From the "Physiologie Cellulaire de la Synapse,"
UMR 5091 CNRS/Université Bordeaux 2, Institut François
Magendie, Rue Camille Saint Saëns, 33077 Bordeaux Cedex, France,
§ "Mécanismes Moléculaires des Communications
Cellulaires," UPR CNRS 9023, CCIPE, 141 Rue de la Cardonille, 34094 Montpellier Cedex 5, France, and ¶ "Analyse d'Images
Quantitative," URA CNRS 1947, Institut Pasteur, 25 Rue du Dr. Roux,
75724 Paris Cedex, France
Received for publication, June 7, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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Metabotropic glutamate receptors 5 (mGluR5)
are members of the growing group C G protein-coupled receptor
family. Widely expressed in mammalian brain, they are involved in
modulation of the glutamate transmission. By means of transfection of
mGluR5 receptors in COS-7 cells and primary hippocampal neurons in
culture followed by immunocytochemistry and quantitative image analysis
and by a biochemical assay, we have studied the internalization of
mGluR5 splice variants. mGluR5a and -5b were endocytosed in COS-7 cells as well as in axons and dendrites of cultured neurons. Endocytosis occurred even in the absence of receptor activity, because receptors mutated in the glutamate binding site were still internalized as well
as receptors in which endogenous activity had been inhibited by
an inverse agonist. We have measured a constitutive rate of endocytosis
of 11.7%/min for mGluR5a. We report for the first time the endocytosis
pathway of mGluR5. Internalization of mGluR5 is not mediated by
clathrin-coated pits. Indeed, inhibition of this pathway by Eps15
dominant negative mutants did not disturb their endocytosis. However,
the large GTPase dynamin 2 is implicated in the endocytosis of mGluR5
in COS-7. mGluR5 is the first shown member of the group C
G-protein coupled receptor family internalized by a nonconventional pathway.
Glutamate is the major excitatory neurotransmitter in the
mammalian central nervous system. It exerts its effects by binding to
ionotropic and metabotropic receptors. Metabotropic glutamate receptors
(mGluRs)1 belong to the
seven-transmembrane domain G protein-coupled receptor (GPCR) family.
Together with calcium-sensing receptors, Currently, very little is known about the targeting and trafficking
mechanisms of mGluRs in cells. During the last few years, attention has
focused mainly on endocytosis of a subtype of ionotropic glutamate
receptors: the We are interested in understanding the endocytic properties of mGluR5.
Alternative splicing of the mGluR5 gene generates two splice variants:
mGluR5a and mGluR5b, which differ only by a 32-amino acid insertion in
the intracellular C tail domain of mGluR5b. There is no functional
difference between them except that mGluR5b seems to be the predominant
form expressed in brain of adult animals (7). Both splice variants as
well as mGluR1a interact with the family of Homer proteins (2). The
first identified member of this family, Homer1a, is a product of an
immediate early gene expression, which is induced during intense
neuronal activities. All Homers can bind to mGluR5 and mGluR1a by their
amino terminus and also to other proteins of the postsynaptic density.
Although most GPCRs undergo endocytosis, the conditions and mechanisms
of this process can vary from receptor to receptor. Many of them are
endocytosed via the clathrin-coated pit pathway but some are not
(8-10). Some have an agonist-induced endocytosis, and some are
continuously endocytosed even in the absence of stimulation. These
different behaviors should serve distinct physiological functions.
By using transfection of epitope-tagged mGluR5 receptors in COS-7 cells
and hippocampal cultured neurons we found that mGluR5a and -5b are
endocytosed constitutively by a clathrin-independent pathway.
Antibodies and Reagents
We obtained anti-Myc monoclonal antibody (9E10) from Roche
Molecular Biochemicals; anti-Myc polyclonal antibody and anti-mGluR5 from Upstate Biotechnology, Inc. (Lake Placid, NY); anti-HA polyclonal antibody from MBL (Medical and Biological Laboratories Co., Ltd.); anti-MAP2 polyclonal from Peninsula Laboratories Europe Ltd.; anti- Constructs
The epitope-tagged rat mGluR5a and mGluR5b expression plasmids
were constructed using, as a template, the pRKG5a and pRKG5b plasmids
that encode mGluR5a and mGluR5b (12). An MluI restriction site was inserted just after the signal peptide for the N-terminal epitope-tagged receptor (between the Ser22 and
Ser23 codons of mGluR5a) using a PCR overlap extension
method. Sense and antisense oligonucleotides coding the c-Myc epitope
(EQKLISEEDL) or the HA epitope (YPYDVPDYA) with MluI
cohesive ends were synthesized and used to introduce the c-Myc or the
HA into the N-terminal MluI site. Using this method, we
generated the plasmids pRKG5aNmyc (mGluR5-myc in Ref. 13), pRKG5bNmyc,
pRKG5aNHA, and pRKG5bNHA encoding mGluR5aNmyc, mGluR5bNmyc,
mGluR5aNHA, and mGluR5bNHA, respectively. By determining inositol
phosphate accumulation in transfected cells as described previously
(14), we verified that the epitope tagging changed neither the G
protein coupling nor the pharmacological profile of the receptor (Table
I). Plasmids encoding mGluR5aNHA mutated in the glutamate binding site
were obtained by using the QuikChange® strategy. Plasmids encoding the GFP-tagged dynamins, Dyn1-GFP, Dyn1K44A-GFP, Dyn2-GFP, and Dyn2K44A-GFP, were kindly provided by M. McNiven (15). To determine the
role played by Eps15, we used two cDNAs kindly provided by A. Benmerah and A. Dautry-Varsat, one control GFP-DIII Cells and Transfection
COS-7--
COS-7 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum, 1% pyruvate
(5.5 g/liter), and 2 mM Glutamax. COS-7 cells were
transiently transfected with the different cDNAs using FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's indications.
Rat Hippocampal Neurons--
Hippocampal cells were prepared
from 18-day-old rat embryos according to the method of Goslin and
Banker (18). Briefly, the hippocampi were dissociated by trypsin and
mechanical treatment, plated on poly-L-lysine-coated glass
coverslips at a density of 9500 cells/cm2, and maintained
in a serum-free medium (N2 medium) suspended above a glia feeder layer.
Adding 5 mM cytosine arabinoside prevented proliferation of
nonneuronal cells. All experiments were performed in cells kept 8-12
days in culture. Neurons were transfected with designated plasmids
using Effectene (Qiagen) according to the manufacturer's indications
except that the cells were left only 1 h in contact with Effectene
and DNA.
Endocytosis Assay and Immunocytochemistry
To follow endocytosis of tagged receptors, antibodies anti-Myc
(5 µg/ml) or anti-HA (1:100), diluted in conditioned culture medium,
were incubated on living cells for 30 min at 37 °C. When specified,
Rh-Tf was added at a concentration of 300 nM. Cells were
either directly fixed by incubation for 20 min at room temperature in
4% paraformaldehyde, 4% sucrose in PBS, or remaining surface antibodies were stripped away with an acid wash (4-min incubation at
4 °C with Dulbecco's modified Eagle's medium, pH 2) before fixation.
After fixation, cells were incubated for 10 min at room temperature
with 50 mM NH4Cl in PBS in order to quench
aldehyde groups and then permeabilized in PBS, 0.2% bovine serum
albumin, 0.05% saponin (permeabilizing buffer) for 40 min at 37 °C.
If necessary, the cells were incubated for 45 min at room temperature
with anti-MAP2 (1:2000) in permeabilizing buffer. After three washes in
permeabilizing buffer, cells were incubated for 45 min at room
temperature with fluorochrome-coupled secondary antibodies (Cy3
anti-mouse or anti-rabbit (1:500), fluorescein isothiocyanate
anti-rabbit (1:100)), washed several times with permeabilizing buffer
then in PBS, and finally mounted in Dabco/Moviol as described by
Hémar et al. (19).
When the remaining surface anti-Myc (1:100) was visualized after having
been incubated on living cells for 30 min at 37 °C, cells were
incubated for 1 h at 4 °C with Cy3 anti-mouse secondary antibodies and then fixed and mounted in Dabco/Moviol.
Immunofluorescence was visualized with a Zeiss Axiophot 2 microscope.
Image acquisitions were done with a Quantix digital cooled CCD camera
(Photometrix) driven by IP-labs (Scanalitics) software. Images were
mounted with Adobe Photoshop.
Image Analysis
The detection and counting of endocytic vesicles was performed
automatically by a computer program that uses an undecimated wavelet
decomposition of the image. The program recognizes the vesicles by
correlating and thresholding wavelet coefficients at different scales
of analysis (20). Region masks defining the area occupied by the cell
body and processes are determined by an automatic thresholding
algorithm when processing fluorescently labeled neurons or by
interactive drawing when analyzing COS-7 cells. Vesicles inside each
cell are validated as the result of a logical AND operator between the
vesicle image and the region masks. The results are presented as the
mean ± S.E. of the number of vesicles per cell surface unit.
Statistical significance was assessed by using a Mann-Whitney test.
Biotinylation Assay of Receptor Endocytosis
The day before the experiment, COS-7 cells transfected with
expression vector encoding untagged mGluR5a were pooled and plated in
six-well plates in order to avoid variability in transfection efficiency between wells. Cells were washed twice in ice-cold PBS (pH
8) and incubated with 1 ml of sulfo-NHS-SS-biotin (Pierce) diluted in
PBS (pH 8) for 30 min at 4 °C. After two washes in PBS, two wells
were kept at 4 °C (t0) in ice-cold PBS, and
the others were incubated different times at 37 °C in prewarmed PBS. Cells were then washed twice in PBS. For each incubation time, one well
was kept at 4 °C while the other was incubated three times for 15 min with 1 ml of cleavage solution (50 mM
2-mercaptoethanesulfonic acid, 100 mM NaCl, 2.5 mM CaCl2, and 50 mM Tris-HCl (pH
8.7 at 4 °C)) and washed twice in ice-cold PBS. All of the wells
were lysed by scraping in 1 ml of lysis buffer (25 mM
Hepes, 150 mM NaCl, 1% Triton X-100, and "Complete"
protease inhibitor mixture (Roche Molecular Biosciences)). After 20 min
on ice, each lysate was spun at 8000 × g for 10 min.
20 µl of each clarified lysate was kept for an estimation of the
total quantity of mGluR5 at each point. 850 µl of each clarified
lysate were incubated overnight at 4 °C with 50 µl of
streptavidin-agarose beads. The beads were washed three times at
4 °C in lysis buffer and once in 50 mM Tris-HCl (pH
7.4). The beads were extracted with SDS-sample buffer containing 1 M dithiothreitol. The lysates and the extracted beads were
analyzed by Western blotting using anti-mGluR5 antibody (0.8 µg/µl). The ECL kit (Amersham Biosciences) was used for
chemiluminescent detection. Quantitative analysis of Western blots was
performed using Metamorph software (Universal Imaging). We measured the
amount of mGluR5 at the surface at t = 0 (i.e. biotinylated receptors at t = 0), the
amount of surface and intracellular receptors (i.e. total biotinylated receptors) at each time of incubation at 37 °C, and the
intracellular receptors (biotinylated receptors resistant to cleavage).
We measured for each point the total amount of mGluR5 in order to
calibrate these data. The percentage of internalized mGluR5 at time
t was then calculated with the equation,
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid receptor
B (GABAB) and pheromone, olfactory and taste receptors, mGluRs
form the third family of GPCRs (group C) (for reviews, see Refs. 1 and
2). They have little homology with other GPCRs from groups A and B and
are characterized by a large N-terminal extracellular domain that binds
glutamate. Eight genes code for mGluRs that generate more than 15 proteins by alternative splicing. On the basis of their sequence
similarity, pharmacology, and signal transduction mechanisms, mGluRs
have been classified into three groups. Group 1 consists of mGluR1 and
mGluR5 that are preferentially positively coupled to phospholipase C
via Gq/11 proteins. mGluRs of group 2 and 3 are negatively
coupled to adenylate cyclase. Group 1 mGluRs are expressed in neuronal
and glial cells within the brain, and receptor activation has been
implicated in several forms of synaptic plasticity.
-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid
(AMPA) receptors. Internalization of AMPA receptors in response to
various stimuli is an important process for the rapid attenuation of
neurotransmission during synaptic plasticity (3). By different approaches, it has been shown that AMPA receptors are endocytosed by a
clathrin-coated pit-mediated process (4-6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adaptin monoclonal clone AP6 from Affinity Bioreagents Inc.;
and Cy3-, Cy5-, and fluorescein isothiocyanate-conjugated secondary antibodies from Jackson Immunoresearch Laboratories. Dulbecco's modified Eagle's medium, minimum essential medium, F-12/Dulbecco's modified Eagle's medium, Glutamax, and fetal bovine serum came from Invitrogen, and other supplements were from
Sigma. 2-Methyl-6-(phenylethynyl)pyridine (MPEP) was a kind gift
from Dr. Rainer Kühn (NovartisPharma, Basel, Switzerland). Human
holotransferrin was coupled to rhodamine (Rh-Tf) as described in
Hémar et al. (11). Sulfo-NHS-SS-biotin was from
Pierce, and the streptavidin-agarose beads were from Sigma.
2 (16) and one
dominant negative mutant GFP-E
95/295 (17).
where surface(t0)
(Eq. 1)
total(t) represents the degraded receptors or endocytosed
receptors which underwent biotin reduction inside the cell.
The endocytosis rate was determined by fitting data to a single
exponential function of the form y = A(1 e(t/
)), where
A is constant, using Kaleidagraph. The endocytosis rate is
1/
.
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RESULTS |
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mGluR5a and -b Are Endocytosed in Transfected COS-7 Cells and in
Rat Hippocampal Neurons--
To investigate internalization of mGluR5
receptors, recombinant receptors were transiently expressed in
heterologous COS-7 cells or in hippocampal neurons. In transfected
COS-7 cells, the receptors are detected at the cell surface and also in
high proportion (90%) in intracellular compartments (putatively of the
biosynthetic and endocytic
pathways).2 Since there is no
available antibody against the extracellular part of mGluR5, we used
mGluR5a and mGluR5b constructs containing the c-Myc or HA epitope tags
at the extracellular N terminus, allowing specific labeling of
surface-expressed receptors on living cells with anti-tag antibodies.
Endocytosis immunoassay was performed as follows: 1) transfected cells
were incubated for 30 min at 37 °C with anti-tag antibodies; 2)
remaining surface antibodies were or not stripped away with an acid
wash; 3) finally, cells were fixed, permeabilized, and incubated with
fluorescent secondary antibodies. Both splice variants, mGluR5aNmyc and
mGluR5bNmyc, were endocytosed as shown by the presence of an acid
wash-resistant punctiform labeling (Fig.
1, E and F). The
labeled compartments are endosomal structures, because they contain
antibodies that have been internalized from culture medium. The
antibody was internalized by receptor-mediated endocytosis, since no
intracellular labeling was observed in nontransfected cells on the same
coverslips (Fig. 1, compare A with C and
B with D). The comparison of the images obtained
with or without acid wash shows that there are still receptors at the
cell surface (Fig. 1, compare E with A and
F with B). The remaining surface labeling appears
very different from that of the endocytic compartments. Remaining
surface antibodies were revealed, after the incubation at 37 °C,
under nonpermeabilizing conditions (i.e. at 4 °C without
fixation and permeabilization (Fig. 1, G and H)).
The labeling appears as smaller puncta compared with the intracellular
labeling (Fig. 1, compare G with E and H with F). This shows that the binding of the
antibody has not induced a severe down-regulation of the receptor at
the surface. In any case, the cross-linking of the receptors by the
bivalent anti-Myc antibody does not seem to be necessary for their
internalization, since we observed the same labeling when anti-Myc Fab
fragments were used.3
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In order to determine whether the mGluR5 receptors are also endocytosed
in neurons, we performed the same experiments in primary cultures of
rat embryonic hippocampal dissociated neurons. In transfected neurons,
mGluR5a and -b are both expressed and endocytosed in dendrites and
axons (Fig. 2). Indeed, after incubation
at 37 °C, anti-Myc labeling appears as large fluorescent acid
wash-resistant puncta in cell body and neurites (Fig. 2, B
and C, and Fig. 2, E and F, with acid
wash, compared with Fig. 2, A and D, without acid
wash). The internalized receptors (in red) are found in
dendrites labeled with the dendritic specific marker MAP2 (in
green; Fig. 2, B and E) and in axons
that do not contain MAP2 (Fig. 2, C and F).
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mGluR5a Is Constitutively Endocytosed-- In the experimental conditions we used, mGluR5a or mGluR5b receptors are endocytosed without the addition of glutamate. This suggested that the receptors could be endocytosed constitutively, which means in the absence of ligand binding and/or activity.
Although the first observations have been done in the absence of added agonist, one cannot rule out that such an agonist is produced by the cells and would induce the internalization of the receptor. To test this hypothesis, we studied the endocytosis of mutated receptors that have a reduced affinity for glutamate. We took advantage of the similarity of the glutamate binding site of another group 1 metabotropic glutamate receptor, mGluR1, to design the mutants. The binding site of mGluR1 has been crystallized, and the amino acids interacting with glutamate have been identified (21). We mutated into alanine the tyrosine at position 222 and the aspartate at position 304 of mGluR5a (mGluR5aNHA-Y222A and mGluR5aNHA-D304A, respectively). The EC50 for glutamate in activating the Y222A and D304A mutated receptors is 30- and >300-fold higher than that measured with the wild-type receptor, respectively (Table I). Both mutated receptors transfected in COS-7 cells were endocytosed (Fig. 3, A and B). We have evaluated the endocytosis level by image analysis. We quantified the number of acid wash-resistant fluorescent spots (endosomes) per cell surface unit (Fig. 3C). The numbers obtained for both mutants are not significantly different from that of the wild type (wild type: 21.4 ± 1.2 spots/100 µm2, 2.1 × 104 µm2 explored in 47 cells analyzed; mGluR5aNHAY222A, 21.2 ± 2.9 spots/100 µm2, 6.2 × 103 µm2 explored in 11 cells analyzed, p = 0.8; mGluR5aNHAD304A, 20.7 ± 3.3 spots/100 µm2, 5.3 × 103 µm2 explored in 10 cells analyzed, p = 0.7). We conclude that the binding of a ligand is not necessary for the endocytosis of mGluR5a.
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It has been shown that mGluR5 receptors display an endogenous constitutive activity that can be inhibited by the inverse agonist MPEP (22, 23). MPEP also inhibits the activity stimulated by the agonist binding. In order to look for endocytosis of mGluR5aNmyc in the absence of activity, we treated cells with 10 µM MPEP for 30 min before and during the anti-Myc incubation in our endocytosis assay. Fig. 3 shows that in COS-7 cells (Fig. 3D) and in neurons (Fig. 3E), mGluR5a is endocytosed even in the presence of MPEP. The quantifications of the images are presented in Fig. 3F. The number of acid wash-resistant fluorescent spots was not decreased and even increased, showing that the endocytosis is not inhibited in the presence of MPEP (COS-7 without MPEP: 19.3 ± 0.9 spots/100 µm2, 3.8 × 104 µm2 explored in 83 cells analyzed; COS-7 treated with MPEP: 28.3 ± 1.9 spots/100 µm2, 2.4 × 104 µm2 explored in 47 cells, p = 0.0002; neuron without MPEP: 42.9 ± 7.4 spots/100 µm2, 2.7 × 103 µm2 explored in five cells; neuron treated with MPEP: 109.4 ± 13.2 spots/100 µm2, 2.7 × 103 µm2 explored in six cells, p = 0.0062). We conclude that neither agonist binding nor intrinsic activity of the receptor is necessary for endocytosis of mGluR5a.
In order to evaluate the amount of receptors that undergo
constitutive endocytosis we performed biotinylation assays of
endocytosis on COS-7 cells transfected with untagged mGluR5a
encoding vector. After biotinylation at 4 °C with
sulfo-NHS-SS-biotin, the cells were incubated at 37 °C in PBS for
different times. After purification on streptavidin-agarose beads and
Western blot revealed with anti-mGluR5 antibodies, we measured the
total (without cleavage of surface biotin) and intracellular (after
cleavage) amounts of biotinylated receptors. Fig.
4A shows the corresponding
mGluR5 labeling on one Western blot. The amount of intracellular
receptors increases with time while the total amount decreases. Since
the experiment was done in PBS, this shows that the receptor is indeed
constitutively endocytosed. We quantified the density of the bands and
calculated the percentage of internalized receptors (Fig.
4B, see "Experimental Procedures" for details). The time
course of endocytosis can be well described by a single exponential and
a time constant () of 8.5 min. The rate of endocytosis is thus
11.7%/min. This indicates that the constitutive endocytosis of mGluR5
is not due to bulk flow endocytosis of the membrane, since this value
is significantly higher than 1%/min (24).
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mGluR5a Is Endocytosed by a Clathrin-independent Pathway-- Several internalization pathways can coexist in a cell. The most extensively characterized mechanism of endocytosis is clathrin-mediated, but it has been recently evidenced that bona fide receptors can enter the cell by other pathways (reviewed in Refs. 9 and 10). In order to discriminate between these pathways, the best tools now available are dominant negative mutants of proteins implicated in clathrin-mediated endocytosis like Eps15 and the dynamins.
Overexpression of an EH-deleted dominant negative mutant of Eps15
(E95/295) that still contains the binding site for the adaptor AP2
inhibits clathrin-coated pit assembly and blocks
clathrin-dependent endocytosis (16, 17). We used this
mutant to see if it could inhibit endocytosis of mGluR5a in COS-7 cells
and in hippocampal neurons. We cotransfected GFP-tagged E
95/295 and
mGluR5aNHA and looked for the endocytosis of anti-HA antibodies. In
order to control the efficiency of the Eps15 mutant, Rh-Tf endocytosis was monitored in the same cells. Whereas Rh-Tf endocytosis is abolished
(Fig. 5G), Fig. 5,
E and F (for COS-7 cells) and Fig. 6, C and D (for
neurons) show that endocytosis of the receptor (Figs. 5F and
6D) is still observed in cells coexpressing GFP-E
95/295 (Fig. 5E and 6C). Quantifications show that the
endocytosis in the presence of dominant negative mutant of Eps15,
GFP-E
95/295, is the same as in cells transfected with a
control Eps15 construct, GFP-DIII
2, lacking the AP2 binding site
(Fig. 5, A and B, for COS-7; Fig. 6, A
and B, for neurons). The values are for COS-7 cells:
GFP-DIII
2, 15.9 ± 1.7 spots/100 µm2, 2 × 104 µm2 explored in 47 cells analyzed;
GFP-E
95/295, 14 ± 0.8 spots/100 µm2, 2.2 × 104 µm2 explored in 54 cells analyzed,
p = 0.8; and for the neurons, GFP-DIII
2, 27.1 ± 6.5 spots/100 µm2, 3.9 × 103
µm2 explored in nine cells analyzed; GFP-E
95/295,
36 ± 4.3 spots/100 µm2, 3.7 × 103
µm2 explored in 10 cells analyzed, p = 0.09. Since Eps15 has not been implicated in pathways other than the
clathrin-coated pits pathway (25), we conclude that mGluR5a is
endocytosed through a nonclathrin-mediated pathway. Nevertheless, after
endocytosis, the receptor is found at least partly in the same
endosomes as transferrin endocytosed by clathrin-coated pits (Fig.
5D). This has been observed also for the interleukin-2
receptor and more recently for the muscarinic acetylcholine receptor M2
(19, 26, 27).
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To confirm that the mGluR5a is not in clathrin-coated pits at the cell
surface, we performed a double labeling of the receptor at 4 °C and
of -adaptin after permeabilization (Fig.
7).
-Adaptin is a protein that is part
of the adaptor complex AP2, which is associated with clathrin-coated
pit at the plasma membrane (28). As expected, very little, if any,
colocalization is observed between the mGluR5aNmyc cell surface
clusters and
-adaptin. In accordance, no colocalization was observed
with clathrin using anti-clathrin heavy chain antibodies (data not
shown).
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Dynamins 1 and 2 are large GTPases that have been implicated in clathrin and nonclathrin-mediated endocytosis (9, 10, 25, 29-31). Dynamin 1 is a presynaptic protein that is used for the recycling of synaptic vesicles, whereas dynamin 2 is ubiquitously expressed (15). To dissect more precisely the machinery used by mGluR5 to be internalized, COS-7 cells or cultured hippocampal neurons have been co-transfected with mGluR5aNHA and dynamin 1 or dynamin 2 wild type or dominant negative mutants coupled to GFP.
The dominant negative mutants, Dyn1K44A-GFP and Dyn2K44A-GFP, are
defective in their GTPase activity and inhibit transferrin uptake in
COS-7 cells (32) (data not shown). In COS-7 cells, we still observe
acid wash-resistant intracellular labeling of endocytosed anti-HA
antibodies when cells are co-transfected with Dyn1K44A-GFP (Fig.
8, C and D). The
slight inhibition observed in the quantitated data is not significant
(Fig. 8I; Dyn1-GFP, 25.1 ± 2.3 spots/100
µm2, 1.6 × 104 µm2
explored in 30 cells analyzed; Dyn1K44A-GFP, 20.5 ± 2.4 spots/100 µm2, 0.8 × 104 µm2
explored in 17 cells analyzed; p = 0.2). Dynamin 1 dominant negative mutant is not able to inhibit mGluR5a internalization
in COS-7 cells, although it inhibits the clathrin-mediated endocytosis of transferrin.3 Conversely, cotransfection with
Dyn2K44A-GFP inhibits mGluR5aNHA endocytosis (86% inhibition (Fig. 8,
G-I); Dyn2-GFP, 20.6 ± 3.6 spots/100
µm2, 1.3 × 104 µm2
explored in 20 cells analyzed; Dyn2K44A-GFP, 2.9 ± 0.7 spots/100 µm2, 0.7 × 104 µm2
explored in 20 cells analyzed, p < 0.0001). In these
cotransfected cells, labeling after acid wash was barely detectable,
although the receptor is present at the cell surface in the presence of Dyn2K44A-GFP (Fig. 8, E and F). Dynamin 2 is thus
necessary for the clathrin-independent endocytosis of mGluR5a. This
illustrates that the dynamin variants do not play the same role and
cannot be indifferently used.
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In hippocampal neurons, although dynamin mutants seem to be well
expressed and localized near the membrane, mGluR5a is still endocytosed
with dynamin 1 or 2 dominant negative mutant (Fig. 9; Dyn1-GFP, 48.2 ± 10.5 spots/100
µm2; 5.3 × 103 µm2
explored in 14 cells analyzed; Dyn1K44A-GFP, 62.7 ± 16.9 spots/100 µm2, 2.4 × 103
µm2 explored in six cells analyzed, p = 0.14; Dyn2-GFP, 43 ± 3.3 spots/100 µm2, 8.4 × 103 µm2 explored in 23 cells analyzed;
Dyn2K44A-GFP, 52 ± 4.3 spots/100 µm2, 7.2 × 103 µm2 explored in 18 cells analyzed,
p = 0.13).
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DISCUSSION |
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Our results show by immunofluorescence and image quantification that the group 1 metabotropic glutamate receptor mGluR5a is endocytosed in COS-7 cells (Fig. 1) and in axons and dendrites of hippocampal neurons in culture (Fig. 2). mGluR5a is endocytosed constitutively, because mutated receptors that cannot bind glutamate are internalized (Fig. 3, A-C) and because the receptor is still internalized in the presence of the inverse agonist MPEP that inhibits the intrinsic and stimulated activity of the receptor (Fig. 3, D-F). We found that the endocytic rate is 11.7%/min as measured by a biochemical assay (Fig. 4). Using dominant negative mutants of proteins involved in clathrin-coated pit-mediated endocytosis (Eps15 and dynamins), we show that mGluR5a is endocytosed by a clathrin-independent pathway (Figs. 5, 6, 8, and 9).
Compartmentalization of Transfected Epitope-tagged mGluR5 Receptors-- Previous studies on the in vivo localization of mGluR5 reported a mainly dendritic but also axonal presence of mGluR5 in hippocampus (33, 34). The immunolocalizations were done with antibodies that could not discriminate between the two splice variants mGluR5a and -b that differ only by 32 amino acids inserted in the intracellular part of the mGluR5b (12, 35). One hypothesis is that one splice variant could be presynaptic and the other postsynaptic. For the first time, we report that both epitope-tagged mGluR5a and mGluR5b are expressed and endocytosed in axons and dendrites of transfected hippocampal neurons in culture. The nonpolarized expression of the transfected mGluR5 receptors in hippocampal neurons in culture contrasts with the polarized dendritic localization in hippocampal neurons of transfected mGluR1a, which shares 60% homology with mGluR5 (data not shown) (36, 37). Hence, our results disfavor the hypothesis that mGluR5 splice variants have different polarized localizations.
Ango et al. (38) have shown that, transfected in cultured cerebellar granule cells, mGluR5a is expressed only in cell bodies. Overexpression of the immediate early gene product Homer1a leads to the nonpolarized expression of the receptor in dendrites and axons as we observed in hippocampal neurons. Overexpression of the long forms of Homer1 (Homer1b and -c) leads to the polarized dendritic expression of mGluR5a. In contrast, they found that, transfected in striatal neurons in culture that express endogenously Homer1b proteins, mGluR5a is expressed in neurites. In accordance with these data, we have observed by reverse transcription-PCR and Western blot that hippocampal neurons in culture express Homer proteins (13) (data not shown).
Endocytosis of mGluR5 has been reported in cell bodies of cultured enteric neurons (39). Here we report that both splice variants are endocytosed in cultured neurons from the central nervous system, in dendrites, and in axons. Axonal endocytosis of receptors is not well documented; to our knowledge, only one report shows the axonal endocytosis of a GPCR (dopaminergic receptor D1) (40). Recently, Garrido et al. (41) showed that the C-terminal tail of the voltage-gated sodium channel (Nav1.2) contains a sequence able to target a reporter protein to axons and dendrites of transfected hippocampal neurons, but it drives endocytosis of this chimera only in the somatodendritic domain. This dissymmetry leads to a polarized axonal expression. This exemplifies that axonal endocytosis should have different molecular requirements than that in dendrites.
Constitutive Endocytosis-- By two different approaches, we show that mGluR5aNHA is endocytosed constitutively (i.e. in absence of ligand binding and phospholipase C activation). We obtained the same results for endocytosis of mGluR5b in the presence of MPEP.3 By a biochemical approach, we showed that the wild type receptor (untagged) is also constitutively endocytosed at a rate around 12%/min. In agreement with our data, Hubert et al. have observed in the substancia nigra that 80% of the labeling of mGluR5 is intracellular, although they have not identified the type of intracellular compartments containing mGluR5, biosynthetic or endocytic (42). Although we show a constitutive endocytosis, it does not mean that endocytosis flux is not regulated by ligand or receptor activity. A regulation of mGluR5 endocytosis by ligand has been described in enteric neurons (39). It has been reported by several groups that the very close cousin of mGluR5, mGluR1a, is endocytosed in heterologous cells. Both a constitutive and a stimulated endocytosis have been observed (43-47). We show here that, surprisingly, the number of intracellular spots is increased after MPEP treatment. This could come from an accelerated endocytosis, a modification of the shape of endosomes, and/or changes in the intracellular pathway followed by mGluR5. We pursue the biochemical experiments in order to measure potential variations of endocytosis parameters (endocytosis rate, proportion of recycling/degradation) that we cannot obtain by image analysis. In the quantification, we count the number of spots/cell surface but not the content and size of each spot.
The constitutive endocytosis of mGluR5 creates an intracellular pool that could permit the cell to rapidly modulate the number of cell surface receptors.
Internalization Pathway-- Using the dominant negative mutant of Eps15, a protein involved in clathrin-coated pit-mediated endocytosis, we show that mGluR5a is endocytosed by a nonclathrin-coated pit pathway. The same results have been obtained for mGluR5b (data not shown). Our results demonstrate for the first time that a member of GPCR group C is constitutively endocytosed by a nonconventional pathway.
If agonist-stimulated mGluR1a endocytosis in HEK293 cells seems to occur trough clathrin-coated pits (45, 47), the constitutive endocytosis pathway is less well established (46). Authors have concluded the use of clathrin-mediated pathway, because the endocytosis was inhibited by hypertonic sucrose treatment and because the receptor is found in the same endosomes as transferrin. Hypertonic sucrose does not inhibit only clathrin-mediated endocytosis. For instance, it inhibits M2 muscarinic acetylcholine and GABAA receptors endocytosis that are not mediated by clathrin-coated pits (48, 49). mGluR5 endocytosis is also inhibited by hypertonic sucrose in COS-7 cells (data not shown), and endocytosed receptors colocalized partly with endocytosed transferrin (Fig. 5). Furthermore, Dale et al. (46) showed that dynamine 1 dominant negative mutant does not inhibit mGluR1a endocytosis as we found for mGluR5. Thus, further experiments using Eps15 mutants on mGluR1a constitutive internalization will be more conclusive for the understanding of the constitutive internalization pathway followed by mGluR1a.
mGluR5a (as well as mGluR5b) (data not shown) endocytosis is insensitive to dynamin 1 mutant in COS-7 cells and in neurons. However, in COS-7 cells, the nonconventional pathway followed by mGluR5 uses dynamin 2, the endogenous isoform in these cells. Surprisingly, dynamin 1 dominant negative mutant is not able to compete with dynamin 2 in this pathway, although it competes with dynamin 2 implicated in clathrin-coated pit endocytosis. Indeed, transferrin endocytosis is inhibited by both dynamin 1 and dynamin 2 dominant negative mutants in COS-7 cells (data not shown) (15). This points out that dynamins may have distinct functions in clathrin and nonclathrin endocytosis. This can be put together with the results of Altschuller et al. (50), showing a different function of dynamins in apical versus basolateral endocytosis in the polarized epithelial cell line Madin-Darby canine kidney cells. Furthermore, in a collaborative work with the laboratory of Mark McNiven (Mayo Clinic, Rochester, MN), we have shown that the dynamin 3aaa isoform (31) can inhibit mGluR5 endocytosis in COS-7 cells (data not shown).
In neurons, we obtained a different result in that dynamin 2 dominant negative mutants does not inhibit mGluR5 endocytosis, although transfected Dynamin 2 mutants are targeted to the membranes close to the site of mGluR5 endocytosis (Fig. 6). Perhaps the experimental paradigm is not adapted to neurons. This hypothesis cannot be ruled out, because there is only one publication using these tools in neurons. The authors show that virally overexpressed dynamin 2 mutants inhibit the ionotropic glutamate receptor AMPA GluR2 subunit internalization (4). Another explanation for such an observation in hippocampal neurons is that there could exist an endocytosis pathway used by mGluR5 that does not depend on dynamins 1 and 2. In general, the role of dynamins in the different neuronal compartment where they are expressed is not clear except for dynamin 1 in synaptic vesicle recycling (51, 52).
The involvement of dynamins in nonclathrin-coated pit-mediated endocytosis has already been described in nonneuronal cells (29, 30, 53, 54). Endocytosis of some GPCRs is insensitive to dynamin 1 dominant negative mutants in heterologous cells (e.g. the muscarinic acetylcholine receptor M2 (55), dopaminergic receptor D2 (56), and angiotensin receptor AT1A (57)). This suggests that they are not endocytosed by a clathrin-coated pit-mediated endocytosis. It would be interesting to know if the pathway used by these receptors needs dynamin 2 as we show for mGluR5. An inhibitory effect of dynamin 1 dominant negative mutants has been observed in some nonclathrin-coated pit endocytosis: in the case of interleukin-2 receptor endocytosis using the regular K44A mutant (53) and in the case of muscarinic receptor M2 using dynamin 1 that lacks the complete GTP binding domain or the phosphatidylinositol 4,5-bisphosphate-stimulated GTPase activity (54). It is interesting to note that in that last case the dynamin 1 K44A mutant was not able to inhibit endocytosis. This suggests that perhaps dynamins do not interact with the same partners in clathrin-dependent and -independent endocytosis.
What could be this clathrin-independent pathway? The main studied
nonclathrin pathway is caveolae-mediated endocytosis, but few
transmembrane receptors have yet been found in such caveolin-coated structures. Moreover, real caveolae have not been observed in lymphocytes and in neurons (58-60). A noncoated, raft-mediated endocytosis could be involved. Rafts are membrane microdomains enriched
in sphingolipids and cholesterol in which there is preferential localization of glycosylphosphatidylinositol-anchored proteins and some
signaling molecules (59). Interleukin-2 receptors have been localized
in detergent-resistant membrane and observed in noncoated areas of the
membrane. A first step to unravel the internalization pathway of mGluR5
will be to know if mGluR5 is present in rafts. There are two
interesting observations: the Gq, the G protein coupled to
mGluR5, is present in rafts (61, 62), and mGluR1a, endocytosed through
clathrin-coated pits, is not found in rafts (63). Elucidation of this
nonclathrin endocytosis is of great interest for understanding the
peculiar role for endocytosis of receptors.
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ACKNOWLEDGEMENTS |
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We sincerely thank Rainer Kühn, Alexandre Benmerah, Alice Dautry-Varsat, and Mark McNiven for the gifts of compounds and plasmids. We are grateful to Alice Dautry-Varsat and Bénédicte Dargent for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Conseil Régional d'Aquitaine, the Association pour la Recherche Médicale en Aquitaine.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
33-5-57-57-40-89; Fax: 33-5-57-57-40-82; E-mail:
ahemar@u-bordeaux2.fr.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M205663200
2 A. Hémar, unpublished data.
3 L. Fourgeaud and A. Hémar, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
mGluR, metabotropic
glutamate receptors;
GABAB, -aminobutyric acid receptor
B;
GPCR, G protein-coupled receptor;
AMPA,
-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid;
MPEP, 2-methyl-6-(phenylethynyl)pyridine;
GFP, green fluorescent protein;
Tf, transferrin;
Rh, rhodamine;
HA, hemagglutinin;
PBS, phosphate-buffered saline;
NHS, N-hydroxysuccinimide.
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