Biochem. J. (2009) 418, 163–172 (Printed in Great Britain)
163
doi:10.1042/BJ20080867
Rab11 regulates the recycling of the β 2 -adrenergic receptor through
a direct interaction
Audrey PARENT, Emilie HAMELIN, Pascale GERMAIN and Jean-Luc PARENT1
Service de Rhumatologie, Département de Médecine, Faculté de Médecine, Université de Sherbrooke, 3001, 12e Avenue Nord, QC, Canada, J1H 5N4, and Centre de Recherche
Clinique-Etienne Lebel, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada, J1H 5N4
The β2ARs (β 2 -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
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 postGolgi 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 Rabinteracting 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
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 [3–5]. 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 [6–8].
The β2ARs (β 2 -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. [11–13]
Key words: β 2 -adrenergic receptor (β2AR), G-protein-coupled
receptor (GPCR), Rab11, recycling, trafficking.
Abbreviations used: AT1 AR, 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.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2009 Biochemical Society
164
A. Parent and others
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 IgAstimulated transcytosis [21]. Interactions between Rab5 and
AT1 AR (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 [22–24]. 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 antimouse 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 β2ARCT 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
c The Authors Journal compilation c 2009 Biochemical Society
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 Na4 P2 O7 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 11 000 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 11 000 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 OverExpressTM
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 GSTtagged recombinant mutants were purified using glutathione–
SepharoseTM 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
A β 2 AR–Rab11 interaction
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 phosphataseconjugated 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) nonfat 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 oilimmersion 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
Figure 1
recycling
165
The dominant-negative Rab11 S25N mutant inhibits β2AR
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.
the perinuclear recycling endosome, where modulation of Rab11
activity dictates the balance between receptor recycling and downregulation 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.
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 HAtagged β2AR or HA–DP (prostaglandin D2 receptor). Cell lysates
were incubated with a HA-specific polyclonal antibody, and coimmunoprecipitated 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 coimmunoprecipitate 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.
c The Authors Journal compilation c 2009 Biochemical Society
166
Figure 2
A. Parent and others
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 β2AR preferentially interacts with GDP-bound Rab11
It has been shown for complexes between Rab GTPases and
receptors such as TPβ and AT1 AR 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
c The Authors Journal compilation c 2009 Biochemical Society
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 GDPbound 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.
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 Cterminally 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
A β 2 AR–Rab11 interaction
Figure 3
167
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).
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.
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
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.
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
c The Authors Journal compilation c 2009 Biochemical Society
168
A. Parent and others
Figure 6 Amino acids Arg333 and Lys348 of the β2AR-CT are involved in the
interaction with Rab11
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).
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
c The Authors Journal compilation c 2009 Biochemical Society
(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).
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.
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
A β 2 AR–Rab11 interaction
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 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.
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
169
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 [29–
32]. 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 wildtype 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
c The Authors Journal compilation c 2009 Biochemical Society
170
Figure 8
A. Parent and others
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.
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
c The Authors Journal compilation c 2009 Biochemical Society
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
A β 2 AR–Rab11 interaction
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
[6–8,36–42]. 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.
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.)
REFERENCES
1 Hoxie, J. A., Ahuja, M., Belmonte, E., Pizarro, S., Parton, R. and Brass, L. F. (1993)
Internalization and recycling of activated thrombin receptors. J. Biol. Chem. 268,
13756–13763
2 Hein, L., Ishii, K., Coughlin, S. R. and Kobilka, B. K. (1994) Intracellular targeting and
trafficking of thrombin receptors. A novel mechanism for resensitization of a G
protein-coupled receptor. J. Biol. Chem. 269, 27719–27726
3 Vickery, R. G. and von Zastrow, M. (1999) Distinct dynamin-dependent and -independent
mechanisms target structurally homologous dopamine receptors to different endocytic
membranes. J. Cell Biol. 144, 31–43
4 Koch, T., Schulz, S., Schroder, H., Wolf, R., Raulf, E. and Hollt, V. (1998)
Carboxyl-terminal splicing of the rat μ opioid receptor modulates agonist-mediated
internalization and receptor resensitization. J. Biol. Chem. 273, 13652–13657
5 Seachrist, J. L., Anborgh, P. H. and Ferguson, S. S. (2000) β2-Adrenergic receptor
internalization, endosomal sorting, and plasma membrane recycling are regulated by rab
GTPases. J. Biol. Chem. 275, 27221–27228
6 Innamorati, G., Le Gouill, C., Balamotis, M. and Birnbaumer, M. (2001) The long and the
short cycle. Alternative intracellular routes for trafficking of G-protein-coupled receptors.
J. Biol. Chem. 276, 13096–13103
7 Theriault, C., Rochdi, M. D. and Parent, J. L. (2004) Role of the Rab11-associated
intracellular pool of receptors formed by constitutive endocytosis of the β isoform of the
thromboxane A2 receptor (TPβ). Biochemistry 43, 5600–5607
8 Fan, G. H., Lapierre, L. A., Goldenring, J. R. and Richmond, A. (2003) Differential
regulation of CXCR2 trafficking by rab GTPases. Blood 101, 2115–2124
9 Morrison, K. J., Moore, R. H., Carsrud, N. D., Trial, J., Millman, E. E., Tuvim, M., Clark,
R. B., Barber, R., Dickey, B. F. and Knoll, B. J. (1996) Repetitive endocytosis and
recycling of the β2-adrenergic receptor during agonist-induced steady state
redistribution. Mol. Pharmacol. 50, 692–699
10 Kallal, L., Gagnon, A. W., Penn, R. B. and Benovic, J. L. (1998) Visualization of agonistinduced sequestration and down-regulation of a green fluorescent protein-tagged
β2-adrenergic receptor. J. Biol. Chem. 273, 322–328
171
11 Moore, R. H., Tuffaha, A., Millman, E. E., Dai, W., Hall, H. S., Dickey, B. F. and Knoll, B. J.
(1999) Agonist-induced sorting of human β2-adrenergic receptors to lysosomes during
downregulation. J. Cell Sci. 112, 329–338
12 Moore, R. H., Hall, H. S., Rosenfeld, J. L., Dai, W. and Knoll, B. J. (1999) Specific
changes in β2-adrenoceptor trafficking kinetics and intracellular sorting during
downregulation. Eur. J. Pharmacol. 369, 113–123
13 Moore, R. H., Millman, E. E., Alpizar-Foster, E., Dai, W. and Knoll, B. J. (2004) Rab11
regulates the recycling and lysosome targeting of β2-adrenergic receptors. J. Cell Sci.
117, 3107–3117
14 Schwartz, S. L., Cao, C., Pylypenko, O., Rak, A. and Wandinger-Ness, A. (2007) Rab
GTPases at a glance. J. Cell Sci. 120, 3905–3910
15 Urbe, S., Huber, L. A., Zerial, M., Tooze, S. A. and Parton, R. G. (1993) Rab11, a small
GTPase associated with both constitutive and regulated secretory pathways in PC12 cells.
FEBS Lett. 334, 175–182
16 Ullrich, O., Reinsch, S., Urbe, S., Zerial, M. and Parton, R. G. (1996) Rab11 regulates
recycling through the pericentriolar recycling endosome. J. Cell Biol. 135, 913–924
17 Novick, P. and Zerial, M. (1997) The diversity of rab proteins in vesicle transport.
Curr. Opin. Cell Biol. 9, 496–504
18 Zerial, M. and McBride, H. (2001) Rab proteins as membrane organizers. Nat. Rev. Mol.
Cell Biol. 2, 107–117
19 Grosshans, B. L., Ortiz, D. and Novick, P. (2006) Rabs and their effectors: achieving
specificity in membrane traffic. Proc. Natl. Acad. Sci.U.S.A. 103, 11821–11827
20 Smythe, E. (2002) Direct interactions between rab GTPases and cargo. Mol. Cell 9,
205–206
21 van IJzendoorn, S. C., Tuvim, M. J., Weimbs, T., Dickey, B. F. and Mostov, K. E. (2002)
Direct interaction between Rab3b and the polymeric immunoglobulin receptor controls
ligand-stimulated transcytosis in epithelial cells. Dev. Cell 2, 219–228
22 Seachrist, J. L., Laporte, S. A., Dale, L. B., Babwah, A. V., Caron, M. G., Anborgh, P. H.
and Ferguson, S. S. (2002) Rab5 association with the angiotensin II type 1A receptor
promotes Rab5 GTP binding and vesicular fusion. J. Biol. Chem. 277, 679–685
23 van de Graaf, S. F., Chang, Q., Mensenkamp, A. R., Hoenderop, J. G. and Bindels, R. J.
(2006) Direct interaction with Rab11a targets the epithelial Ca2+ channels TRPV5 and
TRPV6 to the plasma membrane. Mol. Cell. Biol. 26, 303–312
24 Hamelin, E., Theriault, C., Laroche, G. and Parent, J. L. (2005) The intracellular
trafficking of the G protein-coupled receptor TPβ depends on a direct interaction
with Rab11. J. Biol. Chem. 280, 36195–36205
25 Parent, J. L., Labrecque, P., Orsini, M. J. and Benovic, J. L. (1999) Internalization of the
TXA2 receptor α and β isoforms: role of the differentially spliced COOH terminus in
agonist-promoted receptor internalization. J. Biol. Chem. 274, 8941–8948
26 Parent, A., Laroche, G., Hamelin, E. and Parent, J. L. (2008) RACK1 regulates the cell
surface expression of the G-protein-coupled receptor for thromboxane A2 . Traffic 9,
394–407
27 Olkkonen, V. M. and Stenmark, H. (1997) Role of rab GTPases in membrane traffic.
Int. Rev. Cytol. 176, 1–85
28 Marchese, A., Paing, M. M., Temple, B. R. and Trejo, J. (2008) G protein-coupled receptor
sorting to endosomes and lysosomes. Annu. Rev. Pharmacol. Toxicol. 48, 601–629
29 Cao, T. T., Deacon, H. W., Reczek, D., Bretscher, A. and von Zastrow, M. (1999) A
kinase-regulated PDZ-domain interaction controls endocytic sorting of the β2-adrenergic
receptor. Nature 401, 286–290
30 Cong, M., Perry, S. J., Hu, L. A., Hanson, P. I., Claing, A. and Lefkowitz, R. J. (2001)
Binding of the β2 adrenergic receptor to N -ethylmaleimide-sensitive factor regulates
receptor recycling. J. Biol. Chem. 276, 45145–45152
31 Whistler, J. L., Enquist, J., Marley, A., Fong, J., Gladher, F., Tsuruda, P., Murray, S. R. and
von Zastrow, M. (2002) Modulation of postendocytic sorting of G-protein-coupled
receptors. Science 297, 615–620
32 Hanyaloglu, A. C., McCullagh, E. and von Zastrow, M. (2005) Essential role of hrs in a
recycling mechanism mediating functional resensitization of cell signaling. EMBO J. 24,
2265–2283
33 Gallant, M. A., Slipetz, D., Hamelin, E., Rochdi, M. D., Talbot, S., de Brum-Fernandes,
A. J. and Parent, J. L. (2007) Differential regulation of the signaling and trafficking of the
two prostaglandin D2 receptors, prostanoid DP receptor and CRTH2. Eur. J. Pharmacol.
557, 115–123
34 McLauchlan, H., Newell, J., Morrice, N., Osborne, A., West, M. and Smythe, E. (1998) A
novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits. Curr. Biol. 8,
34–45
35 Carroll, K. S., Hanna, J., Simon, I., Krise, J., Barbero, P. and Pfeffer, S. R. (2001)
Role of Rab9 GTPase in facilitating receptor recruitment by TIP47. Science 292,
1373–1376
36 Roosterman, D., Schmidlin, F. and Bunnett, N. W. (2003) Rab5a and rab11a mediate
agonist-induced trafficking of protease-activated receptor 2. Am. J. Physiol. Cell Physiol.
284, C1319–C1329
c The Authors Journal compilation c 2009 Biochemical Society
172
A. Parent and others
37 Roosterman, D., Cottrell, G. S., Schmidlin, F., Steinhoff, M. and Bunnett, N. W. (2004)
Recycling and resensitization of the neurokinin 1 receptor. Influence of agonist
concentration and rab GTPases. J. Biol. Chem. 279, 30670–30679
38 Mitchell, H., Choudhury, A., Pagano, R. E. and Leof, E. B. (2004) Ligand-dependent and
-independent transforming growth factor-β receptor recycling regulated by
clathrin-mediated endocytosis and Rab11. Mol. Biol. Cell 15, 4166–4178
39 Kreuzer, O. J., Krisch, B., Dery, O., Bunnett, N. W. and Meyerhof, W. (2001) Agonistmediated endocytosis of rat somatostatin receptor subtype 3 involves β-arrestin and
clathrin coated vesicles. J. Neuroendocrinol. 13, 279–287
40 Volpicelli, L. A., Lah, J. J., Fang, G., Goldenring, J. R. and Levey, A. I. (2002) Rab11a and
myosin vb regulate recycling of the M4 muscarinic acetylcholine receptor. J. Neurosci.
22, 9776–9784
Received 30 April 2008/16 October 2008; accepted 4 November 2008
Published as BJ Immediate Publication 4 November 2008, doi:10.1042/BJ20080867
c The Authors Journal compilation c 2009 Biochemical Society
41 Dale, L. B., Seachrist, J. L., Babwah, A. V. and Ferguson, S. S. (2004) Regulation of
angiotensin II type 1A receptor intracellular retention, degradation, and recycling by Rab5,
Rab7, and Rab11 GTPases. J. Biol. Chem. 279, 13110–13118
42 Fan, G. H., Lapierre, L. A., Goldenring, J. R., Sai, J. and Richmond, A. (2004) Rab11family interacting protein 2 and myosin vb are required for CXCR2 recycling and
receptor-mediated chemotaxis. Mol. Biol. Cell 15, 2456–2469
43 Lock, J. G. and Stow, J. L. (2005) Rab11 in recycling endosomes regulates the sorting
and basolateral transport of E-cadherin. Mol. Biol. Cell 16, 1744–1755
44 Ward, E. S., Martinez, C., Vaccaro, C., Zhou, J., Tang, Q. and Ober, R. J. (2005)
From sorting endosomes to exocytosis: association of Rab4 and Rab11 GTPases
with the fc receptor, FcRn, during recycling. Mol. Biol. Cell 16,
2028–2038