1 School of Biology and Petit Institute for Biosciences and Bioengineering,
Georgia Institute of Technology, Atlanta, GA 30332-0363, USA
2 Department of Neurology, Emory University School of Medicine, Atlanta, GA
30322, USA
Author for correspondence (e-mail:
harish.radhakrishna{at}biology.gatech.edu)
Accepted 28 January 2003
![]() |
Summary |
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Key words: LPA1, Endocytosis, Dynamin, Rab5, Lysophosphatidic acid
![]() |
Introduction |
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Molecular cloning studies have identified three mammalian receptors that
belong to the endothelial differentiation gene (EDG) family of
G-protein-coupled receptors (GPCRs) that are activated by LPA:
LPA1/EDG-2, LPA2/EDG-4 and LPA3/EDG-7
(Chun et al., 1999). These
receptors were initially termed EDG receptors since they share sequence
homology with the sphingosine-1-phosphate (S1P)-specific S1P1/EDG-1
receptor (Hla et al., 2001
).
Heterologous expression studies have shown that all three receptors can
activate Gi- and Gq-coupled signaling pathways
(Ishii et al., 2000
).
LPA1 and LPA2, but not LPA3, can additionally
stimulate G12/13-coupled pathways. Recent studies have also
indicated that LPA is a potent mitogen for ovarian cancer epithelial cells and
that increased LPA concentrations in the serum and ascites might serve as a
useful biomarker for ovarian cancer (Fang
et al., 2000
; Moolenaar,
1999
; Xu et al.,
1998
). These studies also suggest that expression of
LPA2 or LPA3, which are not expressed in normal ovarian
epithelial cells, is upregulated in ovarian cancer epithelial cells
(Fang et al., 2000
).
Interestingly, LPA1 has been shown to be a negative regulator of
ovarian cancer cell growth (Furui et al.,
1999
). Of the three known LPA receptors, LPA1 shows the
widest tissue distribution. Human LPA1 is expressed in adult organs
such as brain, heart, ovary, testes, colon, prostate and spleen, but is not
detectably expressed in liver, thymus or lung
(Contos and Chun, 2001
;
Hecht et al., 1996
).
Agonist binding and activation of most GPCRs usually results in the rapid
phosphorylation and endocytosis of the receptor
(Ferguson, 2001). GPCR
endocytosis serves as an entry point for targeting activated GPCRs into a
variety of intracellular compartments including endosomes and lysosomes.
Dephosphorylation of receptors in endosomes and subsequent recycling back to
the cell surface constitutes GPCR resensitization, whereas targeting receptors
to lysosomes for degradation is used for GPCR downregulation. Thus far,
nothing is known about the trafficking or intracellular destinations of any
LPA-coupled receptor.
To gain further insight into how cells regulate the activity of specific LPA receptors, we investigated the agonist-induced trafficking of the human LPA1 receptor in HeLa cells. Our results indicate that LPA1 is rapidly internalized into cells via dynamin2- and Rab5-dependent mechanisms in an LPA-specific and LPA dose-dependent manner. Interestingly, we find that LPA1 is internalized and recycled at a low basal level when cells are cultured in medium that contained 10% FBS, which suggested that LPA levels in serum are sufficient to induce LPA1 activation and endocytosis.
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Materials and Methods |
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Lysophosphatidic acid (1-Oleoyl-2-hydroxy-sn-glycerol-3-phosphate; LPA) and D-erythro sphingosine-1-phosphate (S1P) were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). L-alpha-lysophosphatidylcholine (LPC), fatty acid-free BSA, and all other chemicals were purchased from Sigma. Stock solutions of LPA and LPC were prepared by dissolving in dH2O and sonication, whereas S1P was prepared by dissolving in methanol, followed by evaporation under a stream of nitrogen gas. The dried S1P was then dissolved in 4 mg/ml fatty acid-free BSA (Sigma) in dH2O. For lipid stimulation, cells were grown on glass coverslips for 16-24 hours at 37°C in complete medium and then incubated in serum-free DMEM (SF-DMEM) for an additional 16 hours at 37°C prior to incubation with the appropriate lipid in SF-DMEM.
DNA manipulations and transfections
An expression plasmid encoding the human LPA1 receptor
containing an amino terminal FLAG epitope tag
(Bandoh et al., 1999) was the
kind gift of Junken Aoki (University of Tokyo, Japan). The FLAG epitope tag in
this receptor is exposed to the extracellular environment when LPA1
is at the cell surface. To enhance cell-surface expression of LPA1,
PCR was used to attach a signal leader sequence from the influenza
hemaglutinin protein onto the amino terminus of FLAG-tagged LPA1
cDNA using the following primers:
5'-ATCATGAAGACCATCATCGCCCTGAGCTACATCTTCTGCCTGGTGTTCGCCGACTACAAAGACGATGACGATAAA-3'
and 5'-GATCTCAAACCACAGAGTGATC-3'. Following PCR amplification, the
cDNA product was subcloned into the eukaryotic expression vector pcDNA
3.1/V5-His using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) All DNA
sequences were confirmed by DNA sequencing (Emory DNA Sequencing Core
Facility, Atlanta, GA).
To generate stable HeLa cell transfectants, wild-type (WT) LPA1
was transfected into HeLa cells using the calcium phosphate co-precipitation
method (Radhakrishna and Donaldson,
1997). At 36 hours after transfection, cells were detached and
re-plated at a 1:25 dilution into complete medium containing 600 µg/ml G418
(Life Technologies, Gaithersburg, MD). Approximately two weeks later,
G418-resistant clones were amplified and tested for LPA1 expression
by indirect immunofluorescence microscopy. For immunolocalization studies,
HeLa cells were grown on glass coverslips and transfected in six-well dishes
using the calcium phosphate method. WT and mutant plasmids encoding green
fluorescent protein (GFP)-Rab5 were transiently co-transfected along with
plasmids for FLAG-tagged LPA1 into six-well dishes using 5 µg of
Rab5 DNA. WT and mutant dynamin plasmids were co-transfected with FLAG-tagged
LPA1 transfected using 10 µg of dynamin plasmid per well.
Indirect immunofluorescence
At 22 hours after transfection, the cells were rinsed with SF-DMEM and
incubated in the same medium for 16-24 hours before further treatments. Cells
were treated as described in the figure legends, fixed in 2% formaldehyde in
PBS for 10 minutes, and rinsed with 10% FBS and 0.02% azide in PBS
(PBS-serum). The cells were permeabilized by treating with ice-cold methanol
for 30 seconds at 20°C, rinsing with ice-cold PBS twice and
incubating in PBS-serum for 5 minutes. Fixed cells were incubated with primary
antibodies diluted in PBS-serum containing 0.2% saponin for 45 minutes, and
then washed (three times, 5 minutes each) with PBS-serum. The cells were then
incubated in secondary antibodies diluted in PBS-serum plus 0.2% saponin for
45 minutes, washed with PBS-serum (three times, 5 minutes each) and once with
PBS, and mounted on glass slides. Samples were observed using an Olympus BX40
epifluorescence microscope equipped with a 60x Plan pro lens and
photomicrographs were prepared using a Spot RT monochrome `C' digital camera
(Diagnostic Instruments, Sterling Hts, MI). The fluorescence images were
photographed using the same exposure time and processed identically using
Adobe Photoshop 5.0.
Quantitation of LPA1 internalization
HeLa cells expressing FLAG-tagged LPA1 were treated as described
in the figure legends and fixed as described above. The fixed cells were
labeled with 10 µg/ml concentration of Alexa488-labeled conconavalin A
(ConA), which was obtained from Molecular Probes in the absence of detergent
permeabilization to label the plasma membrane uniformly. The cells were then
washed with PBS/serum and labeled with mouse anti-FLAG antibodies (M1) and
Alexa594-labeled goat anti-mouse antibodies in the presence of 0.2% saponin as
described above. Photomicrographs of 24 cells per time point or experimental
treatment were obtained from a total of three independent experiments using a
Zeiss (Heidelberg, Germany) LSM 510 laser scanning confocal microscope
equipped with a 63x PlanApochromat oil immersion lens. The percentage of
cell-surface receptors was determined by measuring the extent of
LPA1 co-localization with the cell-surface marker Alexa594-labeled
ConA. Quantitation of co-localization was performed as described previously
using Metamorph Imaging System Software (Universal Imaging Corporation, West
Chester, PA) (Volpicelli et al.,
2001). Briefly, background was substracted from unprocessed images
and the percentage of LPA1 pixels (red) overlapping ConA pixels
(green) was measured. The data was normalized to untreated cells (time=0) and
the percentage of internalized receptors was calculated by subtracting the
percentage of cell-surface receptors from 100%. The data is presented as mean
(± s.e.m.) and statistical analysis was performed using ANOVA followed
by a Dunnett's post-hoc test.
Immunoblotting
At 30-36 hours after plating, cells were detached from a T-75 flask with
trypsin/EDTA or scraped from culture dishes after the indicated treatment,
washed twice with ice-cold PBS, and pelleted by centrifugation at 300
g for 5 minutes at 4°C. The pellets were resuspended in
100-200 µl of cell lysis buffer (1% NP-40, 1% sodium deoxycholate, 0.1%
SDS, 0.15 M NaCl, 0.01 M sodium phosphate pH 7.2, 2 mM EDTA, 50 mM NaF, 0.2 M
sodium orthovanadate, 0.02% azide, 100 µg/ml leupeptin and 0.1 mM PMSF) and
incubated on ice for 15 minutes. Detergent-insoluble material was removed by
centrifugation at 13,000 g for 10 minutes at 4°C. The
samples (30 µg of protein per lane) were then separated by SDS-PAGE on 10%
gels and transferred to nitrocellulose paper. MAP kinase activation was
detected using the PhosphoPlus p44/42 MAP Kinase antibody kit (Cell Signaling,
Beverly, MA) and LPA1 was detected using a polyclonal rabbit
anti-FLAG antibody (Sigma). The binding of primary antibodies was detected by
enhanced chemifluorescence detection (Amersham Biosciences, Piscataway,
NJ).
Measurement of serum response factor (SRF) activity
A transcriptional reporter gene assay (Clontech) was used to monitor the
activity of SRF. For these studies, we used the HepG2 human hepatoma cell line
since this cell does not contain functional LPA receptors
(Fischer et al., 1998).
Approximately 7x104 HepG2 cells were plated in 96-well dishes
and transfected with 0.2 µg plasmid encoding FLAG-LPA1, 0.2
µg pSRE-luc, 0.05 µg pRL-TK and either 0.2 µg of pBluescript
KS+ or 0.2 µg of the GTPase construct. Cells were transfected in
SF-DMEM using lipofectamine (Invitrogen) at 1 µl lipofectamine per 0.4
µg DNA. pSRE-luc encodes firefly luciferase and contains three tandem
copies of the serum response element upstream of a basal promoter; luciferase
expression is strongly stimulated by SRF. The pRL-TK construct constitutively
encodes Renilla reniformis luciferase whose expression is controlled
by a thymidine kinase promoter; Renilla luciferase expression serves
to monitor transfection efficiency. After incubation with the DNA complexes
for 24 hours, the cells were rinsed with SF-DMEM and incubated in the same
medium with either no additions or 1 µM LPA for an additional 16 hours.
Both firefly luciferase and Renilla luciferase activity was measured
using the Dual Luciferase Reporter Assay System (Promega, Madison, WI) and
data were collected with a TD-20/20 luminometer (Turner Designs, Sunnyvale,
CA). Normalized luciferase activity was calculated by dividing the firefly
luciferase activity by the Renilla luciferase activity. Statistical
analysis was performed using a single-factor ANOVA followed by a Tukey's
statistical test.
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Results |
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Since HeLa cells are known to express endogenous LPA1 and
LPA2 receptors, we wanted to determine the time course of
LPA-induced activation of signaling for later comparison with the time course
of agonist-induced LPA1 endocytosis. Stimulation of many cell types
with LPA induces a rapid, but transient, activation of the mitogen-activated
protein kinase (MAPK) pathway (van Corven
et al., 1992). Thus, we examined the time course of LPA-induced
activation of the endogenous MAP kinases ERK1/2
(Fig. 1B) both in stably
transfected HeLa cells expressing FLAG-tagged LPA1 and in
untransfected HeLa cells. ERK1/2 activation was assessed using commercially
available antibodies that recognize the dually phosphorylated, active form of
ERK1/2. In cells stably expressing FLAG-LPA1, the levels of
activated ERK1/2 increased rapidly from 1 to 5 minutes following treatment
with 10 µM LPA, with peak activation occurring between 5 and 10 minutes,
and then steadily decreased such that very little activated ERK1/2 could be
detected after 30 minutes of LPA treatment. In the absence of LPA treatment,
ERK phosphorylation was not detected. Untransfected HeLa cells also exhibited
a rapid increase in activated ERK1/2; however, we consistently observed that
the peak ERK activation occurred between 10 and 30 minutes. This response was
slightly slower than that observed in cells over-expressing
FLAG-LPA1 and was most probably due to enhanced ERK activation
through the elevated levels of LPA1 present in the
FLAG-LPA1-expressing cells. Taken together, these results indicated
that LPA stimulation induced a rapid but transient activation of MAPK in HeLa
cells.
Agonist-dependent internalization and recycling of
LPA1
We next determined the effects of LPA stimulation on the cellular
distribution of LPA1 (Fig.
2) using indirect immunofluorescence. Treatment with 10 µM LPA
resulted in a time-dependent redistribution of LPA1 from a
predominantly plasma membrane (PM) localization, observed in unstimulated
cells (Fig. 2, 0 min), to small
punctate intracellular structures. These structures are likely to be
intracellular endosomal compartments since they were not observed if
immunofluorescence labeling was performed without detergent permeabilization
(data not shown). In the absence of permeabilization, anti-FLAG antibodies
only labeled the LPA1 receptors at the cell surface by binding to
the externally oriented FLAG epitope. Furthermore, the anti-FLAG antibodies
did not label untransfected HeLa cells (data not shown). Endosomal staining
was first observed within 10 minutes after LPA treatment and increased in
fluorescence intensity such that, after 30 minutes of stimulation,
LPA1 localized predominantly to these vesicular structures. There
was also a noticeable decrease in plasma membrane labeling after 30 minutes of
LPA treatment (Fig. 2, 30 min).
This pattern of localization was the same after 60 minutes of LPA treatment
(Fig. 2, 60 min).
|
To quantify LPA1 internalization, we used a quantitative
immunocytochemical approach that was recently used to analyze muscarinic
acetylcholine receptor internalization in PC12 cells
(Volpicelli et al., 2001).
This approach takes advantage of the observation that unstimulated receptors
show greater co-localization with a plasma membrane marker than internalized
receptors. Internalization is quantified by measuring the extent of
fluorescence overlap between the receptor and plasma membrane marker using
fluorescence imaging software (see Materials and Methods). In the case of the
M4 muscarinic acetylcholine receptor, data obtained from the
fluorescence quantitation of receptor endocytosis was indistinguishable from
data obtained through radioactive ligand binding experiments
(Volpicelli et al., 2001
). We
measured the effects of LPA treatment on the extent of fluorescent pixel
overlap between a plasma membrane marker, Alexa488-labeled ConA, and
LPA1, which was stained with mouse anti-FLAG antibodies and
Alexa594-labeled secondary antibodies. Fig.
3A shows a representative panel of images, obtained by confocal
microscopy, which compares the distribution of LPA1 and
Alexa488-ConA in stably transfected HeLa cells either before or after
treatment with 10 µM LPA. Both Alexa488-ConA and LPA1 are
extensively co-localized at the PM in untreated cells. Following LPA
treatment, there is a significant reduction in the extent of co-localization
between LPA1 and Alexa488-ConA. Quantification of the overlap in
fluorescence showed that approximately 40% of surface LPA1
receptors are internalized within 15 minutes after LPA treatment and that
there is no further increase in internalization
(Fig. 3B). This is comparable
with the extent of internalization of b2-adrenergic receptors
(b2ARs) (Oakley et al.,
1999
; Seachrist et al.,
2000
).
|
We next sought to determine the identity of the LPA1+ endosomal structures. We co-localized the internalized LPA1 with different endocytic organelle markers using double-label immunofluorescence staining (Fig. 4). The internalized LPA1 showed extensive overlap with both transferrin receptor (TfR) and the early endosomal marker EEA1. Interestingly, LPA1 appeared to coincide more with TfR+ compartments than with EEA1+ compartments. Since TfR labeling includes small transport vesicles, sorting endosomes, as well as juxtanuclear recycling endosomes, these observations are consistent with the possibility that internalized LPA1 traverses the same endocytic pathway as the TfR. By contrast, LPA1 did not co-localize with the lysosomal marker LAMP-2, indicating that following short-term exposure to LPA, these receptors are not transported to lysosomes. This raised the possibility that internalized LPA1 might recycle back to the cell surface.
|
Internalization of other GPCRs, such as the b2AR, is thought to
be required for receptor resensitization and subsequent recycling
(Oakley et al., 1999).
Internalized b2ARs have been shown to be dephosphorylated in an
early endosomal compartment prior to recycling back to the cell surface
(Pitcher et al., 1995
;
Seachrist et al., 2000
). We
investigated whether internalized LPA1 could recycle back to the PM
upon removal of LPA (Fig. 5).
Cells were first treated with 10 µM LPA for 30 minutes to induce
internalization of LPA1 into endosomal compartments. The cells were
rinsed to remove LPA and then incubated at 37°C for various times prior to
fixation and immunofluorescence localization of LPA1. In the
absence of LPA treatment, LPA1 was predominantly localized to the
PM (Fig. 5A, Untreated). After
30 minutes treatment with 10 µM LPA, LPA1 localized to numerous
endosomal structures (Fig. 5A,
+LPA). Upon removal of agonist (Fig.
5B), LPA1 first localized to large juxtanuclear
endosomes after 5 minutes and then began to appear at the PM after 15 minutes
with a corresponding decrease in endosomal labeling. Within 30 to 60 minutes
after removal of agonist, LPA1 was predominantly localized to the
PM. These observations indicated that internalized LPA1 rapidly
recycled back to the PM upon removal of LPA.
|
LPA1 internalization is both dose dependent and LPA
specific
To determine whether LPA-induced internalization of LPA1
occurred at physiologically relevant concentrations of LPA, we determined the
dose dependence of LPA treatment on LPA1 internalization
(Fig. 6). Concentrations of LPA
in the range of 1-10 µM have been reported to be required for growth
stimulation of fibroblasts (van Corven et
al., 1992). Following 30 minutes incubation with different
concentrations of LPA, we observed that LPA1 internalization was
dose dependent and that labeling of small punctate endosomal structures was
first observed after treatment with 10 nM LPA. We observed a steady increase
in the number and fluorescence intensity of these endosomal structures as the
concentration of LPA was increased up to 100 µM.
|
To determine whether internalization of LPA1 was specific for
LPA, we examined the effects of two related bioactive lipids, S1P and LPC. S1P
(100 nM) has been shown to potently and specifically activate the closely
related S1P1/EDG-1, S1P3/EDG-3, S1P2/EDG-5,
S1P4/EDG-6 and S1P5/EDG-8 receptors
(Hla et al., 2001). Treatment
of LPA1-expressing HeLa cells with either S1P (0.1 µM or 10
µM) or LPC (1 µM) did not induce the internalization of LPA1.
Rather, LPA1 remained at the cell surface, suggesting that neither
of these related lipids stimulated LPA1 internalization
(Fig. 7). Although cells
treated with S1P appeared to have larger puncta, these were not internal
structures since immunofluorescence labeling in the absence of detergent
permeabilization was the same as that observed in permeabilized cells (data
not shown). At higher concentrations of LPC, the cells became rounded and
detached from the substratum (not shown). Taken together, these results
indicated that LPA1 internalization was dependent upon LPA
concentration and was specifically stimulated by LPA and not by other related
lipids.
|
Agonist-induced internalization of LPA1 is dependent upon
functional dynamin2 and Rab5 proteins
Since internalized LPA1 co-localized with endosomal markers of
the clathrin-mediated endocytic pathway, we investigated whether
LPA1 was perhaps internalized by clathrin-dependent mechanisms. To
address this question, we examined the effects of either WT or
dominant-inhibitory mutants of dynamin2 and Rab5, which are known regulators
of clathrin-dependent endocytosis (Bucci et
al., 1995; Cao et al.,
1998
; Damke et al.,
1994
). Stably transfected HeLa cells expressing LPA1
were transiently transfected with GFP-tagged mutants of dynamin2, K44A
(Dyn2-GFP K44A) (Fig. 8), or
Rab5a, S34N (GFP-Rab5a S34N) (Fig.
9), as well as GFP-tagged WT forms of these GTPases, and assessed
for agonist-induced endocytosis.
|
|
The 100 kDa GTPase dynamin2 is ubiquitously expressed and is involved in
the severing of deeply invaginated clathrin-coated pits to form
clathrin-coated vesicles (Damke et al.,
1994). In cells expressing Dyn2-GFP K44A, agonist-stimulated
internalization of LPA1 was completely inhibited and
LPA1 remained at the cell surface
(Fig. 8B). In contrast to
Dyn2-GFP K44A, cells transfected with WT Dyn2-GFP displayed agonist-induced
internalization of LPA1 that was indistinguishable from cells
expressing LPA1 alone. Both WT and mutant Dyn2 localized in a
diffuse cytoplasmic pattern in the transfected cells. This suggested that
LPA1 internalization followed a dynamin-dependent pathway.
The Ras-related Rab5 GTPase is another regulator of early endocytic traffic
between the PM and early endosomes (Bucci
et al., 1995). Rab5 is known to stimulate homotypic endosomal
fusion following endocytosis. A recent study by Seachrist et al.
(Seachrist et al., 2000
) has
shown the dominant-inhibitory GFP-tagged Rab5a S34N mutant potently inhibits
agonist-induced internalization of b2-adrenergic receptors.
Similarly, expression of GFP-Rab5a S34N in LPA1-expressing HeLa
cells strongly inhibited agonist-induced internalization
(Fig. 9B). In these cells,
Rab5a S34N showed a diffuse cytosolic distribution throughout the cell. In
these same cells, LPA1 was localized at the cell surface and showed
no vesicular labeling as observed in cells that were not transfected with
GFP-Rab5a S34N. Transfection with WT GFP-Rab5a did not alter agonist-induced
internalization of LPA1, which localized to punctate internal
structures as observed in cells expressing LPA1 alone. To quantify
the phenotypic effects of Dyn2-GFP K44A and GFP-Rab5a S34N on LPA1
internalization, we scored the percentage of cells expressing these mutants
for the presence of LPA1+ endocytic structures
(Fig. 9C). In the absence of
these mutant proteins, 71±4% of the cells contained
LPA1+ endocytic structures following treatment with 10
µM LPA for 30 minutes. However, the results from three independent
experiments indicated that expression of either Dyn2-GFP K44A (7±4%) or
GFP-Rab5a S34N (2±1%) almost completely inhibited the appearance of
LPA1+ endocytic structures following LPA treatment.
Taken together, these results indicated that LPA-stimulated endocytosis of
LPA1 is strongly dependent upon both dynamin2 and Rab5a.
To test if either Rab5 S34N or Dyn2 K44A affected LPA1-mediated
signaling, we examined the effects of these mutants on
LPA1-mediated stimulation of transcription via SRF activation
(An, 2000). These experiments
were performed in HepG2 human hepatoma cells since these cells are
nonresponsive to LPA (Fig.
10A) and do not express any known LPA receptors
(Fischer et al., 1998
). SRF
activity was monitored using a firefly luciferase reporter gene plasmid that
contains three tandem copies of the serum response element upstream of a basal
promoter (see Materials and Methods). HepG2 cells were transiently transfected
in serum-free medium with plasmids encoding the firefly luciferase reporter
plasmid, the Renilla luciferase reference plasmid (to normalize for
transfection efficiency), and the FLAG-tagged LPA1 expression
plasmid. In addition, the cells were also transfected with either WT or mutant
Rab5 or Dyn2 expression plasmids. At 24 hours following transfection, the
cells were incubated either in the presence or absence of 1 µM LPA for 16
hours prior to determination of luciferase activity. In cells expressing the
SRE-luciferase plasmid alone, LPA treatment did not induce luciferase
expression, which is consistent with the absence of LPA receptors in these
cells (Fig. 10A, SRE-Luc
Alone). By contrast, cells that co-expressed LPA1 and the
SRE-luciferase construct exhibited a 1.5- to 2-fold increase in firefly
luciferase activity when treated with 1 µM LPA
(Fig. 10A, SRE-Luc +
LPA1). The data in Fig. 10B and
10C show that neither expression of WT Rab5 nor WT Dyn2
significantly affected the LPA1-mediated induction of firefly
luciferase activity in response to agonist treatment. Induction of SRF
activity was mildly inhibited in cells expressing dominant-inhibitory Rab5
S34N. Co-expression of dominant-inhibitory Dyn2 K44A greatly elevated SRF
activity in both untreated and LPA-treated cells; however, this increase was
observed in cells expressing Dyn2 K44A alone and thus was independent of
LPA1 expression (data not shown). Analysis of the LPA-dependent
increase in SRF activity (Fig.
10C) showed that both Rab5 S34N (28% inhibition) and Dyn2 K44A
(26% inhibition) slightly diminished LPA1-dependent SRF activation
(P<0.05). The fold increase in LPA-stimulated SRF activity was 72%
(cells co-expressing LPA1 and Rab5 S34N) and 74% (cells
co-expressing LPA1 and Dyn2 K44A) of that observed in cells
expressing LPA1 alone. These data indicate that Rab5 and Dyn2 are
critical for the agonist-induced internalization of LPA1 and can
also influence LPA1-dependent SRF activation.
|
Basal internalization and recycling of LPA1 in
serum-containing medium
Given that cells in culture are constantly bathed in serum-containing
medium, we examined whether the LPA present in medium containing 10% FBS was
sufficient to trigger LPA1 internalization. It has been estimated
that normal serum levels of LPA range from 0.1 to 10 µM
(Xu et al., 1998).
Immunofluorescence localization of LPA1 in cells grown in 10% serum
showed that it was primarily localized to the PM with little vesicular
labeling (Fig. 11A, No
treatment). To investigate whether LPA1 was internalized at a low
level, we determined the localization of LPA1 in the presence of
10% serum in the presence of the proton ionophore monensin Monensin has been
shown to disrupt the low pH environment of endosomal compartments and, as a
consequence, disrupt receptor recycling to the PM
(Basu et al., 1981
). Incubation
of the LPA1 stable HeLa transfectants with 25 µM monensin
resulted in a time-dependent accumulation of LPA1 in endosomal
structures (Fig. 11A, +25
µM monensin, 15 min and 30 min). Labeling of these structures was first
observed after 5 minutes of treatment and then steadily increased such that,
after 30 minutes of treatment, the pattern of LPA1 labeling was
similar to that of cells treated with 10 µM LPA in serum-free medium for 30
minutes (Fig. 2, 30 min).
Monensin treatment itself did not induce LPA1 internalization since
monensin treatment in serum-free medium did not stimulate LPA1
internalization; internalization in serum-free medium required the addition of
LPA (Fig. 11B). Furthermore,
monensin treatment inhibited LPA1 recycling in serum-free medium
upon removal of LPA (data not shown). These results suggest that
LPA1 undergoes a low basal internalization and most probably
recycles back to the cell surface when cells are cultured in serum-containing
medium.
|
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Discussion |
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LPA1 internalization is a consequence of receptor
activation
Several lines of evidence indicate that LPA1 internalization is
a consequence of agonist-induced receptor activation. First, LPA1
internalization was dependent upon LPA concentration. We observed that
concentrations as low as 10 nM LPA could induce modest LPA1
internalization (Fig. 6).
Internalization continued to increase as the LPA concentration was increased
up to 100 µM LPA. This is consistent with published reports that have shown
LPA concentrations in this range (i.e. 0.1-20 µM) potently induce
intracellular signaling pathways such as stress fiber formation, inhibition of
forskolin-stimulated adenylate cyclase activity and growth stimulation
(Fukushima et al., 1998;
Ishii et al., 2000
;
van Corven et al., 1992
).
Second, comparison of the time course of LPA1 internalization with that of LPA-induced MAPK activation showed that LPA1 internalization coincides with signal desensitization. Analysis of the time course of LPA stimulation of MAPK activity (Fig. 1B) showed that maximal MAPK activation occurred after approximately 5 minutes of LPA treatment and MAPK activity then decreased between 10 to 30 minutes of LPA treatment. By contrast, LPA1 was primarily localized to the PM after 5 minutes of LPA treatment (Fig. 2). LPA1 was first observed in endosomal structures after 10 minutes of LPA stimulation and this endosomal labeling steadily increased thereafter. This is consistent with the internalization of LPA1 occurring after signal desensitization. Finally, LPA1 internalization was specific for LPA treatment. Neither S1P (10 µM) nor LPC (0.1 µM) stimulated the internalization of LPA1. Thus, these observations suggest that LPA1 internalization is a consequence of receptor activation, similar to other GPCRs that undergo agonist-induced internalization.
LPA1 is likely to be internalized via clathrin-dependent
endocytosis
Several observations from this study are consistent with LPA1
internalization occurring via clathrin-dependent endocytosis. First, we
observed that internalized LPA1 showed extensive co-localization
with the clathrin-dependent endosomal markers EEA1 and TfR
(Fig. 4). TfRs are internalized
via clathrin-dependent endocytosis and EEA1 is a Rab5 effector that is
recruited to early endosomal membranes by activated Rab5
(Bucci et al., 1995).
Second, our findings that LPA1 internalization is dependent upon
the function of dynamin2 and Rab5a suggest that LPA1 might be
internalized via clathrin-dependent endocytosis. Both dynamin2 and Rab5
GTPases are known regulators of clathrin-dependent endocytosis
(Bucci et al., 1995;
Damke et al., 1994
). Dynamin2
is ubiquitously expressed and has been shown to be required for the severing
of deeply invaginated clathrin-coated pits to form coated vesicles and also
for the severing of invaginated caveolae
(Damke et al., 1994
;
Henley et al., 1998
).
Following coated vesicle formation, the clathrin coats rapidly dissociate from
coated vesicles in an ATP-dependent fashion. The Rab5a GTPase then stimulates
the homotypic fusion of these uncoated vesicles by regulating the formation of
the proper v-SNARE/t-SNARE associations and by recruiting the components of
the vesicle fusion machinery (Miaczynska
and Zerial, 2002
).
Dominant-inhibitory mutants of both dynamin2 (Dyn2 K44A) and Rab5a (Rab5 S34N) both strongly inhibited the LPA-induced internalization of LPA1 (Figs 8 and 9). In cells expressing these GTPase mutants, LPA1 was confined to the cell surface. Given that Rab5a and dynamin2 are known regulators of clathrin-dependent endocytosis, these results suggest that LPA1 is likely to be internalized via clathrin-dependent endocytosis. However, since Dyn2 K44A inhibits both clathrin-coated vesicle formation as well as formation of caveolae-dependent transport structures; it remains possible that LPA1 is internalized by either clathrin- or caveolae-dependent mechanisms.
Interestingly, we observed that LPA1 was confined to the cell
surface in cells expressing GFP-Rab5a S34N. The best-described role for Rab5
is in mediating the homotypic fusion of early endosomes
(Miaczynska and Zerial, 2002).
However, several recent studies have also shown a role for Rab5 in the
sequestration of receptorligand complexes into clathrin-coated pits
(McLauchlan et al., 1998
;
Seachrist et al., 2000
). A
complex of Rab5 and Rab guanine nucleotide dissociation inhibitor (Rab-GDI)
has been shown to be a necessary cytosolic component for the sequestration of
TfRs into coated pits (McLauchlan et al.,
1998
). Thus, failure to internalize LPA1 in cells
expressing GFP-Rab5a S34N may be a consequence of a defect in receptor
localization to coated pits.
Liu et al. (Liu et al.,
1999), have previously shown that the S1P-coupled receptor,
S1P1/EDG-1, also undergoes agonist-stimulated internalization and
extensively co-localizes with internalized transferrin and also partially
co-localizes with lysosomal markers suggesting that S1P1/EDG-1 is
internalized via clathrin-dependent endocytosis. Together with our results on
LPA1 trafficking, these observations suggest that perhaps other
lysophospholipid receptors may also undergo agonist-induced internalization.
Whether or not internalization of these other family members occurs via
clathrin-mediated mechanisms or perhaps non-clathrin-dependent pathways
remains to be determined.
Role of endocytosis in regulation of LPA1 function
GPCR endocytosis in many instances occurs subsequently to ligand-induced
G-protein activation and involves receptor phosphorylation and the binding of
arrestin proteins (Ferguson,
2001). Internalization is thought to contribute to either signal
desensitization and/or resensitization once the internalized GPCR is
dephosphorylated in an endosomal compartment. Thus, one role for
LPA1 internalization might be to facilitate its dephosphorylation
and subsequent resensitization.
In addition to receptor resensitization, several observations suggest a
broader role for GPCR endocytosis in receptormediated signaling events.
Several recent studies suggest that activated GPCRs can assemble multi-protein
signaling complexes to initiate secondary signaling events from endosomal
compartments within cells. Studies of the thrombin receptor, PAR1, the
neurokinin-1 receptor, and the angiotensin 1a receptor have shown that
following agonist treatment, these internalized GPCRs form complexes, via
b-arrestin, with downstream components of the MAPK signaling pathway including
Raf1, MEK1 and ERK2 (DeFea et al.,
2000; Luttrell et al.,
2001
). Interestingly, these MAPK components co-localize with the
internalized GPCRs on endosomal structures. It has been suggested that this
may provide a G-protein-independent mechanism to target activated ERKs to
specific intracellular compartments to phosphorylate cytoplasmic targets
selectively.
The data in Fig. 10
indicate that inhibition of LPA1 internalization slightly decreased
LPA-dependent induction of SRF-mediated transcription; dominant-inhibitory
Rab5a S34N and Dyn2 K44A reduced LPA1-dependent activation of SRF
by 28% and 26%, respectively. However, these mutants strongly inhibited
LPA1 internalization (Fig.
9), suggesting that the primary effect of these mutants was to
impede LPA1 endocytosis. LPA-dependent activation of SRF is
mediated through the stimulation of Ras- and Rho-dependent signaling
(Hill et al., 1995;
van Corven et al., 1993
)
through Gbg and G12/13 signaling pathways. Others have
shown that dynamin mutants can inhibit LPA-induced ERK activation via the Ras
pathway (Daaka et al., 1998
;
Kranenburg et al., 1999
).
Thus, one possible explanation for the slight reduction in
LPA1-dependent SRF activation is that dyn2 K44A and Rab5a S34N
might inhibit the Ras/ERK-dependent component of SRF activation.
It is also possible that LPA1 internalization may be important for other LPA-dependent signaling processes. The data in Fig. 11 suggests that LPA1 is internalized and most probably recycled at a low basal level in cells cultured in serum-containing medium. Given that serum contains LPA, this basal internalization is likely to represent agonist-induced uptake. If so, then this raises the question of what the long-term signaling consequence of such basal uptake is on cells. Further studies of the role of LPA1 localization in the regulation of LPA-stimulated signaling, as well as other LPA-coupled receptors, is likely to provide important information about the role of endocytosis in regulating LPA-induced cellular responses.
Finally, an important implication of our finding that LPA1 is
internalized in serum-containing medium is that LPA1
internalization may be a useful diagnostic measure of the relative levels of
LPA present in clinical serum samples. Recent observations indicate that serum
LPA levels are increased in patients with ovarian cancer even at early stages
(Xu et al., 1998). Measurement
of LPA1 internalization could be adapted into a simple bioassay for
screening patient serum and/or ascites samples for LPA.
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Acknowledgments |
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References |
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