Agonist-induced Sequestration, Recycling, and Resensitization of
Platelet-activating Factor Receptor
ROLE OF CYTOPLASMIC TAIL PHOSPHORYLATION IN EACH PROCESS*
Isao
Ishii
§¶,
Erika
Saito
,
Takashi
Izumi
,
Michio
Ui§, and
Takao
Shimizu
From the
Department of Biochemistry and Molecular
Biology, Faculty of Medicine, The University of Tokyo,
Hongo 7-3-1, Bunkyo, Tokyo 113 and § Ui Laboratory, The
Institute of Physical and Chemical Research (RIKEN),
Hirosawa 2-1, Wako, Saitama 351-01, Japan
 |
ABSTRACT |
Agonist-induced sequestration, recycling, and
resensitization of platelet-activating factor (PAF) receptor were
characterized in transfected Chinese hamster ovary cells. Exposure of
the cells to PAF led to rapid sequestration of the receptors into the
intracellular compartment and desensitization of the response to PAF.
The sequestration was inhibited by pretreatments that perturbed the
clathrin-mediated pathway. Subsequent removal of PAF by washing with
receptor antagonists led to rapid recycling of the sequestered
receptors to the cell surface accompanied by resensitization to PAF. To
evaluate the potential role of phosphorylation in the receptor
cytoplasmic tail during these processes, mutant receptors in which the
tails were truncated or substituted, so as to lack serine/threonine residues, were created. PAF phosphorylated the wild-type receptor rapidly and strongly, but the mutants did not. The maximal extent of
sequestration of each mutant was lower than that of the wild-type, and
one of the substituted mutants showed no sequestration. Furthermore, the sequestration-defective mutant showed evidence of desensitization after agonist stimulation but not resensitization after agonist removal. Thus, agonist-induced phosphorylation of the cytoplasmic tail
facilitates but is not essential for receptor sequestration, and
sequestration/recycling appears important in receptor
resensitization.
 |
INTRODUCTION |
Exposure of G protein-coupled receptors
(GPCRs)1 to their agonists is
generally followed by a rapid desensitization of signaling. The
mechanism of the desensitization involves a series of distinct steps
including a functional uncoupling from G proteins and their effector
systems, receptor sequestration into the intracellular compartment, and
a net loss of receptors (receptor down-regulation). Among these steps,
the receptor sequestration and following processes are considered to
play an important role both in desensitization and resensitization of
GPCRs (1-3).
The molecular mechanisms of GPCR sequestration have been extensively
studied using a
2-adrenergic receptor
(
2AR) as the main model (1-6). Functional uncoupling of
the
2AR from Gs proteins is a consequence of
agonist-induced phosphorylation of the receptor cytoplasmic tail by the
following two types of protein kinases: cAMP-dependent
protein kinase and G protein-coupled receptor kinases (GRKs) (7).
Recent studies have shown that this phosphorylation promotes binding of
-arrestins to the
2AR and that
-arrestins subsequently act as adapters of clathrin, a major structural protein of
coated pits, which suggests that
2AR sequestration is
mediated by a physical interaction between receptor-associated
-arrestins and clathrin-coated pits (8-13). Despite the large
amount of information concerning
2AR sequestration, much
less is known of sequestration of other types of GPCRs, and available
information is only fragmentary.
Platelet-activating factor (PAF,
1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)
is a potent phospholipid mediator involved in the pathogenesis of
allergic disorders, inflammation, anaphylactic shock, and various other
physiological events (14, 15). PAF drives multiple signaling pathways
through a single type of GPCR, PAF receptor (PAFR) (15). Repetitive or
long lasting application of PAF leads to a rapid functional uncoupling
of the PAFR from effector systems (16-18). We and other groups
(18-22) suggested that agonist-induced phosphorylation of the PAFR by
GRKs may be involved in the rapid uncoupling process. However, the
information available on the subsequent sequestration process is
limited (23, 24), and the fate of the sequestered PAFR has remained
unknown. Furthermore, the potential role of receptor phosphorylation in the sequestration remains to be elucidated.
We expressed epitope-tagged guinea pig PAFRs with cytoplasmic tail
mutations in Chinese hamster ovary (CHO) cells. Immunohistochemistry with monoclonal antibodies directed against the epitopes enabled us to
quantitate cell surface expression of the PAFR, to visualize PAFR
movement, and to immunoprecipitate PAFR proteins for receptor phosphorylation assays. We gained novel information on PAFR dynamics during agonist-induced sequestration, recycling, and resensitization. The role of phosphorylation of the receptor cytoplasmic tail in each
process was also evaluated using the mutant receptors.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[acetyl-3H]PAF C16
(1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine)
(370 GBq/mmol), [3H]WEB 2086 (499.5 GBq/mmol),
[3H]arachidonic acid (3.7 TBq/mmol),
125I-labeled sheep anti-mouse IgG F(ab')2
fragment (300 kBq/µg), [32P]orthophosphate (330 TBq/mmol), and [35S]EXPRESS Methionine/Cysteine Protein
Labeling Mix (37 TBq/mmol) were purchased from NEN Life Science
Products. [3H]Folic acid (1.04 TBq/mmol) was from
Amersham Pharmacia Biotech. PAF C16,
1-O-hexadecyl-2-(methylcarbamyl)-sn-glycero-3-phosphocholine (mcPAF), anti-FLAG M5 monoclonal antibody, and anti-HA
12CA5 monoclonal antibody were obtained from Cascade Biochem (Reading,
UK), Cayman Chemical (Ann Arbor, MI), Kodak, and Boehringer Mannheim,
respectively. PAFR antagonists, WEB 2086 and TCV-309, were generous
gifts from Boehringer Ingelheim and Takeda (Osaka, Japan),
respectively. All other reagents, unless otherwise stated, were of
analytical grade and were from Wako (Osaka) or Sigma.
Plasmid Construction--
All mutations were introduced into a
pBluescript SK(
) vector (Stratagene) containing the entire coding
region of guinea pig PAFR cDNA (25) using a Transformer
Site-directed Mutagenesis Kit (CLONTECH). For
construction of epitope-tagged receptors, nucleotide sequences that
encoded 8- or 9-amino acid peptides (DYKDDDDK for FLAG and YPYDVPDYA
for HA) were inserted between the amino-terminal initiator methionine
and the second amino acid of the wild-type (WT) or mutant PAFRs by
polymerase chain reaction using Pfu DNA polymerase
(Stratagene), as described elsewhere (20, 26). The coding region of
each construct was sequenced on both strands, using an ABI 373 DNA
Sequencer (Perkin-Elmer) and was subcloned into a mammalian expression
vector, pcDNA I Neo (Invitrogen) for stable expression in CHO
cells.
Construction of Stable CHO Transformants--
CHO cells were
maintained as monolayer cultures in Ham's F12 medium (Nissui
Pharmaceutical; Tokyo) supplemented with 10 mM Hepes (pH
7.4) and 10% fetal bovine serum (FBS) (Upstate Biotechnology, Lake
Placid, NY). The cells plated onto 12-well dishes were transfected with
plasmid constructs using TRANSFECTAM (BioSepra, Marlborough, MA)
according to the manufacturer's protocol. Cells resistant to 1 mg/ml
Geneticin (Life Technologies, Inc.) were collected after 2 weeks of
culture, cloned by limiting dilution, and screened for specific
[3H]WEB 2086-binding activity (data not shown). The
clones used in this study were selected based on PAFR expression level
and were maintained in the presence of 0.3 mg/ml Geneticin.
Radioligand Binding Assay--
CHO cells were seeded onto
12-well dishes at 48 h prior to assay. The cells were washed twice
with Tyrode's buffer (26) containing 10 mM Hepes (pH 7.4)
and 0.1% of fatty acid-free bovine serum albumin (Hepes/Tyrode's/BSA
buffer) and were incubated at 25 °C for 1 h with graded
concentrations of [3H]PAF in the presence (for
nonspecific binding) or absence (for total binding) of 10 µM WEB 2086 in the same buffer. The cells were washed
twice with the buffer and solubilized in 1% (w/v) Triton X-100. The
radioactivity associated with the cells was measured by liquid
scintillation counting. The specific binding activity (total binding
minus nonspecific binding) was determined for Scatchard plot analysis,
and ligand-binding parameters, Kd (nM)
and Bmax (fmol/well) were calculated. A
Bmax value of 100 fmol/well was roughly equal to
100,000 binding sites per single CHO cell (data not shown).
Measurement of Arachidonic Acid Release and IP3
Production--
CHO cells prelabeled with
[3H]arachidonic acid were subjected to release assay, as
described (26). Quantitation of inositol 1,4,5-triphosphate
(IP3) production after PAF stimulation was performed as
described (26), using an IP3 radioreceptor assay kit (NEN
Life Science Products).
Analysis of Receptor Sequestration--
Quantitation of cell
surface expression of the PAFR was carried out as described by Gerard
and Gerard (23), with some modifications. CHO cells were seeded onto
CulturPlate 24 (Packard Instrument Co.) at 48 h prior to assay.
The cells were washed twice with Hepes/Tyrode's/BSA buffer, subjected
to various treatments at 37 °C for the indicated time, and then
fixed with 2% freshly prepared paraformaldehyde in
Ca2+,Mg2+-free Dulbecco's phosphate-buffered
saline (PBS) (Nissui) for 10 min. After washing twice with PBS, the
cells were incubated for 1 h in Tyrode's buffer containing 10 mM Hepes (pH 7.4) and 20% (v/v) FBS (Hepes/Tyrode's/FBS
buffer) to reduce nonspecific binding, and for 30 min with or without
10 µg/ml anti-FLAG M5 or 5 µg/ml anti-HA 12CA5 antibody in the same
buffer. After washing twice with PBS, the cells were incubated for 30 min with Hepes/Tyrode's/FBS buffer and for 30 min with 33.3 kBq of
125I-labeled sheep anti-mouse IgG F(ab')2
fragment in the same buffer. The cells were then washed three times
with PBS, dissolved in MicroScint 20 scintillator (Packard), and the
radioactivity was counted in a TopCount Microplate Scintillation
Counter (Packard). All procedures after fixing were carried out at room
temperature. Antibody-dependent binding was used to show
cell surface expression of the PAFR.
Immunofluorescence Microscopy--
Microscopy was done basically
as described by Trapaidze et al. (27). CHO cells were grown
for 48 h on glass coverslips. After stimulation with agonists or
antagonists, the cells were fixed with 2% freshly prepared
paraformaldehyde in PBS. Fixed cells were washed twice with TBS buffer
(20 mM Tris-Cl (pH 7.5), 150 mM NaCl, and 1 mM CaCl2), permeabilized with 0.05% Triton X-100 in Blotto buffer (3% nonfat dry milk in 50 mM
Tris-Cl (pH 7.5)) for 15 min, washed twice with Blotto, and incubated
for 1 h in Blotto. The cells were then incubated for 1 h with
10 µg/ml anti-FLAG antibody in Blotto, washed three times with TBS,
and incubated for 30 min with 5 µg/ml fluorescein
isothiocyanate-conjugated goat anti-mouse IgG (Life Technologies, Inc.)
in Blotto. Then, they were washed three times with TBS and mounted with
SlowFade antifade reagents (Molecular Probes). The cells were viewed
under a Nikon Diaphot TMD300 fluorescence microscope with a NCF Fluor 100 × oil-immersion objective lens (Nikon) set at aperture number 1.3.
Measurement of Folic Acid Incorporation--
Folic acid
incorporation was measured as described (28, 29) but with some
modification. Cells were grown in 12-well dishes for 48 h in
hand-prepared folic acid-free Dulbecco's modified Eagle's medium
containing 5% FBS. Next, the cells were subjected to various
treatments in folic acid-free Dulbecco's modified Eagle's medium to
inhibit the clathrin- or caveolae-mediated pathways. Following these
treatments, the cells were incubated with 50 nM [3H]folic acid in each medium for 90 min. After
incubation, the cells were quickly washed with ice-cold
Hepes/Tyrode's/BSA buffer, chilled on ice for 20 min, and washed twice
with ice-cold acidic saline (150 mM NaCl adjusted to pH 3.0 with glacial acetic acid) to remove radioactive folic acid bound to the
intrinsic cell surface folic acid receptors. The cells were then
solubilized in 1% Triton X-100 and subjected to scintillation
counting.
Inhibition of the Clathrin-mediated Pathway--
CHO cells were
subjected to hypertonic shock with 0.45 M sucrose,
potassium depletion, and acidification with Dulbecco's modified Eagle's medium containing 10 mM acetic acid (pH 5.0), as
described in detail elsewhere (30), followed by analysis of PAFR
sequestration and folic acid incorporation.
Measurement of Protein Synthesis--
CHO-FLAG-WT cells were
seeded onto 6-well dishes at 48 h prior to assay. The cells were
washed twice with methionine/cystine-free Dulbecco's modified Eagle's
medium (Sigma) and incubated for 2 h in the same medium containing
graded concentrations of cycloheximide. The cells were then incubated
for 90 min in medium containing each concentration of cycloheximide and
3.7 MBq of [35S]EXPRESS Methionine/Cysteine Protein
Labeling Mix. Next, the cells were washed twice with ice-cold PBS,
scraped into Eppendorf tubes with PBS, and precipitated in 10% (w/v)
trichloroacetic acid for 20 min on ice. The precipitates were washed
twice with ice-cold 10% trichloroacetic acid, solubilized in 0.5 N NaOH, and subjected to gamma counting.
Receptor Phosphorylation--
Agonist-induced phosphorylation of
the PAFR was examined as described (21, 22, 31) but with some
modification. CHO cells were seeded onto 100-mm dishes at 48 h
prior to assay. On the day of assay, the cells were washed twice with
phosphate-free Dulbecco's modified Eagle's medium (Sigma), incubated
at 37 °C for 2 h in the same medium and for an additional
2 h in the medium containing 18.5 MBq
[32P]orthophosphate. The cells were stimulated by ligands
for the specified time, and the reaction was stopped by rapid washing with ice-cold PBS. All subsequent procedures were carried out at
4 °C. Adherent cells were scraped into Eppendorf tubes with a
suspension buffer of the following composition: 20 mM Hepes (pH 7.4), 5 mM EDTA, 1 mM EGTA, 10 mM sodium fluoride, 10 mM sodium pyrophosphate,
and 1 mM phenylmethylsulfonyl fluoride. After washing twice
with the suspension buffer, the cell pellets were solubilized in the
solubilization buffer (the suspension buffer containing 0.5% Triton
X-100, 0.05% SDS, and 150 mM NaCl) by vigorous vortexing and rotational agitation for 1 h. Insoluble materials were removed by centrifugation, and the supernatants were precleared with 100 µl
of Protein A/G Plus-Agarose (Santa Cruz). The supernatants were
incubated with 10 µg of anti-HA 12CA5 antibody for 2 h, followed by incubation overnight with 100 µl of Protein A/G Plus-Agarose to
precipitate the epitope-tagged receptors. The next day, the samples
were washed 4 times with washing buffer (the suspension buffer
containing 0.5% Triton X-100, 150 mM NaCl, and 0.1%
ammonium sulfate) and then once with the same buffer lacking ammonium
sulfate. SDS sample buffer was added to the pellets, and sonication was done in a water bath for 5 min. The solubilized proteins were separated
on 10% SDS-polyacrylamide gel and analyzed by autoradiography using a Bio-Imaging Analyzer BAS2000 (Fuji Photo Film).
 |
RESULTS |
Agonist-induced Sequestration of the PAF Receptor in CHO
Cells--
PAFR sequestration was analyzed in transfected CHO cells
expressing guinea pig receptors with amino-terminal and cytoplasmic tail mutations (Fig. 1). To detect the
receptors immunochemically, an 8- or 9-amino acid epitope (FLAG or HA)
was introduced into the extracellular amino-terminal domain of each
receptor (Fig. 1, B-F). The wild-type receptor
and mutant receptors, in which the cytoplasmic tail was truncated
(HA-Del, His303
termination codon) or substituted to
lack Ser/Thr residues (HA-Tail-1, Ser318,
Thr321, Thr322, Thr324,
Thr326, Thr335
Ala; HA-Tail-2, mutations in
HA-Tail-1 plus Ser305, Ser313,
Ser314
Gly), were stably expressed in CHO cells. The
mutations in HA-Tail-1 were restricted to the carboxyl-terminal domain
following Cys317, a potential palmitoylation site (25).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 1.
Diagrams showing the mutant PAF receptors
used in this study. Putative seven-transmembrane topology of
mutant PAF receptors indicating the amino-terminal epitope tag (FLAG or
HA), point mutations in the cytoplasmic tail Ser/Thr residues, and the
potential palmitoylation site at Cys317 are shown.
|
|
In all CHO transformants, saturation experiments with
[3H]PAF revealed the existence of a single binding site
class on Scatchard plot analysis (data not shown). There was no
detectable specific PAF binding in untransfected or vector (pcDNA I
Neo)-transfected CHO cells (data not shown). The ligand-binding
parameters of each transformant, Kd (nM)
and Bmax (fmol/well), were as follows: CHO-WT (a
CHO transformant expressing the WT receptor shown in Fig.
1A), Kd = 1.71 ± 0.24, Bmax = 319.4 ± 52.2; CHO-FLAG-WT, Kd = 2.17 ± 0.19, Bmax = 450.7 ± 28.7; CHO-HA-WT, Kd = 1.34 ± 0.24, Bmax = 216.9 ± 36.4; CHO-HA-Del,
Kd = 1.06 ± 0.06, Bmax = 142.6 ± 12.6; CHO-HA-Tail-1, Kd = 2.27 ± 0.42, Bmax = 111.2 ± 16.1;
CHO-HA-Tail-2, Kd = 1.50 ± 0.30, Bmax = 117.1 ± 14.6 (mean ± S.E. of
three separate experiments done in duplicate). Thus, each transformant
displayed PAF-binding parameters comparable to those of CHO-WT cells.
PAF-induced arachidonic acid release in 6 min was comparable between
these transformants (data not shown).
In CHO-FLAG-WT cells, exposure to agonists (PAF or mcPAF) initiated
receptor sequestration as assessed by anti-FLAG antibody binding to the
cell surface receptors (Fig. 2). The
sequestration occurred within 5 min after stimulation and reached a
plateau after 1 h of incubation showing a half-life of 30 min
(Fig. 2A). PAF as well as mcPAF induced sequestration in a
concentration-dependent manner, from a low concentration of
0.25 nM reaching a maximal effect at 10 nM with
an EC50 value of 1-2 nM (Fig. 2B).
A second application of 100 nM PAF at 10, 30, or 60 min
after the first application induced no further sequestration (Fig.
2C). Immunofluorescence microscopy revealed bright staining
of the receptors all over the cell surface, including numerous
punctuate patterns (Fig. 2D, left). Following 60 min of incubation with 100 nM PAF, the fluorescence
intensity of the cell surface was markedly reduced, and small areas of
bright staining emerged in some intracellular compartments (Fig.
2D, middle).

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 2.
Agonist-induced sequestration of the PAF
receptor in CHO-FLAG-WT cells. Receptor sequestration was
determined by anti-FLAG antibody binding to the cell surface receptors.
A, time course of sequestration induced by 100 nM PAF or 100 nM mcPAF. B, PAF (or
mcPAF) concentration-dependent sequestration of the PAFR
over 90 min. C, second application of 100 nM PAF
to 100 nM PAF-pretreated cells. Arrows below
indicate the time (10, 30, and 60 min) of the second application after
the first application was performed. D, immunofluorescence
of FLAG-WT receptors in untreated cells (left), PAF (100 nM for 1 h)-treated cells (middle), and PAF
(100 nM for 1 h) and then TCV-309 (10 µM
for 1 h)-treated cells (right). Data are representative
of three independent experiments that gave similar results.
|
|
Effects of Pretreatments to Inhibit the Clathrin- or
Caveolae-mediated Pathways on Sequestration--
To explore the
mechanism of PAFR sequestration, CHO-FLAG-WT cells were subjected to
pretreatments that inhibited the clathrin-mediated pathway such as
hypertonic sucrose shock, potassium depletion, and acidification (30),
followed by the sequestration assay (Table
I). In parallel with these experiments,
the pretreated cells were also assayed for folic acid incorporation, an
event mediated by caveolae but not by clathrin (32). Folic acid
incorporation was concentration-dependent up to 100 nM [3H]folic acid and was
time-dependent within 4 h of incubation (data not
shown), so incorporation was measured during 90 min of incubation with
50 nM [3H]folic acid.
View this table:
[in this window]
[in a new window]
|
Table I
PAF-induced sequestration of the PAF receptor but not folic acid
incorporation was suppressed by treatments inhibiting the
clathrin-mediated pathway
CHO-FLAG-WT cells were subjected to hypertonic sucrose shock, potassium
depletion, acidification, and 100 nM PMA treatment. PAF
(100 nM)-induced sequestration of the PAF receptor and
folic acid incorporation were measured at 90 min, and both activities
were expressed as percentages relative to the control as 100.
|
|
Hypertonic shock with 0.45 M sucrose prevented about half
of PAF-induced PAFR sequestration but had no effect on folic acid incorporation. Potassium depletion caused almost complete inhibition (97.5%) of sequestration but slightly inhibited folic acid
incorporation (only 12.5%). Acidification inhibited sequestration by
80.3% but had no obvious effect on folic acid incorporation. In
addition, effect of 100 nM phorbol 12-myristate 13-acetate
(PMA) on both activities was examined, because protein kinase C
activators such as PMA reduced the number of caveolae and thus
inhibited folic acid incorporation (33, 34). Stimulation with PMA
resulted in 35.6% loss of folic acid incorporation but caused no
change in PAFR sequestration. No treatment caused marked interference with PAF binding to its receptor and PAF-induced signal transduction (data not shown). These data suggest that PAF-induced receptor sequestration is mostly through clathrin-coated pits.
Phosphorylation and Sequestration of the PAF
Receptor--
Agonist-induced phosphorylation of the PAFR at 10 min
was compared among four kinds of CHO transformants (CHO-HA-WT,
CHO-HA-Del, CHO-HA-Tail-1, and CHO-HA-Tail-2) (Fig.
3A). Exposure of CHO-HA-WT cells to 100 nM PAF for 10 min resulted in phosphorylation
of both 40-50- and 80-100-kDa proteins. Because we detected no
corresponding bands in untransfected CHO cells or CHO-WT cells (data
not shown), the immunoprecipitated radioactive proteins were presumed
to be the HA-WT receptors. The 80-100-kDa bands may represent receptor dimers, as suggested by Ali et al. (20). PAF induced
phosphorylation of both bands in a concentration-dependent
manner; it was detectable in 10 min at a concentration of 0.1 nM and reached a maximal level at 1 nM (data
not shown). Phosphorylation was rapid and lasting; it was detectable
15 s after 100 nM PAF stimulation, reached a maximal
level within 2.5 min, and lasted for at least 15 min (data not shown).
In any case, both 40-50- and 80-100-kDa bands were phosphorylated
proportionally (data not shown).

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 3.
PAF-, PMA-, or Bt2-cAMP-induced
phosphorylation and sequestration of the PAF receptor in CHO
transformants. A, phosphorylation of mutant PAF receptors in
the four CHO transformants induced by 10 min of stimulation with 100 nM PAF, 100 nM PMA, or 1 mM
Bt2-cAMP. The numbers indicate the estimated
molecular masses (kDa) on 10% SDS-polyacrylamide gel electrophoresis.
B, time course curves for PAF (100 nM)-induced
sequestration of the HA-tagged mutant PAF receptors. C,
effect of PMA or dibutyryl cAMP on PAF
concentration-dependent sequestration of the PAFR at 90 min
in CHO-FLAG-WT cells. Data are representative of three independent
experiments that gave similar results (A) or the mean of
duplicate samples and representative of two independent experiments
(B and C).
|
|
PMA and dibutyryl cAMP (Bt2-cAMP), which activate protein
kinase C and cAMP-dependent protein kinase, respectively,
have been shown to phosphorylate the human PAFR in RBL-2H3 cells (20). Treatment of CHO-HA-WT cells with 100 nM PMA or 1 mM Bt2-cAMP for 10 min also caused
phosphorylation of the guinea pig receptor (Fig. 2A). The
maximal extent of the phosphorylation induced by PMA was about half
that caused by PAF, whereas Bt2-cAMP was much less
effective than PAF even though its effect was obvious by radioactive
counting and reproducible. PAF, PMA, and Bt2-cAMP induced
much less phosphorylation in the three cytoplasmic tail mutant
receptors as in the HA-WT receptor. Thus, most of the phosphorylation targets in the PAF receptor for these different stimuli exist in the
cytoplasmic tail Ser/Thr residues (especially the carboxyl-terminal six
Ser/Thr residues).
These four transformants were also examined for receptor sequestration.
PAF induced sequestration of HA-Del and HA-Tail-2 receptors, although
the extent of sequestration was much less than that of the HA-WT
receptor (Fig. 2B). The HA-Tail-1 receptor did not show
sequestration. Six different CHO transformants for each receptor were
analyzed with almost identical results (data not shown). Furthermore,
treatment of the cells with PMA or Bt2-cAMP neither induced
sequestration nor affected PAF-induced sequestration in CHO-FLAG-WT
cells (Fig. 2C). Thus, PAF induced phosphorylation of the
receptors through enzymes other than protein kinase C or cAMP-dependent protein kinase, and it is related to
receptor sequestration.
PAF Receptor Recycling and Resensitization--
In some GPCRs,
sequestered receptors return to the cell surface after agonist removal
in the process called "recycling." To observe PAFR recycling, PAF
(100 nM)-treated CHO-FLAG-WT cells were quickly washed
twice with the Hepes/Tyrode's/BSA buffer containing an excess (10 µM) of the hydrophobic PAFR antagonist WEB 2086 at 10, 60, or 90 min after PAF application, followed by incubation in the same
buffer containing 10 µM WEB 2086 (Fig.
4A). The sequestered receptors
after 60 or 90 min of incubation with 100 nM PAF began to
recycle rapidly (<10 min) after agonist removal by antagonist washing,
but receptors sequestered after 10 min of incubation needed a longer
time (>50 min) to begin recycling. We obtained almost the same results
using a highly hydrophilic PAFR antagonist, TCV-309 (Fig.
4B), which has a similar IC50 value to that of
WEB 2086, as seen in experiments on [3H]PAF replacement
(data not shown). In immunofluorescence microscopy, the sequestered
receptors were seen to return to the cell surface which included
numerous punctuate patterns (Fig. 2D, right).
Neither of the PAFR antagonists had any effect on receptor distribution in the absence of PAF (Fig. 4, A and B,
open triangles). Such recycling was also observed in
experiments with CHO-HA-Tail-2 cells (data not shown). In
sequestration-defective CHO-HA-Tail-1 cells, agonist removal did not
increase cell surface expression of the receptor (data not shown).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Recycling of the PAF receptor after agonist
removal by antagonist washing. A, CHO-FLAG-WT cells were
stimulated with 100 nM PAF followed by washing and
incubation with buffer containing 10 µM WEB 2086, a PAFR
antagonist. Arrows indicate washing by antagonist at each
time (10, 60, and 90 min) after PAF application. B, the same
experiment as in A using a hydrophilic PAFR antagonist
(TCV-309) instead of hydrophobic WEB 2086. In both figures, open
circles and open triangles indicate the untreated cells
and antagonist (10 µM)-treated cells, respectively.
Closed symbols indicate the antagonist (10 µM)-treated cells after 10 (circles), 60 (triangles), and 90 (squares) min of incubation
with 100 nM PAF. Data are the mean of duplicate samples and
are representative of three independent experiments that gave similar
results.
|
|
To examine the roles of sequestration and recycling in
desensitization/resensitization to PAF, we carried out the following experiments. CHO-FLAG-WT cells were stimulated by 100 nM
PAF for 1 h followed by washing and 1 h of incubation with
buffer containing 10 µM TCV-309, and then were exposed
again to 100 nM PAF. The recycled receptors regained the
potential to undergo sequestration (Fig.
5A). We next examined PAFR
desensitization and resensitization based on the IP3
response to PAF. PAF-dependent IP3 production was measured in CHO-FLAG-WT cells at each step indicated by the arrows in Fig. 5A (Fig. 5B).
Incubation for 1 h with 100 nM PAF shifted the
concentration-dependent curve to the right by about 2 orders of magnitude, thereby indicating desensitization. Subsequent incubation for 1 h with 10 µM TCV-309 led to a 1 order curve shift to the left, indicating restoration of the
PAF-induced IP3 response. We obtained similar results with
CHO-HA-Tail-2 cells (Fig. 5D) but not with CHO-HA-Tail-1
cells (Fig. 5C). Sequestration-defective CHO-HA-Tail-1 cells
displayed desensitization but not resensitization.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 5.
Resensitization of the PAF receptor after
recycling. A, cell surface expression of FLAG-WT receptors
was measured. CHO-FLAG-WT cells were incubated with 100 nM
PAF for 1 h followed by washing and 1 h of incubation with 10 µM TCV-309. Cells were then stimulated again with 100 nM PAF. B, PAF-dependent
IP3 production over 15 s in CHO-FLAG-WT cells
collected at each step indicated by arrows in A. C and D, the same experiment as in B
performed with CHO-Tail-1 cells (C) and CHO-Tail-2 cells
(D) instead. Data are the mean of duplicate samples and are
representative of two experiments that gave similar results.
|
|
 |
DISCUSSION |
Recently, Le Gouill et al. (24) reported the
agonist-induced sequestration of the human PAFR expressed in COS-7 and
CHO cells. They measured the activity of [3H]PAF
incorporation as an indicator of PAFR sequestration. In this present
study, we used immunochemical approaches with antibodies to directly
detect PAFR, and this action facilitated a quantitative analysis of the
dynamic PAFR sequestration/recycling process. Since suitable anti-PAFR
antibodies for this purpose were not available, FLAG or HA epitope was
introduced into the amino-terminal extracellular domain of PAFR.
Because CHO cells have endogenous proteins recognized by anti-HA
antibody (data not shown), we utilized FLAG tagging for precise
characterization of PAFR sequestration. No detectable endogenous
proteins on the CHO cell surface were recognized by anti-FLAG antibody
(data not shown). However, as anti-FLAG antibody did not efficiently
immunoprecipitate the FLAG-tagged receptor in the presence of
solubilizing detergents (0.5% Triton X-100 and 0.05% SDS), we used HA
tagging in some experiments. The anti-HA 12CA5 antibody binds to its
epitope with an extremely high affinity, and interactions were stable
under the immunoprecipitation conditions we used (data not shown).
Sequestration and Recycling of PAF Receptors--
In CHO-FLAG-WT
cells, PAF induced PAFR sequestration in both a
time-dependent and a concentration-dependent
manner (Fig. 2, A and B). Thrombin, which
activates CHO cells via endogenous GPCR, did not induce PAFR
sequestration, and pretreatment with PAFR antagonists completely
inhibited PAFR sequestration (data not shown). Thus, this phenomenon is
agonist-specific. Initiation of sequestration was rapid after agonist
stimulation (<5 min). Fig. 2A shows no apparent lag period
between agonist (PAF or mcPAF) addition and the onset of sequestration.
The PAF concentration-dependent profile of sequestration
over 90 min was similar to that observed in the assay of arachidonic
acid release in 6 min (data not shown). Not all of the cell surface
receptors were sequestered even after stimulation with a high
concentration of PAF (Fig. 2B) or a second application of
100 nM PAF (Fig. 2C), and some (30-40% of the
total) remained on the cell surface. PAF might be metabolized by PAF acetylhydrolases (35) after receptor-mediated incorporation (23, 24).
However, such metabolism did not affect sequestration because mcPAF, a
synthetic analog of PAF resistant to the PAF acetylhydrolases, had a
time-dependent or a concentration-dependent effect similar to that of PAF (Fig. 2, A and B).
The cell surface-remaining receptors could transmit signals in response
to PAF, even though some desensitization was observed (Fig.
5B).
Initiation of recycling also began rapidly after agonist removal (Fig.
4, A and B). The rapid onset of recycling of the
FLAG-WT receptor may exclude the possibility that PAFR protein
synthesis is accelerated after agonist removal. In agreement with this, recycling was not blocked by 2 h of preincubation with a high concentration of cycloheximide, which effectively inhibits protein synthesis (85, 94, and 97% inhibition at concentrations of 10, 30, and
60 µg/ml cycloheximide, respectively). It is important to note here
that shut-down of the signals from cell surface PAFR was needed to
trigger the recycling after receptor sequestration. Incubation with a
hydrophobic PAFR antagonist (WEB 2086) or a hydrophilic antagonist
(TCV-309) could induce recycling, suggesting that some signals
triggering recycling may be transmitted through the cell surface PAFR
detached from agonists. Thus, sequestered receptors may be sorted into
early endosomal compartments and prepared to recycle back to the cell
surface waiting for signal shut-down.
Microscopic analysis revealed PAFR movement into some intracellular
compartments after PAF stimulation (Fig. 2D,
middle). Interestingly, recycled receptors regained their
cell surface distribution patterns, including numerous dots (Fig.
2D, right). Characterization and identification
of these intracellular compartments and dots is an on-going study.
Sequestration via the Clathrin-coated Pits-mediated
Pathway--
Many plasma membrane proteins have been shown to
internalize via the clathrin-mediated pathway or other pathways such as
the caveolae (36, 37). It has long been controversial whether or not
GPCRs are sequestered via clathrin-coated pits. Most but not all of the
data suggest the involvement of clathrin in GPCR sequestration (9,
38-42). It should also be noted that heterotrimeric G proteins are
concentrated within the caveolar fraction (43, 44) and interact with
caveolin in a regulated form (45). It seems reasonable to assume that
sequestration mechanisms may vary among GPCRs and cell types in which
they are expressed.
In our experiments, cells were subjected to hypertonic sucrose shock,
potassium depletion, and acidification to inhibit the clathrin-mediated
pathway in a specific manner (30, 46). Sucrose shock and potassium
depletion prevent the interaction of clathrin and adapter proteins,
whereas acidification interferes with the budding of clathrin-coated
vesicles from the cell membrane (30). After these pretreatments, we
measured PAF-induced PAFR sequestration and folic acid incorporation.
Intrinsic folic acid receptors in the plasma membrane belong to the
family of glycosyl phosphatidylinositol-anchored proteins localized in
caveolae (47-50), and folic acid is known to be incorporated via the
caveolae (36). We used incorporation of folic acid as a control for a
clathrin-independent pathway. Each pretreatment inhibited PAF-induced
sequestration but not folic acid incorporation (Table I). In contrast,
PMA treatment, which inhibits folic acid incorporation by inactivating
caveolar internalization (33, 34), did not affect PAFR sequestration (Fig. 3C and Table I). Thus, PAFR sequestration seems to be
mediated by clathrin rather than by caveolae.
Resensitization of the PAF Receptor--
Recently, Krueger
et al. (3) reported that phosphorylated
2ARs
are dephosphorylated by some GPCR-specific protein Ser/Thr phosphatases
in acidic environments like the endosomal compartments, events which
lead to receptor recycling and resensitization. As shown in Fig. 5,
A and B, the recycled receptors regained the potential to respond efficiently to PAF. In contrast,
sequestration-defective CHO-HA-Tail-1 cells (Fig. 3B) showed
desensitization but not resensitization of the IP3 response
to PAF (Fig. 5C), suggesting that sequestration/recycling may play a role in the resensitization.
Role of Receptor Cytoplasmic Tail Phosphorylation--
In the
cytoplasmic domain of guinea pig PAFR, there are 14 Ser/Thr residues in
intracellular portions (25). Among them, nine cluster in the
cytoplasmic tail. We reported that a synthetic 18-amino acid peptide
corresponding to the cytoplasmic tail of guinea pig PAFR containing the
six Ser/Thr residues (Ser318-Thr335) was
phosphorylated in vitro by recombinant GRK2 (18). Moreover, other investigators reported that PAF induced the translocation of
transfected GRK2 from the cytosol to the cell membrane in human leukocytes (19). In the present study, we used cytoplasmic tail mutant
PAFRs (HA-Del, HA-Tail-1, and HA-Tail-2) to examine the role of
receptor cytoplasmic tail phosphorylation.
PAF as well as PMA and Bt2-cAMP induced phosphorylation of
the wild-type receptor but not those of the mutants. Guinea pig PAFR
possesses only two consensus motifs (RX(S/T)) for
phosphorylation by cAMP-dependent protein kinase (51) in
the intracellular domain, which may reflect the low extent of
phosphorylation by Bt2-cAMP. Most of the phosphorylation
sites related to these stimuli existed in the cytoplasmic tail Ser/Thr
residues (Fig. 3A). Both PMA and Bt2-cAMP failed
to induce PAFR sequestration (Fig. 3C). All cytoplasmic tail
mutations created in this study more or less impaired the ability of
the receptor to undergo sequestration (Fig. 3B). We speculate that agonist-induced phosphorylation of the receptor cytoplasmic tail by some GRKs rather than protein kinase C or cAMP-dependent protein kinase may facilitate but not be
essential for PAFR sequestration. The phosphorylated Ser/Thr residues
should vary between different kinases. Ferguson et al. (6)
reported that a sequestration-defective mutant of the
2AR was rescued in sequestration by GRK2 overexpression.
In that case, receptor phosphorylation by GRK2 facilitated
sequestration of the mutant
2AR. The HA-Tail-1 receptor
were not sequestered after PAF stimulation (Fig. 3B) even
though it did transmit signals (Fig. 5C), a result which
agrees well with a report that human PAFR with a cytoplasmic tail
deletion (Cys317
termination) did not show PAF-induced
sequestration (24). As described for other GPCRs (52), PAFR
sequestration may be independent from signal transduction.
Phosphorylation of six Ser/Thr residues following Cys317
may play a role to facilitate sequestration. Additional substitution of
three Ser residues in the cytoplasmic tail (in HA-Tail-2 receptor) or a
large deletion of the cytoplasmic tail (in HA-Del receptor) rescued
sequestration activity, indicating that there may be both positive and
negative structural determinants regulating sequestration in the
cytoplasmic tail, as described for other GPCRs (53). Finally, at least
in the PAFR, it seems that the receptor
phosphorylation/dephosphorylation cycle is not the sole or critical
determinant governing receptor sequestration/recycling/resensitization
processes, because HA-Tail-2 receptor, which lacked all phosphorylation
targets in its cytoplasmic tail, still underwent all these processes
(Fig. 3B and Fig. 5D).
Conclusion--
In this paper, we extensively examined the
dynamics (sequestration, recycling, and resensitization) of the PAFR.
We found that phosphorylation facilitates but is not essential for
receptor sequestration and that receptor sequestration/recycling is
important in resensitization. For a better understanding of the
molecular mechanisms that govern GPCR desensitization and dynamic
redistribution, more information is needed on GPCR protein modification
such as receptor dimerization and receptor palmitoylation in addition to current knowledge of receptor phosphorylation.
 |
ACKNOWLEDGEMENTS |
We thank Drs. K. Kameyama and H. Kurose (The
University of Tokyo) and members of our laboratories at The University
of Tokyo and RIKEN for valuable discussions. We also thank M. Ohara for comments on the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid from the
Ministry of Education, Science, Sports and Culture (Scientific Research on Priority Areas number 09273105) and the Ministry of Health and
Welfare of Japan, and by grants from Sankyo Foundation of Life
Science, Yamanouchi Foundation for Metabolic Disorders, Human Science Foundation, and Senri Life Science Foundation.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.
¶
Present address: Laboratory of Biomedical Genetics, Graduate
School of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo 113, Japan.
To whom correspondence should be addressed. Tel.:
81-3-5802-2925; Fax: 81-3-3813-8732; E-mail:
tshimizu{at}m.u-tokyo.ac.jp.
1
The abbreviations used are: GPCR, G
protein-coupled receptor; PAF, platelet-activating factor
(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine);
2AR,
2-adrenergic receptor; GRK, G
protein-coupled receptor kinase; PAFR, PAF receptor; CHO,
Chinese hamster ovary; mcPAF, 1-O-hexadecyl-2-(methylcarbamyl)-sn-glycero-3-phosphocholine; WT, wild-type; FBS, fetal bovine serum; IP3, inositol
1,4,5-triphosphate; PBS, Ca2+,Mg2+-free
Dulbecco's phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; Bt2-cAMP, dibutyryl cAMP; HA,
hemagglutinin.
 |
REFERENCES |
-
Yu, S. S.,
Lefkowitz, R. J.,
and Hausdorff, W. P.
(1993)
J. Biol. Chem.
268,
337-341[Abstract/Free Full Text]
-
Pippig, S.,
Andexinger, S.,
and Lohse, M. J.
(1995)
Mol. Pharmacol.
47,
666-676[Abstract]
-
Krueger, K. M.,
Daaka, Y.,
Pitcher, J. A.,
and Lefkowitz, R. J.
(1997)
J. Biol. Chem.
272,
5-8[Abstract/Free Full Text]
-
von Zastrow, M.,
and Kobilka, B. K.
(1992)
J. Biol. Chem.
267,
3530-3538[Abstract/Free Full Text]
-
von Zastrow, M.,
and Kobilka, B. K.
(1994)
J. Biol. Chem.
269,
18448-18452[Abstract/Free Full Text]
-
Ferguson, S. S. G.,
Menard, L.,
Barak, L. S.,
Koch, W. J.,
Colapietro, A.-M.,
and Caron, M. G.
(1995)
J. Biol. Chem.
270,
24782-24789[Abstract/Free Full Text]
-
Hausdorff, W. P.,
Caron, M. G.,
and Lefkowitz, R. J.
(1990)
FASEB J.
4,
2881-2889[Abstract]
-
Pippig, S.,
Andexinger, S.,
Daniel, K.,
Puzicha, M.,
Caron, M. G.,
Lefkowitz, R. J.,
and Lohse, M. J.
(1993)
J. Biol. Chem.
268,
3201-3208[Abstract/Free Full Text]
-
Goodman, O. B.,
Krupnick, J. G.,
Santini, F.,
Gurevich, V. V.,
Penn, R. B.,
Gagnon, A. W.,
Keen, J. H.,
and Benovic, J. L.
(1996)
Nature
383,
447-450[CrossRef][Medline]
[Order article via Infotrieve]
-
Zhang, J.,
Ferguson, S. G.,
Barak, L. S.,
Menard, L.,
and Caron, M. G.
(1996)
J. Biol. Chem.
271,
18302-18305[Abstract/Free Full Text]
-
Gurevich, V. V.,
Dion, S. B.,
Onorato, J. J.,
Ptasienski, J.,
Kim, C. M.,
Sterne-Marr, R.,
Hosey, M. M.,
and Benovic, J. L.
(1995)
J. Biol. Chem.
270,
720-731[Abstract/Free Full Text]
-
Ferguson, S. S. G.,
Downey, W. E.,
Colapietro, A. M.,
Barak, L. S.,
Menard, L.,
and Caron, M. G.
(1996)
Science
271,
363-366[Abstract]
-
Ruiz-Gomez, A.,
and Mayor, F., Jr.
(1997)
J. Biol. Chem.
272,
9601-9604[Abstract/Free Full Text]
-
Hanahan, D. J.
(1986)
Annu. Rev. Biochem.
55,
483-509[CrossRef][Medline]
[Order article via Infotrieve]
-
Izumi, T.,
and Shimizu, T.
(1995)
Biochim. Biophys. Acta
1259,
317-333[Medline]
[Order article via Infotrieve]
-
Morrison, W. J.,
Dhar, A.,
and Shukla, S. D.
(1989)
Life Sci.
45,
333-339[Medline]
[Order article via Infotrieve]
-
Nakamura, M.,
Honda, Z.,
Izumi, T.,
Sakanaka, C.,
Mutoh, H.,
Minami, M.,
Bito, H.,
Seyama, Y.,
Matsumoto, T.,
Noma, M.,
and Shimizu, T.
(1991)
J. Biol. Chem.
266,
20400-20405[Abstract/Free Full Text]
-
Takano, T.,
Honda, Z.,
Sakanaka, C.,
Izumi, T.,
Kameyama, K.,
Haga, K.,
Haga, T.,
Kurokawa, K.,
and Shimizu, T.
(1994)
J. Biol. Chem.
269,
22453-22458[Abstract/Free Full Text]
-
Chuang, T. T.,
Sallese, M.,
Ambrosini, G.,
Parruti, G.,
and De Blasi, A.
(1992)
J. Biol. Chem.
267,
6886-6892[Abstract/Free Full Text]
-
Ali, H.,
Richardson, R. M.,
Tomhave, E. D.,
DuBose, R. A.,
Haribabu, B.,
and Snyderman, R.
(1994)
J. Biol. Chem.
269,
24557-23563[Abstract/Free Full Text]
-
Richardson, R. M.,
Haribabu, B.,
Ali, H.,
and Snyderman, R.
(1996)
J. Biol. Chem.
271,
28717-28724[Abstract/Free Full Text]
-
Ali, H.,
Fisher, I.,
Haribabu, B.,
Richardson, R. M.,
and Snyderman, R.
(1997)
J. Biol. Chem.
272,
11706-11709[Abstract/Free Full Text]
-
Gerard, N. P.,
and Gerard, C.
(1994)
J. Immunol.
152,
793-800[Abstract/Free Full Text]
-
Le Gouill, C.,
Parent, J.-L.,
Rola-Pleszczynski, M.,
and Stankova, J.
(1997)
J. Biol. Chem.
272,
21289-21295[Abstract/Free Full Text]
-
Honda, Z.,
Nakamura, M.,
Miki, I.,
Minami, M.,
Watanabe, T.,
Seyama, Y.,
Okado, H.,
Toh, H.,
Ito, K.,
Miyamoto, T.,
and Shimizu, T.
(1991)
Nature
349,
342-346[CrossRef][Medline]
[Order article via Infotrieve]
-
Ishii, I.,
Izumi, T.,
Tsukamoto, H.,
Umeyama, H.,
Ui, M.,
and Shimizu, T.
(1997)
J. Biol. Chem.
272,
7846-7854[Abstract/Free Full Text]
-
Trapaidze, N.,
Keith, D. E.,
Cvejic, S.,
Evans, C. J.,
and Devi, L. A.
(1996)
J. Biol. Chem.
271,
29279-29285[Abstract/Free Full Text]
-
Kamen, B. A.,
and Capdevila, A.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
5983-5987[Abstract]
-
Kamen, B. A.,
Wang, M. T.,
Streckfuss, A. J.,
Peryea, X.,
and Anderson, R. G. W.
(1988)
J. Biol. Chem.
263,
13602-13609[Abstract/Free Full Text]
-
Hansen, S. H.,
Sandvig, K.,
and van Deurs, B.
(1993)
J. Cell Biol.
121,
61-72[Abstract]
-
Ali, H.,
Richardson, R. M.,
Tomhave, E. D.,
Didsbury, J. R.,
and Snyderman, R.
(1993)
J. Biol. Chem.
268,
24247-24254[Abstract/Free Full Text]
-
Anderson, R. G. W.,
Kamen, B. A.,
Rothberg, K. G.,
and Lacey, S. W.
(1992)
Science
255,
410-411[Medline]
[Order article via Infotrieve]
-
Smart, E. J.,
Foster, D. C.,
Ying, Y.-S.,
Kamen, B. A.,
and Anderson, R. G. W.
(1994)
J. Cell Biol.
124,
307-313[Abstract]
-
Smart, E. J.,
Ying, Y.-S.,
and Anderson, R. G. W.
(1995)
J. Cell Biol.
131,
929-938[Abstract]
-
Stafforini, D. M.,
McIntyre, T.,
Zimmerman, G. A.,
and Prescott, S. M.
(1997)
J. Biol. Chem.
272,
17895-17898[Free Full Text]
-
Hurtley, S. M.
(1991)
Trends Biochem. Sci.
16,
165-166[Medline]
[Order article via Infotrieve]
-
Robinson, M. S.,
Watts, C.,
and Zerial, M.
(1996)
Cell
84,
13-21[Medline]
[Order article via Infotrieve]
-
Raposo, G.,
Dunia, I.,
Delavier-Klutchko, C.,
Kaveri, S.,
Strosberg, A. D.,
and Benedetti, E. L.
(1989)
Eur. J. Cell Biol.
50,
340-352[Medline]
[Order article via Infotrieve]
-
Dupree, P.,
Parton, R. G.,
Raposo, G.,
Kurzchalia, V.,
and ans Simons, K.
(1993)
EMBO J.
12,
1597-1605[Abstract]
-
Raposo, G.,
Dunia, I.,
Marullo, S.,
Andre, C.,
Guillet, J. G.,
Strosberg, A. D.,
Benedetti, E. L.,
and Hoebeke, J.
(1987)
Biol. Cell
60,
117-123[Medline]
[Order article via Infotrieve]
-
Tolbert, L. M.,
and Lameh, J.
(1996)
J. Biol. Chem.
271,
17335-17342[Abstract/Free Full Text]
-
Chun, M.,
Liyanage, U. K.,
Lisanti, M. P.,
and Lodish, H. F.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11728-11732[Abstract/Free Full Text]
-
Lisanti, M. P.,
Scherer, P. E.,
Vidugiriene, J.,
Tang, Z.-L.,
Hermanoski-Vosatka, A.,
Tu, Y.-H.,
Cook, R. F.,
and Sargiacomo, M.
(1994)
J. Cell Biol.
126,
111-126[Abstract]
-
Chang, W. J.,
Ying, Y.,
Rothberg, K.,
Hooper, N.,
Turner, A.,
Gambliel, H.,
De Gunzburg, J.,
Mumby, S.,
Gilman, A.,
and Anderson, R. G. W.
(1994)
J. Cell Biol.
126,
127-138[Abstract]
-
Li, S.,
Okamoto, T.,
Chun, M.,
Sargiacomo, M.,
Casanova, J. E.,
Hansen, S. H.,
Nishimoto, I.,
and Lisanti, M. P.
(1995)
J. Biol. Chem.
270,
15693-15701[Abstract/Free Full Text]
-
Sandvig, K.,
Olsnes, S.,
Petersen, O. W.,
and van Deurs, B.
(1987)
J. Cell Biol.
105,
679-689[Abstract]
-
Sadasivan, E.,
and Rothenberg, S. P.
(1989)
J. Biol. Chem.
264,
5806-5811[Abstract/Free Full Text]
-
Ratnam, M.,
Marquardt, H.,
Duhring, J. L.,
and Freisheim, J. H.
(1989)
Biochemistry
28,
8249-8254[Medline]
[Order article via Infotrieve]
-
Elwood, P. C.
(1989)
J. Biol. Chem.
264,
14893-14901[Abstract/Free Full Text]
-
Luhrs, C. A.,
and Slomiany, B. L.
(1989)
J. Biol. Chem.
264,
21446-21449[Abstract/Free Full Text]
-
Kennelly, P. J.,
and Krebs, E. G.
(1991)
J. Biol. Chem.
266,
15555-15558[Free Full Text]
-
Hunyady, L.,
Baukal, A. J.,
Balla, T.,
and Catt, K. J.
(1994)
J. Biol. Chem.
269,
24798-24804[Abstract/Free Full Text]
-
Huang, Z.,
Chen, Y.,
and Nissenson, R. A.
(1995)
J. Biol. Chem.
270,
151-156[Abstract/Free Full Text]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.