selectin, a transmembrane cell adhesion protein involved in inflammation, is sorted from recycling cell surface receptors (e.g., low density lipoprotein [LDL] receptor) in endosomes, and is transported from the cell surface to the TGN with a half-time of 20–25 min, six to seven times faster than LDL receptor. Native P-selectin colocalizes with LDL, which is efficiently transported to lysosomes, for 20 min after internalization, but a deletion mutant deficient in endosomal sorting activity rapidly separates from the LDL pathway. Thus, P-selectin is sorted from LDL receptor in early endosomes, driving P-selectin rapidly into late endosomes. P-selectin then recycles to the TGN as efficiently as other receptors. Thus, the primary effect of early endosomal sorting of P-selectin is its rapid delivery to the TGN, with rapid turnover in lysosomes a secondary effect of frequent passage through late endosomes. This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors.

Rab5a and rab11a mediate agonist-induced trafficking of protease-activated receptor 2

2003 ◽  
Vol 284 (5) ◽  
pp. C1319-C1329 ◽  
Author(s):  
Dirk Roosterman ◽  
Fabien Schmidlin ◽  
Nigel W. Bunnett
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Calcium Mobilization ◽  
Dominant Negative ◽  
Wild Type ◽  
Early Endosomes ◽  
Protease Activated Receptor 2

We evaluated the contribution of rab5a and rab11a to trafficking and signaling of protease-activated receptor 2 (PAR2), a receptor for trypsin and tryptase. Agonists stimulated internalization of PAR2 into early endosomes containing rab5a. Dominant negative rab5aS34N disrupted early endosomes and inhibited agonist-stimulated endocytosis of PAR2. Internalized PAR2 was sorted to lysosomes, and rab5a remained in early endosomes. Rab5a promoted and rab5aS34N impeded resensitization of trypsin-induced calcium mobilization. Rab11a was detected in the Golgi apparatus with PAR2, and PAR2 agonists stimulated redistribution of rab11a into vesicles containing PAR2 that migrated to the cell surface. Dominant negative rab11aS25N was mostly confined to the Golgi apparatus. Although expression of rab11aS25N caused retention of PAR2 in the Golgi apparatus, it did not abolish trafficking of PAR2 to the cell surface. However, expression of wild-type rab11a accelerated both recovery of PAR2 at the cell surface and resensitization of PAR2 signaling. Thus rab5a is required for PAR2 endocytosis and resensitization, whereas rab11a contributes to trafficking of PAR2 from the Golgi apparatus to the plasma membrane.

scholarly journals The post-endocytotic fate of the gonadotropin receptors is an important determinant of the desensitization of gonadotropin responses

2005 ◽  
Vol 34 (2) ◽  
pp. 447-457 ◽  
Author(s):  
R S Bhaskaran ◽  
M Ascoli
Keyword(s):  
Cell Surface ◽  
Leydig Cells ◽  
Degradation Pathway ◽  
Cell Surface Receptors ◽  
Wild Type ◽  
Gonadotropin Receptors ◽  
Surface Receptors ◽  
Receptor Complexes ◽  
Mutant Receptors ◽  
Stimulation Of

Internalization of the ligand/receptor complexes is a consequence of the activation of the gonadotropin receptors. Since the recycling or degradation of the internalized receptors results in the maintenance or loss of cell surface receptors respectively and this contributes to the loss of responsiveness, we hypothesized that the fate of the internalized receptors could be an important component of desensitization. We examined this hypothesis using the wild-type and mutants of the human LH (hLHR) receptors and follitropin receptors expressed in MA-10 and KK-1 cells respectively. The receptor mutants were chosen because they are routed mostly to a lysosomal degradation pathway whereas the wild-type receptors are recycled back to the surface. We have shown that agonist stimulation of cells expressing the mutant receptors results in a more pronounced loss of cell surface receptors and agonist responses than stimulation of cells expressing the wild-type receptors. We concluded that receptor recycling promotes the maintenance of cell surface receptors and preserves hormonal responsiveness. This property of the hLHR is likely to be physiologically important because there at least two hLHR-expressing tissues in pregnant women, the maternal corpus luteum and the fetal Leydig cells, where a loss of hormonal responsiveness induced by the elevated levels of human chorionic gonadotropin that occur during pregnancy is not desirable.

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.

scholarly journals Constitutive Activating and Inactivating Mutants of Eel Luteinizing Hormone Receptor

2020 ◽  
Author(s):  
Munkhzaya Byambaragchaa ◽  
Dong-An Kim ◽  
Dae-Jung Kim ◽  
Sun-Mee Hong ◽  
Myung-Hwa Kang ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Luteinizing Hormone ◽  
Cell Surface ◽  
Hormone Receptor ◽  
Luteinizing Hormone Receptor ◽  
Cell Surface Receptors ◽  
Wild Type ◽  
Surface Receptors ◽  
Agonist Stimulation ◽  
In Cells

We analyzed signal transduction of three constitutively activating mutants (M410T, L469R, and D590Y) and two inactivating mutants (D417N and Y558F) of the eel luteinizing hormone receptor (eel LHR), known to occur in human LHR. The objective of this study was to assess the functional effects of these mutations in signal transduction and cell surface loss of receptor. Mutant receptors were transiently expressed in Chinese hamster ovary (CHO-K1) cells. Eel LH-stimulated accumulation of cyclic adenosine monophosphate (cAMP) was measured by homogeneous time-resolved fluorescence (HTRF) assays. The loss of receptors from the cells surface was measured using an enzyme-linked immunosorbent assay (ELISA) in human embryonic kidney (HEK) 293 cells. The cAMP response in cells expressing the wild type eel LHR was increased in a dose-dependent manner using eel LH ligand stimulation. Compared with the wild type, cells expressing the activating mutants (M410T, L469R, and D590Y), exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response without agonist stimulation, respectively. Their maximal responses to agonist stimulation were approximately 65%, 52%, and 98%, respectively, of those of the wild type. The inactivating mutants (D417N and Y558F) did not completely impair signal transduction, and their maximal responses were only 33% and25 % of those of wild type. These data clearly showed that the eel LHR-L469R and D590Y, activating mutants enhanced the rate of the loss of cell surface receptors following treatment with eel LH. Thus, the loss of cell surface receptors in cells expressing mutant eel LHRs was consistent with the eel LH agonist-induced production of cAMP. Our results suggested that the activation of the eel LHR requires appropriate loss of LHR-ligand complexes from the cell surface.

Extracellular Calcium and cAMP: Second Messengers as “Third Messengers”?

Physiology ◽  
2007 ◽  
Vol 22 (5) ◽  
pp. 320-327 ◽  
Author(s):  
Aldebaran M. Hofer ◽  
Konstantinos Lefkimmiatis
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Second Messengers ◽  
Signaling Molecules ◽  
Extracellular Calcium ◽  
Cell Surface Receptors ◽  
Surface Receptors

Calcium and cyclic AMP are familiar second messengers that typically become elevated inside cells on activation of cell surface receptors. This article will explore emerging evidence that transport of these signaling molecules across the plasma membrane allows them to be recycled as “third messengers,” extending their ability to convey information in a domain outside the cell.

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