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.

INTRODUCTION

Following agonist binding, many GPCRs (G-protein-coupled receptors) undergo endocytosis to a common population of early endosomes, from which they are sorted into different pathways in a highly specific manner. Although some GPCRs, such as the PAR (protease-activated receptor) 1, are targeted predominantly towards lysosomes for degradation [1,2], many of them recycle back to the plasma membrane to replenish the biologically active cell-surface receptor population. Some receptors recycle rapidly, probably directly from the early endosomal compartment, where the receptors are sent back to the plasma membrane after being dephosphorylated [35]. However, other GPCRs, such as the V2R (V2 vasopressin receptor), TPβ (thromboxane A2 receptor β-isoform) and the CXCR2 (CXC chemokine receptor 2), undergo slower recycling, in which they traffic from early endosomes through the perinuclear recycling endosomal compartment and then back to the plasma membrane [68].

The β2ARs (β2-adrenergic receptors) undergo ligand-induced endocytosis into early endosomes via clathrin-coated vesicles, but are then rapidly and efficiently recycled back to the plasma membrane, restoring surface receptor numbers and promoting the rapid functional recovery of cellular responsiveness to ligands [5]. With prolonged agonist exposure, β2ARs undergo continuous rounds of endocytosis and recycling [9], but, over time, some receptors are redirected to lysosomes, where they are degraded [10,11]. Results published by Moore et al. [1113] raised the possibility that, during prolonged exposure to agonist, some β2ARs also utilize a slow recycling pathway through the perinuclear recycling endosomal compartment. Their results demonstrate that the trafficking of β2ARs through the perinuclear recycling endosome is regulated by the small GTPase Rab11 [13].

Rab11 is part of the subfamily of Ras-like small GTPases termed Rab GTPases, which are implicated in the regulation of intracellular trafficking. Over 60 members of the Rab GTPase family have been identified, and each is believed to be associated specifically with a particular organelle or pathway [14]. Among them, Rab11 was shown to be associated with post-Golgi membranes, including the trans-Golgi network, and the perinuclear recycling endosome, which is proposed to regulate the slow return of recycling receptors to the plasma membrane [15,16]. Like all GTPases of the Ras family, Rab11 functions as a molecular switch. Rabs associate with membranes in the GDP-bound form and switch to a GTP-bound conformation in a reaction catalysed by a GEF (guanine-nucleotide-exchange factor) [17]. In the active GTP-bound state, they interact with downstream effector proteins, co-ordinating the consecutive stages of transport, such as vesicle formation, vesicle and organelle motility, and the tethering of vesicles to their target compartment [18]. Although Rab proteins are known to interact with a large variety of effectors [19], a new class of Rab-interacting proteins is now emerging, based on results showing that cargo proteins can also bind Rabs and can therefore regulate their own trafficking by direct interactions with the transport machinery [20]. For example, Mostov and co-workers were the first to demonstrate a direct interaction between a Rab protein and a cargo molecule: they showed that that the pIgR (polymeric IgA receptor) interacts directly with Rab3b, controlling IgA-stimulated transcytosis [21]. Interactions between Rab5 and AT1AR (angiotensin II type 1A receptor), and between Rab11a and the Ca2+-selective channels TRPV5/6 (transient receptor potential vanilloid 5/6) as well as the TPβ, were reported previously [2224]. In the present study, we investigated whether an interaction between the Rab11 GTPase and the β2AR is implicated in the intracellular sorting and recycling of the receptor.

EXPERIMENTAL

Reagents

The polyclonal anti-HA (haemagglutinin) antibody and Protein A–agarose were purchased from Santa Cruz Biotechnology. A polyclonal anti-GST (glutathione transferase) antibody was from Bethyl Laboratories. A monoclonal anti-HA antibody was from Covance, and monoclonal FLAG-specific antibodies (M1 and M2) were from Sigma. Rhodamine Red goat anti-mouse antibody was from Molecular Probes. HRP (horseradish peroxidase)-conjugated anti-HA and anti-Rab11 antibodies were bought from Roche and BD Transduction Laboratories respectively. Isoprenaline (isoproterenol) and propranolol were purchased from Sigma. An alkaline phosphatase-conjugated goat anti-mouse antibody and the alkaline phosphatase substrate kit were purchased from Bio-Rad.

Plasmid constructions

cDNA fragments coding for the first and second ICLs (intracellular loops) of the β2AR were created by annealing two oligonucleotides which corresponded to amino acids Ala59–Phe71 for ICL1 and Asp130–Ala150 for ICL2. The annealed fragments were then inserted in the pGEX-4T-1 vector (Amersham Biosciences) using the EcoRI and XhoI sites. cDNA fragments coding for ICL3 (Arg221–Thr274) and the CT (C-terminal tail) of β2AR (Pro330–Leu413) were generated by PCR using the Phusion High-Fidelity PCR system (New England Biolabs). The PCR products were purified and digested with EcoRI and Xho I and ligated into the pGEX-4T vector. Fragments and point mutations of the β2AR-CT were generated using the above methods. Mutation of residues Arg333 and Lys348 to alanine to produce the β2AR RK/AA (R333A/K348A) mutant was carried out by site-directed mutagenesis. The mutant receptor was subcloned into pcDNA3-FLAG [25] digested with EcoRI and XhoI. The integrity of the coding sequences of all of the constructs was confirmed by dideoxy DNA sequencing.

Cell culture and transfection

HEK-293 cells (human embryonic kidney cells) were maintained in DMEM (Dulbecco's modified Eagle's medium) (Invitrogen) supplemented with 10% (v/v) fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. Transient transfections of HEK-293 cells grown to 50–70% confluence were performed using the TransIT-LT1 Reagent (Mirus) according to the manufacturer's instructions.

Immunoprecipitation

Immunoprecipation experiments were performed as described previously [26]. Briefly, 9×105 HEK-293 cells were grown overnight in 60-mm-diameter plates. On the following day, cells were transfected with the indicated constructs as described above and were maintained for 48 h. The cells were then washed with ice-cold PBS and harvested in 500 μl of lysis buffer [50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM Na4P2O7 and 5 mM EDTA] supplemented with protease inhibitors (9 nM pepstatin, 9 nM antipain, 10 nM leupeptin and 10 nM chymostatin; Sigma). After incubation in lysis buffer for 60 min at 4 °C, the lysates were clarified by centrifugation for 20 min at 11000 g at 4 °C. Specific antibodies (1 μg) were then added to the supernatant. After incubation for 60 min at 4 °C, 40 μl of 50% (v/v) Protein A–agarose or Protein G–agarose was added, followed by an overnight incubation at 4 °C. Samples were then centrifuged at 11000 g for 1 min in a microcentrifuge and washed three times with 1 ml of lysis buffer. Immunoprecipitated proteins were eluted by the addition of 40 μl of SDS sample buffer, followed by a 60 min incubation at room temperature (20 °C). Initial lysates and immunoprecipitated proteins were analysed by SDS/PAGE and immunoblotting using specific antibodies.

Recombinant protein production and binding assays

The pRSETa-HA-Rab11 construct [24] was used to produce a His6-tagged HA–Rab11 fusion protein in the OverExpress™ C41(DE3) Escherichia coli strain (Avidis) following the manufacturer's instructions. The recombinant HA–Rab11 protein was purified using Ni-NTA (Ni2+-nitrilotriacetate)–agarose resin (Qiagen) following the manufacturer's instructions. All of the constructs in the pGEX-4-T1 vector listed above were used to produce GST-tagged mutants of β2AR-CT or -ICL fusion proteins in the Over-ExpressTM C41(DE3) E. coli strain. All of the GST-tagged recombinant mutants were purified using glutathione–Sepharose™ 4B (Amersham Biosciences) following the manufacturer's instructions. Purified recombinant proteins were analysed by SDS/PAGE, followed by Coomassie Brilliant Blue R-250 staining. For this, 5 μg of glutathione–Sepharose-bound GST-tagged β2AR-CT or -ICL fusion proteins was incubated with 5 μg of purified His6-tagged HA–Rab11 in binding buffer [10 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol and 0.5% Igepal] supplemented with protease inhibitors (9 nM pepstatin, 9 nM antipain, 10 nM leupeptin and 10 nM chymostatin) and 2 mM dithiothreitol. The binding reactions were then washed four times with binding buffer. SDS sample buffer was added to the binding reactions, and the tubes were boiled for 5 min. The binding reactions were analysed by SDS/PAGE, and immunoblotting was performed using the indicated specific antibodies.

Measurement of cell-surface receptor loss and recycling

For quantification of cell-surface receptor loss and recycling, 8.5×105 HEK-293 cells were plated in 24-well plates pre-coated with 0.1 mg/ml poly-L-lysine (Sigma). Cells were transfected with the indicated constructs and then maintained for an additional 48 h. The cells were then treated with 5 μM isoprenaline for 6 h at 37 °C. The cells were washed extensively and incubated in DMEM containing 5 μM propranolol to allow recycling to occur. At various time points up to 60 min, the cells were fixed in 3.7% (v/v) formaldehyde/TBS (Tris-buffered saline) [20 mM Tris/HCl (pH 7.5) and 150 mM NaCl] for 5 min at room temperature. Cells were then washed three times with TBS, and non-specific binding was blocked by incubation with TBS containing 1% BSA for 30 min. A monoclonal FLAG-specific antibody was then added at a dilution of 1:1000 in TBS and 1% BSA for 60 min. Following the incubation with the primary antibody, cells were washed three times with TBS and blocked again with TBS and 1% BSA for 15 min. Cells were then incubated with an alkaline phosphatase-conjugated goat anti-mouse antibody at 1:1000 dilution in TBS and 1% BSA for 60 min. The cells were washed three times with TBS, and 250 μl of a colorimetric alkaline phosphatase substrate was added. The plates were then incubated at 37 °C until a yellow colour appeared. A 100 μl aliquot of the colorimetric reaction was taken, stopped by the addition of 100 μl of 0.4 M sodium hydroxide, and the absorbance (A) was measured at 405 nm using a Titertek Multiskan MCC/340 spectrophotometer. Cells transfected with pcDNA3 alone were studied concurrently to determine the background absorbance. The percentage of receptor recycling was calculated from the proportion of internalized receptor that was recovered at the cell surface following agonist removal.

Immunofluorescence staining and confocal microscopy

For recycling and co-localization experiments, HEK-293 cells were plated in 6-well plates at a density of 3×105 cells per well. The following day, the cells were transiently transfected with either pcDNA3-FLAG-β2AR and GFP (green fluorescent protein)–Rab11 (0.1 μg) or pcDNA3-FLAG-β2AR RK/AA and GFP–Rab11. On the following day, 2×105 cells were transferred on to coverslips coated with 0.1 mg/ml poly-L-lysine (Sigma) and grown overnight. The cells were then treated or not with 5 μM isoprenaline for 6 h at 37 °C in DMEM. Following agonist stimulation, cells were either fixed immediately or washed extensively and incubated in DMEM containing 5 μM propranolol for 30 min to allow recycling. Cells were then fixed with 4% (v/v) paraformaldehyde in PBS for 10 min at room temperature. The cells were then washed again with PBS, permeabilized by incubation for 20 min with 0.1% Triton X-100 in PBS and blocked with 0.1% Triton X-100 in PBS containing 5% (w/v) non-fat dried skimmed milk powder for 30 min at room temperature. Cells were incubated with primary antibodies diluted in blocking solution for 60 min at room temperature. Then cells were washed twice with PBS, blocked again with 0.1% Triton X-100 in PBS containing 5% (w/v) non-fat dried skimmed milk powder for 30 min at room temperature and incubated with appropriate secondary antibodies diluted in blocking solution for 60 min at room temperature. The cells were washed twice with the permeabilization buffer and twice with PBS, and the coverslips were mounted using Vectashield mounting medium (Vector Laboratories). Confocal microscopy was performed using a scanning confocal microscope (FV1000; Olympus) coupled to an inverted microscope with a ×63 oil-immersion objective lens (Olympus), and images were processed using Image-Pro Plus 6.0 (Media Cybernetics).

Statistical analysis

Statistical analyses were performed using Prism version 4.0 (GraphPad Software) using the Student's t test. Data were considered significant when P values were <0.05 (*) or <0.01 (**).

RESULTS

Rab11 is a β2AR-interacting protein

Previous results presented by Moore et al. [13] suggested a role for Rab11 in regulating the traffic of the β2AR at the level of the perinuclear recycling endosome, where modulation of Rab11 activity dictates the balance between receptor recycling and down-regulation during prolonged exposure to agonist. We first wanted to assess whether Rab11 was involved in the recycling of the β2AR in our system. The activity of endogenous Rab11 was competed by expression of the Rab11 S25N dominant-negative mutant in HEK-293 cells expressing HA-tagged β2ARs. A time course of reappearance of cell-surface receptors after agonist removal was performed by ELISA. The cells were stimulated with 5 μM isoprenaline for 6 h before being thoroughly washed, placed in warm medium containing the antagonist propranolol to prevent any additional internalization and incubated at 37 °C for various time periods to allow receptor recycling [13]. As shown in Figure 1, recycling of the β2AR was strongly inhibited when Rab11 S25N was co-expressed with the β2AR compared with β2AR expression alone. Co-expression of wild-type Rab11 and of the constitutively active Rab11 Q70L mutant failed to alter the recycling of the β2AR in the same system (Figure 1). The Rab11 S25N results thus confirm that Rab11 is involved in β2AR recycling.

The dominant-negative Rab11 S25N mutant inhibits β2AR recycling

Figure 1
The dominant-negative Rab11 S25N mutant inhibits β2AR recycling

HEK-293 cells expressing FLAG–β2AR alone (with pcDNA3) or in combination with wild-type Rab11, Rab11 Q70L or Rab11 S25N were treated for 6 h with 5 μM isoprenaline. Cells were then washed and incubated in DMEM containing 5 μM propranolol for the indicated time periods to prevent further receptor internalization and to allow receptor recycling. Quantification of surface receptors was performed by ELISA and the percentage of receptor recycling was calculated as described in the Experimental section. Results are means±S.E.M. (n=3). intern., internalization.

Figure 1
The dominant-negative Rab11 S25N mutant inhibits β2AR recycling

HEK-293 cells expressing FLAG–β2AR alone (with pcDNA3) or in combination with wild-type Rab11, Rab11 Q70L or Rab11 S25N were treated for 6 h with 5 μM isoprenaline. Cells were then washed and incubated in DMEM containing 5 μM propranolol for the indicated time periods to prevent further receptor internalization and to allow receptor recycling. Quantification of surface receptors was performed by ELISA and the percentage of receptor recycling was calculated as described in the Experimental section. Results are means±S.E.M. (n=3). intern., internalization.

We have reported previously that the intracellular trafficking of the TPβ depends on a direct interaction with Rab11 [24]. To determine whether Rab11 can also associate with other GPCRs, we performed immunoprecipitation experiments in HEK-293 cells transiently expressing FLAG–Rab11 with either the HA-tagged β2AR or HA–DP (prostaglandin D2 receptor). Cell lysates were incubated with a HA-specific polyclonal antibody, and co-immunoprecipitated Rab11 was detected by Western-blot analysis with a monoclonal anti-FLAG antibody. The results of the present study demonstrate that Rab11 does co-immunoprecipitate with the β2AR (Figure 2A). Co-immunoprecipitation of Rab11 was specific to the β2AR, because Rab11 was not co-immunoprecipitated with HA–DP (Figure 2B). The specificity of the Rab11–β2AR interaction was also demonstrated using Rab4, another member of the Rab family of GTPases, which failed to co-immunoprecipitate with the β2AR under the same experimental conditions (Figure 2C). Immunoprecipitation of endogenous Rab11 resulted in the co-immunoprecipitation of the β2AR in significantly higher amounts compared with control, showing that the receptor interacts with endogenous Rab11.

Rab11 associates with the β2AR

Figure 2
Rab11 associates with the β2AR

HEK-293 cells were transiently transfected with HA-tagged β2AR (A) or HA-tagged DP (B) and FLAG-tagged Rab11. Immunoprecipitation (IP) of the receptors was performed using a HA-specific polyclonal antibody, and immunoblotting (IB) was performed with an HA-specific or a FLAG-specific monoclonal antibody. Molecular masses are indicated on the left-hand side (in kDa). (C) HEK-293 cells were transiently transfected with the indicated constructs. Immunoprecipitation (IP) of the receptors was performed using a FLAG-specific monoclonal antibody, and immunoblotting (IB) was performed with HA- or FLAG-specific monoclonal antibodies. Molecular masses are indicated on the left-hand side (in kDa). (D) Cell lysates of HEK-293 cells transiently expressing the HA-tagged β2AR were immunoprecipitated (IP) in the presence or absence [control (IP Ctl)] of a mouse monoclonal antibody against endogenous Rab11. Immunoblotting (IB) was performed with anti-Rab11 and HRP (horseradish peroxidase)-conjugated anti-HA antibodies. The blots shown are representative of three independent experiments.

Figure 2
Rab11 associates with the β2AR

HEK-293 cells were transiently transfected with HA-tagged β2AR (A) or HA-tagged DP (B) and FLAG-tagged Rab11. Immunoprecipitation (IP) of the receptors was performed using a HA-specific polyclonal antibody, and immunoblotting (IB) was performed with an HA-specific or a FLAG-specific monoclonal antibody. Molecular masses are indicated on the left-hand side (in kDa). (C) HEK-293 cells were transiently transfected with the indicated constructs. Immunoprecipitation (IP) of the receptors was performed using a FLAG-specific monoclonal antibody, and immunoblotting (IB) was performed with HA- or FLAG-specific monoclonal antibodies. Molecular masses are indicated on the left-hand side (in kDa). (D) Cell lysates of HEK-293 cells transiently expressing the HA-tagged β2AR were immunoprecipitated (IP) in the presence or absence [control (IP Ctl)] of a mouse monoclonal antibody against endogenous Rab11. Immunoblotting (IB) was performed with anti-Rab11 and HRP (horseradish peroxidase)-conjugated anti-HA antibodies. The blots shown are representative of three independent experiments.

To corroborate further the association between Rab11 and β2AR and to determine if there is a direct interaction between the two proteins, we performed in vitro binding assays using the purified recombinant GST–β2AR-CT along with the purified recombinant His6–HA–Rab11 (where His6 is hexahistidine). We also wanted to ascertain if Rab11 could interact with the other intracellular domains of β2AR. The three ICLs of β2AR were thus individually expressed in fusion with GST (GST–ICLs) and also used in the pulldown assays. The results presented in Figure 3 show that Rab11 binds to glutathione–Sepharose-bound GST–β2AR-CT, but not to glutathione–Sepharose-bound GST and GST–ICLs, indicating that there is a direct interaction between Rab11 and the CT of the β2AR.

The C-terminus of the β2AR interacts directly with Rab11

Figure 3
The C-terminus of the β2AR interacts directly with Rab11

Binding assays were carried out using purified glutathione–Sepharose-bound GST–β2AR-CT and GST–ICLs, which were incubated with purified recombinant His6–HA–Rab11. The binding of Rab11 was detected by immunoblotting (IB) using a HA-specific monoclonal antibody (top and middle panels), and the GST-fusion proteins present in the binding reaction were detected using Ponceau S red staining (bottom panel). The blots shown are representative of three independent experiments. Molecular masses are indicated on the left-hand side (in kDa).

Figure 3
The C-terminus of the β2AR interacts directly with Rab11

Binding assays were carried out using purified glutathione–Sepharose-bound GST–β2AR-CT and GST–ICLs, which were incubated with purified recombinant His6–HA–Rab11. The binding of Rab11 was detected by immunoblotting (IB) using a HA-specific monoclonal antibody (top and middle panels), and the GST-fusion proteins present in the binding reaction were detected using Ponceau S red staining (bottom panel). The blots shown are representative of three independent experiments. Molecular masses are indicated on the left-hand side (in kDa).

The β2AR preferentially interacts with GDP-bound Rab11

It has been shown for complexes between Rab GTPases and receptors such as TPβ and AT1AR that the nucleotide status of the GTPase plays a pivotal role in the interaction between the two proteins [22,24]. We were thus interested in studying whether the interaction between β2AR and Rab11 was regulated by the nature of the nucleotide bound to the small G-protein. To address this question, we performed GST pull-down assays using purified recombinant GST–β2AR-CT on extracts of HEK-293 cells overexpressing HA–Rab11, HA–Rab11 S25N or HA–Rab11 Q70L. Rab11 S25N is a Rab11 mutant that displays a lower affinity for GTP than for GDP, leading to a GDP-bound Rab11, whereas Rab11 Q70L is a mutant with reduced GTPase activity, which stabilizes the GTP-bound and active conformation of Rab11 [27]. The results obtained in Figure 4(A) show that Rab11 and Rab11 S25N, but not Rab11 Q70L, could bind to glutathione–Sepharose-bound GST–β2AR-CT. Similarly, immunoprecipitation experiments in HEK-293 cells revealed that β2AR interacted with Rab11 and Rab11 S25N, but interacted only weakly with Rab11 Q70L (Figure 4B). Interestingly, in both the GST pull-down assays and immunoprecipitation experiments, more Rab11 S25N bound to β2AR than Rab11, suggesting a preferential interaction of the receptor with the GDP-bound form of Rab11.

The β2AR–Rab11 interaction is dependent on the GDP/GTP nucleotide status of Rab11

Figure 4
The β2AR–Rab11 interaction is dependent on the GDP/GTP nucleotide status of Rab11

(A) GST pulldown assays were performed using purified recombinant GST–β2AR-CT protein and extracts of HEK-293 cells overexpressing HA–Rab11, HA–Rab11 S25N or HA–Rab11 Q70L as detailed in the Experimental section. Bound Rab11 proteins (top panel) and the amount of HA–Rab11 proteins present in the reaction (middle panel) were detected with an anti-HA monoclonal antibody. The amount of GST fusion proteins in the binding reactions was evaluated with a GST-specific polyclonal antibody (bottom panel). IB, immunoblot. (B) Co-immunoprecipitation experiments were performed in HEK-293 cells transfected with pcDNA3-FLAG-β2AR and pcDNA3-HA-Rab11, pcDNA3-HA-Rab11S25N or pcDNA3-HA-Rab11Q70L. Immunoprecipitation (IP) of receptors was carried out using a FLAG-specific monoclonal antibody (M2) and immunoblotting (IB) was performed with HA-specific or FLAG-specific monoclonal antibodies as described in the Experimental section. Molecular masses are indicated on the left-hand side (in kDa). The blots shown are representative of three independent experiments.

Figure 4
The β2AR–Rab11 interaction is dependent on the GDP/GTP nucleotide status of Rab11

(A) GST pulldown assays were performed using purified recombinant GST–β2AR-CT protein and extracts of HEK-293 cells overexpressing HA–Rab11, HA–Rab11 S25N or HA–Rab11 Q70L as detailed in the Experimental section. Bound Rab11 proteins (top panel) and the amount of HA–Rab11 proteins present in the reaction (middle panel) were detected with an anti-HA monoclonal antibody. The amount of GST fusion proteins in the binding reactions was evaluated with a GST-specific polyclonal antibody (bottom panel). IB, immunoblot. (B) Co-immunoprecipitation experiments were performed in HEK-293 cells transfected with pcDNA3-FLAG-β2AR and pcDNA3-HA-Rab11, pcDNA3-HA-Rab11S25N or pcDNA3-HA-Rab11Q70L. Immunoprecipitation (IP) of receptors was carried out using a FLAG-specific monoclonal antibody (M2) and immunoblotting (IB) was performed with HA-specific or FLAG-specific monoclonal antibodies as described in the Experimental section. Molecular masses are indicated on the left-hand side (in kDa). The blots shown are representative of three independent experiments.

Identification of the Rab11-binding sites on the β2AR-CT

To identify the regions of the β2AR-CT which contribute to Rab11 binding, we performed GST pull-down assays using several C-terminally truncated mutant constructs of the receptor that we introduced in the pGEX-4T-1 vector (Figure 5A). Binding assays were performed using the purified recombinant GST–β2AR-CT mutant proteins along with the purified recombinant His6–HA–Rab11. The binding reactions were analysed by immunoblotting with a HA-specific monoclonal antibody to detect the presence of the His6–HA–Rab11. Figure 5(B) shows that Rab11 bound to GST–β2AR-CT truncated at amino acids 395, 370, 362 and 352, but not to GST–β2AR-CT containing only amino acids 330–344, narrowing the binding site to a region of eight amino acids comprising residues 345–352. Our results also demonstrate that constructs containing amino acids 345–413, 340–413 and 335–413 of β2AR-CT did not bind to Rab11 (Figure 5B), demonstrating that amino acids 330–335 are also essential for Rab11 binding. Taken together, these results suggest that there are two domains within β2AR-CT that are required for Rab11 binding and that these domains are located between amino acids 330 and 335 and amino acids 345 and 352. To define further the residues which are implicated in Rab11 binding, we mutated the three charged residues located within the two identified domains, Asp331, Arg333 and Lys348, to alanine residues (see Figure 6A). The D331A mutation did not impair Rab11 binding to the β2AR-CT significantly, whereas the R333A and K348A mutations reduced Rab11 binding drastically (Figure 6B). The two mutations that affected Rab11 binding were then combined to generate the double mutant GST–β2AR-CT RK/AA construct. As can be seen in Figure 6(B), this mutant was unable to interact with Rab11, showing that residues Arg333 and Lys348 are required for the association between Rab11 and the β2AR-CT.

Amino acids 330–335 and 345–352 of the β2AR-CT are essential for Rab11 binding

Figure 5
Amino acids 330–335 and 345–352 of the β2AR-CT are essential for Rab11 binding

(A) Schematic representation of the GST-tagged β2AR-CT deletion mutants used in binding assays. The Rab11 binding properties of the constructs are summarized on the right-hand side. +, binding; −, no binding. (B) The binding assays were carried out using purified glutathione–Sepharose-bound GST, GST-tagged β2AR-CT and GST-tagged β2AR-CT mutants, which were incubated with purified recombinant His6–HA–Rab11. The binding of Rab11 was detected by immunoblotting (IB) using a HA-specific monoclonal antibody, and the GST-fusion proteins present in the binding reaction were detected using Ponceau S red staining. The blots shown are representative of three independent experiments. Molecular masses are indicated on the left-hand side (in kDa).

Figure 5
Amino acids 330–335 and 345–352 of the β2AR-CT are essential for Rab11 binding

(A) Schematic representation of the GST-tagged β2AR-CT deletion mutants used in binding assays. The Rab11 binding properties of the constructs are summarized on the right-hand side. +, binding; −, no binding. (B) The binding assays were carried out using purified glutathione–Sepharose-bound GST, GST-tagged β2AR-CT and GST-tagged β2AR-CT mutants, which were incubated with purified recombinant His6–HA–Rab11. The binding of Rab11 was detected by immunoblotting (IB) using a HA-specific monoclonal antibody, and the GST-fusion proteins present in the binding reaction were detected using Ponceau S red staining. The blots shown are representative of three independent experiments. Molecular masses are indicated on the left-hand side (in kDa).

Amino acids Arg333 and Lys348 of the β2AR-CT are involved in the interaction with Rab11

Figure 6
Amino acids Arg333 and Lys348 of the β2AR-CT are involved in the interaction with Rab11

(A) Schematic representation of the β2AR-CT mutants used in binding assays. The Rab11 binding properties of the constructs are summarized on the right-hand side. ++, strong binding; +, moderate binding; −, no binding. (B) The binding assays were carried out using purified glutathione–Sepharose-bound GST-tagged β2AR-CT mutants, which were incubated with purified recombinant His6–HA–Rab11. The binding of Rab11 was detected by immunoblotting (IB) using a HA-specific monoclonal antibody, and the GST-fusion proteins present in the binding reaction were detected using a GST-specific polyclonal antibody. The blots shown are representative of three independent experiments. Molecular masses are indicated on the left-hand side (in kDa).

Figure 6
Amino acids Arg333 and Lys348 of the β2AR-CT are involved in the interaction with Rab11

(A) Schematic representation of the β2AR-CT mutants used in binding assays. The Rab11 binding properties of the constructs are summarized on the right-hand side. ++, strong binding; +, moderate binding; −, no binding. (B) The binding assays were carried out using purified glutathione–Sepharose-bound GST-tagged β2AR-CT mutants, which were incubated with purified recombinant His6–HA–Rab11. The binding of Rab11 was detected by immunoblotting (IB) using a HA-specific monoclonal antibody, and the GST-fusion proteins present in the binding reaction were detected using a GST-specific polyclonal antibody. The blots shown are representative of three independent experiments. Molecular masses are indicated on the left-hand side (in kDa).

Impaired recycling of the β2AR RK/AA mutant following prolonged exposure to agonist

We hypothesized that a β2AR mutant deficient in Rab11 binding would have impaired recycling following prolonged treatment with agonist. To verify our hypothesis, we generated a β2AR mutant in which the residues required for the Rab11 interaction (Arg333 and Lys348) were mutated to alanine residues (β2AR RK/AA). First, HEK-293 cells which were transiently expressing FLAG–β2AR or FLAG–β2AR RK/AA were exposed to 5 μM isoprenaline for 30 min to evaluate if the mutant and wild-type receptors displayed similar internalization properties at an early time point. As seen in Figure 7(A), no significant difference was detected in the internalization of both receptors after 30 min of agonist stimulation. This indicates that the receptor mutant is not affected with regard to processes involved in the internalization of the β2AR, such as receptor activation, phosphorylation and the recruitment of arrestin [28]. The same cells were then stimulated with 5 μM isoprenaline for 6 h before being thoroughly washed, placed in warm medium containing the antagonist propranolol to prevent any additional internalization, and incubated at 37 °C for various time points to allow receptor recycling, as stated above. Interestingly, we observed a modest but significant increase in the loss of the β2AR RK/AA mutant receptors from the cell surface following agonist stimulation for 6 h when compared with wild-type β2AR (62.5±4.0% compared with 51.0±3.5% for the mutant and wild-type receptors respectively, representing an increase of ∼22%) (Figure 7B). The apparent increase in β2AR RK/AA mutant receptor internalization could be due to decreased receptor recycling, leading to a greater disappearance of mutant receptors than wild-type β2AR from the cell surface over time. This idea was supported by the results from Figure 7(C), which show that the recycling of the β2AR RK/AA mutant receptor was impaired significantly compared with wild-type β2AR. Indeed, 15.3±3.3%, 30.0±1.7% and 32.5±2.9% of the wild-type receptors returned to the cell surface after 5, 20 and 60 min, following the removal of agonist. In contrast, only 3.6±0.9%, 8.2±1.4% and 13.8±3.0% of the β2AR RK/AA mutant receptors recycled back to the cell surface for the same time points after agonist washout respectively. These results suggest that abolishing Rab11 binding to the β2AR impairs the ability of the receptor to recycle back to the cell surface following prolonged exposure to agonist.

The role of the Rab11–β2AR interaction in receptor internalization and recycling

Figure 7
The role of the Rab11–β2AR interaction in receptor internalization and recycling

HEK-293 cells expressing either FLAG–β2AR or FLAG–β2AR RK/AA were treated for 30 min (A) or 6 h (B) with 5 μM isoprenaline (ISO). The percentage of cell-surface receptor loss following treatment was measured by ELISA as described in the Experimental section. (C) HEK-293 cells expressing either FLAG-β2AR or FLAG-β2AR RK/AA were treated for 6 h with 5 μM isoprenaline. Cells were then washed and incubated in DMEM containing 5 μM propranolol for the indicated times to allow receptor recycling. Quantification of surface receptors was done by ELISA, and the percentage of receptor recycling was calculated as described in the Experimental section. Results are means±S.E.M. (n=3). intern., internalization.

Figure 7
The role of the Rab11–β2AR interaction in receptor internalization and recycling

HEK-293 cells expressing either FLAG–β2AR or FLAG–β2AR RK/AA were treated for 30 min (A) or 6 h (B) with 5 μM isoprenaline (ISO). The percentage of cell-surface receptor loss following treatment was measured by ELISA as described in the Experimental section. (C) HEK-293 cells expressing either FLAG-β2AR or FLAG-β2AR RK/AA were treated for 6 h with 5 μM isoprenaline. Cells were then washed and incubated in DMEM containing 5 μM propranolol for the indicated times to allow receptor recycling. Quantification of surface receptors was done by ELISA, and the percentage of receptor recycling was calculated as described in the Experimental section. Results are means±S.E.M. (n=3). intern., internalization.

To confirm the results obtained by ELISA, further analysis of β2AR and β2AR RK/AA recycling after prolonged exposure to agonist was done by confocal immunofluorescence microscopy. In doing so, we also wanted to assess whether any co-localization between the β2AR and Rab11 could be detected and whether this co-localization was affected for the β2AR RK/AA mutant receptor. HEK-293 cells transiently expressing FLAG–β2AR or FLAG–β2AR RK/AA with EGFP (enhanced GFP)–Rab11 were exposed to agonist or vehicle for 6 h, and then immediately fixed or washed to allow receptor recycling for 30 min, as described above. Cells were stained to visualize the FLAG-tagged receptors, and co-localization with GFP–Rab11 was assessed by the examination of merged images of red-labelled receptors with green-labelled Rab11. In non-stimulated cells, both β2AR and β2AR RK/AA were seen to be localized predominantly at the plasma membrane (Figure 8A). Treatment with isoprenaline for 6 h promoted the internalization of the β2AR into intracellular vesicles, where a significant overlap with Rab11 was observed (Figure 8B, upper panels). On the other hand, the β2AR RK/AA receptor was promoted to internalize in intracellular vesicles, but these vesicles failed to converge to the Rab11-positive perinuclear compartment after 6 h of agonist stimulation (Figure 8B, lower panels). Recycling of the β2AR and β2AR RK/AA was also studied by removing the agonist and allowing the receptors to recycle for 30 min. Consistent with the recycling results obtained by ELISA, wild-type β2ARs largely recycled back to the cell surface (Figure 8C, upper panels), whereas the majority of β2AR RK/AA mutant receptors remained in intracellular vesicles 30 min after isoprenaline washout (Figure 8C, bottom panels). Taken together, our results demonstrate that mutating the β2AR residues involved in the interaction with Rab11 impairs the targeting of the receptor to perinuclear recycling endosomes and the subsequent recycling of the receptor to the plasma membrane following prolonged exposure to agonist.

The β2AR RK/AA mutant receptor recycles inefficiently following prolonged exposure to agonist

Figure 8
The β2AR RK/AA mutant receptor recycles inefficiently following prolonged exposure to agonist

HEK-293 cells expressing either FLAG–β2AR (upper panels) or FLAG–β2AR RK/AA (lower panels) with Rab11–GFP were treated with vehicle (A) or 5 μM isoprenaline (Iso) for 6 h (B and C). Cells were then fixed immediately (B) or washed and incubated in DMEM containing 5 μM propranolol to allow recycling for 30 min (C). The receptors were visualized using an anti-FLAG antibody (M1) and a Rhodamine Red-conjugated anti-mouse antibody (red fluorescence, left-hand panels); Rab11–GFP is shown in green (centre panels); and overlays of staining patterns are shown in the right-hand panels (Merge; yellow fluorescence, co-localization of staining). Scale bars, 10 μm.

Figure 8
The β2AR RK/AA mutant receptor recycles inefficiently following prolonged exposure to agonist

HEK-293 cells expressing either FLAG–β2AR (upper panels) or FLAG–β2AR RK/AA (lower panels) with Rab11–GFP were treated with vehicle (A) or 5 μM isoprenaline (Iso) for 6 h (B and C). Cells were then fixed immediately (B) or washed and incubated in DMEM containing 5 μM propranolol to allow recycling for 30 min (C). The receptors were visualized using an anti-FLAG antibody (M1) and a Rhodamine Red-conjugated anti-mouse antibody (red fluorescence, left-hand panels); Rab11–GFP is shown in green (centre panels); and overlays of staining patterns are shown in the right-hand panels (Merge; yellow fluorescence, co-localization of staining). Scale bars, 10 μm.

DISCUSSION

Even though the intracellular trafficking pathways following the internalization of GPCRs have been the subject of intense research, our understanding of the signals that target these molecules to the proper pathway is still incomplete. Much effort has been made to discover the intracellular factors that regulate the targeting of receptors to the different intracellular compartments. A variety of amino acid motifs within the receptors themselves have been identified as important determinants in regulating the trafficking of internalized receptors [28]. Those motifs are thought to interact with proteins whose functions are to direct the sorting of internalized receptors for either recycling or degradation [2932]. In the present study, we report that the small GTPase Rab11 interacts with the C-terminus of the β2AR and that this interaction is required for the accurate intracellular trafficking of the receptor.

Only a few studies have demonstrated direct interactions between a Rab GTPase and a cargo molecule. For example, an association between Rab5 and the angiotensin II type 1A receptor was reported [22]. Rab11a was shown to interact with the Ca2+-selective channels TRPV5/6 and the TPβ [23,24]. Yet this is the first report of a direct interaction between a Rab GTPase and the β2AR. Interestingly, Rab11 did not interact with the prostanoid receptor DP, a receptor whose recycling is not mediated by Rab11 [33]. Specificity in the interaction between the β2AR and Rab11 is also illustrated by the fact that the receptor did not interact with Rab4, a related member of the Rab family of GTPases which is also implicated in the recycling of GPCRs. Moreover, Seachrist et al. [22] have reported that Rab5 is an interacting protein of the angiotensin II type 1A receptor, but not of the β2AR. Taken together, this suggests that the association between Rab11 and the β2AR is specific.

To identify the consequences of the interaction of Rab11 with the β2AR, we mapped the Rab11-binding site on the receptor. Two amino acids located in the C-terminus of the β2AR (Arg333 and Lys348) were demonstrated to be essential for Rab11 binding. Since previous results link Rab11 to the slow recycling of β2ARs during prolonged treatment with agonist [13], we measured the amount of internalized β2AR and β2AR RK/AA after a 6 h treatment with agonist, as well as the amount of receptors that recycled back to the cell surface following the washout of agonist. Mutation of the residues involved in Rab11 binding resulted in impaired trafficking of the receptor, which showed drastically reduced recycling to the plasma membrane after the removal of agonist. The β2AR RK/AA mutant also displayed a significant increase in receptor disappearance from the cell surface when compared with wild-type β2AR after 6 h of agonist stimulation, which is consistent with a reduction in receptor recycling. Confocal immunofluorescence microscopy revealed that, in contrast with the wild-type β2AR, the β2AR RK/AA mutant did not localize in the Rab11-positive perinuclear recycling endosomes following prolonged exposure to agonist. This suggests that the direct interaction with Rab11 is necessary for the proper targeting of the β2AR to this recycling compartment, similar to what we observed for the TPβ previously [24]. These microscopy experiments also confirmed that the association between Rab11 and the receptor is required for β2AR recycling to the plasma membrane, since the majority of the mutant receptor was retained in intracellular vesicles after agonist washout.

A role for Rab proteins in cargo recruitment during transport vesicle formation was suggested by previous studies. For example, Rab5 was identified as essential for the sequestration of transferrin receptor into newly formed clathrin-coated pits [34]. It was proposed that a Rab5–GDI (guanine-nucleotide-dissociation inhibitor) complex may have a direct role in promoting sequestration, either by driving invagination or by receptor recruitment. Another example is the mannose 6-phosphate receptor transport from endosomes to the Golgi, which is mediated by a protein named TIP47 (47 kDa tail-interacting protein). Carroll et al. [35] proposed a model in which Rab9 facilitates the recruitment of TIP47 on to mannose 6-phosphate-containing endosomes. These authors showed that the incorporation of the cargo protein into transport vesicles is enhanced by the formation of the ternary complex TIP47–Rab9–mannose 6-phosphate receptor. One possible explanation for the impaired recycling of the β2AR RK/AA mutant thus could be that Rab11 binding to the receptor is required for its efficient incorporation into Rab11-associated transport vesicles.

Seachrist et al. [22] reported that the activation of the angiotensin II type 1A receptor, which associates with Rab5a through a direct interaction, increases the amount of GTP loading on to the GTPase, suggesting that the receptor may function as a GEF for Rab5a or that it may recruit protein complexes that contain a GEF for the GTPase. On the basis of these results, they proposed that GPCRs are not simply passive cargo molecules, but that their activation may influence Rab GTPase activity directly and, as such, GPCRs may control directly their own targeting between intracellular compartments. Our results showed that the β2AR did not interact with the GTP-bound Rab11, but rather did so preferentially with the GDP-bound form of Rab11, analogously to what was reported previously for the TPβ [24]. Our results demonstrated that the GDP-bound form of Rab11 (Rab11 S25N), but not the wild-type and the constitutively active Rab11 forms, affected the recycling of the β2AR. This difference could be explained by the levels of expression and activation of endogenous Rab11 that would be sufficient for the recycling of the β2AR. In this context, overexpression of Rab11 or Rab11 Q70L would thus have no effect on β2AR recycling. On the other hand, expression of the dominant-negative Rab11 S25N mutant would compete with endogenous Rab11 function and slow receptor recycling. The results of the present study not only show that the β2AR interacts with the GDP-bound form of Rab11 preferentially, but also that Rab11 must be able to be activated to regulate β2AR recycling. It is currently difficult to understand why the β2AR and the TPβ [24] interact preferentially with the GDP-bound form of Rab11. One possible reason could be that the GDP-bound Rab11 is the form that associates with a particular set of proteins found at membranes, making it available to interact with receptors. These other proteins could be involved in membrane association and post-translational modification of Rab11, such as the Rab escort proteins or the Rab geranylgeranyl transferases. One could also speculate that proteins involved in Rab11 activation, such as a GEF, could be recruited into a Rab11–β2AR complex to activate the small GTPase: the receptor could act as a scaffold to recruit proteins which are involved in the localization and activation of the GDP-bound Rab11, triggering mechanisms regulating its own trafficking. Experiments are underway in our laboratory to identify proteins found in a GDP-bound Rab11–β2AR complex to better understand how Rab11 directs β2AR trafficking.

In conclusion, our results provide new insight into the molecular machinery involved in β2AR trafficking. We have demonstrated a direct interaction between Rab11 and the β2AR, which is involved in the regulation of the receptor targeting to the perinuclear recycling endosome and receptor recycling to the cell surface following prolonged treatment with agonist. The angiotensin II type 1A receptor, CXCR2, V2R, m4 muscarinic acetylcholine receptor, somatostatin receptor, TGF-β (transforming growth factor-β) receptor, neurokinin 1 receptor and PAR2 were all shown to recycle through the Rab11-dependent slow-recycling pathway [68,3642]. Similarly, E-cadherin was demonstrated previously to be targeted to the basolateral membrane of epithelial cells by a Rab11-dependent pathway [43]. The targeting of the Fc receptor, FcRn, to the cell surface was also observed to occur through a Rab11-mediated mechanism [44]. It will be interesting to study whether the trafficking of these other membrane proteins also requires a direct interaction with Rab11. The results of the present study and previous work on the TPβ [24] support the idea that transmembrane-receptor molecules may form a new class of Rab11-interacting proteins and could thus be able to fine-tune the intracellular transport machinery in order to control their own itinerary through the cell.

Abbreviations

     
  • AT1AR

    angiotensin II type 1A receptor

  •  
  • β2AR

    β2-adrenergic receptor

  •  
  • CT

    C-terminal tail

  •  
  • CXCR2

    CXC chemokine receptor 2

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • DP

    prostaglandin D2 receptor

  •  
  • GEF

    guanine-nucleotide-exchange factor

  •  
  • GFP

    green fluorescent protein

  •  
  • GPCR

    G-protein-coupled receptor

  •  
  • GST

    glutathione transferase

  •  
  • HA

    haemagglutinin

  •  
  • HEK-293

    cell, human embryonic kidney cell

  •  
  • His6

    hexahistidine

  •  
  • ICL

    intracellular loop

  •  
  • PAR

    protease-activated receptor

  •  
  • RK/AA

    R333A/K348A

  •  
  • TBS

    Tris-buffered saline

  •  
  • TIP47

    47 kDa tail-interacting protein

  •  
  • TPβ

    thromboxane A2 receptor β-isoform

  •  
  • TRPV5/6

    transient receptor potential vanilloid 5/6

  •  
  • V2R

    V2 vasopressin receptor

FUNDING

This work was supported by a studentship award from the Natural Sciences and Engineering Research Council of Canada (to A. P.); a studentship award from the Fonds de la Recherche en Santé du Québec (to E. H.); a salary award from the Fonds de la Recherche en Santé du Québec; and by the Canadian Institutes of Health Research [grant number MOP-69085] (to J.-L. P.)

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