From the Departments of Surgery and
Physiology, University of California,
San Francisco, California 94143-0660 and the ¶ CURE
Digestive Diseases Research Center, West Los Angeles Veterans Affairs
Medical Center, and Department of Medicine, UCLA Medical School,
Los Angeles, California 90073-1792
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ABSTRACT |
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Agonist-induced redistribution of
G-protein-coupled receptors (GPCRs) and Biological responses to agonists of G-protein-coupled receptors
(GPCRs)1 are attenuated by
receptor phosphorylation and uncoupling from heterotrimeric G-proteins
and receptor endocytosis (1). G-protein-coupled receptor kinases (GRKs)
and Alterations in the subcellular distribution of GPCRs, GRKs, and
The purpose of the present investigation was to examine substance P
(SP)-induced trafficking of In the present study, we used green fluorescent protein (GFP) and
specific antibodies to localize NK1R, Reagents--
Enhanced GFP (EGFP) expression vector pEGFP-N1 and
JM109 bacteria were from CLONTECH (Palo Alto, CA).
Restriction enzymes, T4 ligase, Lipofectin, and enzyme-free cell
dissociation buffer were from Life Technology, Inc., or New England
Biolabs, Inc. (Beverly, MA). Pfu DNA polymerase was from
Stratagene (La Jolla, CA). A QiaEx extraction kit was from Qiagen
(Hilden, Germany). G418 was from Gemini Bio-Products, Inc. (Calabasas,
CA). Kanamycin and protease inhibitor mixture were from Calbiochem.
Glutathione-agarose was from Amersham Pharmacia Biotech. The sources of
other reagents have been described (24, 32, 37).
Antibodies--
The sources of primary antibodies are shown in
Table I. An antibody to GFP was raised in
rabbits to a GST-GFP fusion protein. A polymerase chain reaction
fragment of cDNA encoding enhanced GFP (EGFP) was cloned into the
HindIII/XbaI restriction sites of pGEX-2T. The
resultant plasmid, pGEXGFP, was transfected into Escherichia
coli strain BL21DE3. The GST-GFP protein was purified using
glutathione-agarose. An emulsion was prepared from equal parts of GFP
and complete Freund's adjuvant. Two New Zealand rabbits (female, 8 weeks) were immunized at intervals of 6-8 weeks. At each immunization,
both rabbits received 2.0 ml of emulsion containing approximately 1 mg
of GFP, divided into 8-10 intradermal sites. At the first
immunization, 0.5 ml of Tri-immunol vaccine was injected intramuscularly. Antibody 9708 had a titer of 1:100,000 by
enzyme-linked immunosorbent assay and was used in all experiments. Goat
anti-mouse IgG coupled with (R)-phycoerythrin was from
Caltag Laboratories (Burlingame, CA). The sources of other secondary
antibodies have been described (37).
Generation of NK1R-GFP, ARR-GFP Constructs--
Constructs with
EGFP at the C terminus of rat NK1R, Transfection and Cell Culture--
The generation and
characterization of KNRK cells stably expressing rat NK1R (KNRK-NK1R
cells) with N-terminal Flag (DYKDDDDK) epitope has been described (38).
The Flag epitope does not affect binding, signaling, or trafficking of
the NK1R. To obtain a cell line expressing NK1R-GFP, KNRK cells were
transfected with cDNA encoding NK1-GFP, and colonies were screened
for expression of GFP by fluorescence microscopy and flow cytometry
(38). To generate a cell line expressing FlagNK1R plus ARR-GFP,
KNRK-NK1R cells (expressing FlagNK1R) were transfected with cDNA
encoding ARR-GFP. To verify coexpression of FlagNK1R plus ARR-GFP,
clones were screened by immunofluorescence microscopy and flow
cytometry to detect NK1R and Flow Cytometry--
Flow cytometry was used to assess expression
of constructs and to enrich populations of cells. The GFP signal was
used to detect NK1R-GFP. In cells coexpressing FlagNK1R and ARR-GFP,
NK1R was detected using an M2 antibody to the extracellular Flag
epitope and a secondary antibody coupled with
(R)-phycoerythrin, and the GFP signal was used to detect
ARR-GFP. Cells were dissociated in enzyme-free buffer, and 1.5 × 106 cells were resuspended in 200 µl of Iscove's medium
containing 1 mg/ml bovine serum albumin (BSA) and 1 mM
CaCl2. Cells were incubated with the M2 antibody (3 µg/ml) for 2 h at 4 °C, washed, and incubated with goat
anti-mouse antibody coupled to phycoerythrin (2 µg/ml) for 2 h
at 4 °C. Cells were resuspended in 1 ml of DMEM with 5% enzyme-free
cell dissociation buffer, 0.3% fetal bovine serum, and 2 µg/ml
propidium iodide and analyzed with a Facscan Flow Cytometer (Becton
Dickinson). Fluorophores were excited at 488 nm, and emission was
collected at 530/30 nm for GFP and 575/25 nm for phycoerythrin.
Viability was assessed by exclusion of propidium iodide.
Western Blotting--
Western blotting was used to confirm
expression of GFP-tagged proteins, determine whether Measurement of [Ca2+]i--
To examine
signaling by cells expressing GFP-tagged proteins,
[Ca2+]i was measured with Fura-2/AM (24).
Fluorescence was measured at 340 and 380 nm excitation and 510 nm
emission, and results were expressed as the ratio of the fluorescence
at the two excitation wavelengths, which is proportional to the
[Ca2+]i. To generate concentration-response
curves, cells were exposed once to varying concentrations of SP. To
examine desensitization, cells were exposed to SP, washed, and then
re-exposed to SP. All observations were in n > 3 experiments.
Endocytosis of 125I-SP--
The rate of NK1R
endocytosis was quantified with 125I-SP as described (32).
Cells were incubated in Hanks' balanced salt solution containing 50 pM 125I-SP, 0.1% BSA, 0.2 mg/ml bacitracin, 20 µg/ml leupeptin, and 20 µg/ml chymostatin for 60 min at 4 °C.
They were washed and incubated at 37 °C for 0-30 min. Cells were
washed with ice-cold PBS and incubated in 250 µl of ice-cold 0.2 M acetic acid containing 50 mM NaCl, pH 2.5, on
ice for 5 min to separate acid-labile (cell surface) from
acid-resistant (internalized) label. Nonspecific binding was measured
in the presence of 1 µM SP and was subtracted to give
specific binding. Observations were in triplicate in n > 3 experiments.
Microscopy and Immunofluorescence--
To examine trafficking of
GFP-labeled proteins in real time, cells were maintained at 37 °C in
DMEM containing 0.1% BSA (DMEM/BSA). Cells were observed before and
after addition of 10 nM SP. To detect proteins by
immunofluorescence, cells were incubated in DMEM/BSA containing 10 nM SP, 100 nM cyanine-3-conjugated SP (Cy3-SP), or no addition (control) for 60 min at 4 °C (for equilibrium
binding), washed at 4 °C, and either fixed immediately or incubated
in SP-free medium at 37 °C for 1 min to 8 h (for trafficking to
proceed). Cells were fixed with 4% paraformaldehyde in 100 mM PBS, pH 7.4, for 20 min at 4 °C. NK1R, Generation of Cell Lines Stably Expressing NK1R-GFP and NK1R + ARR-GFP
KNRK cells were stably transfected with cDNA encoding NK1R-GFP
(KNRK-NK1R-GFP cells). Clone 3W was used for all experiments because a
large proportion of cells expressed NK1R-GFP at a high and uniform
level, assessed by flow cytometry (Fig.
1A) and microscopy (Fig.
1C). To generate cells coexpressing FlagNK1R and ARR-GFP (KNRK-NK1R + ARR-GFP cells), KNRK-NK1R cells were transfected with
cDNA encoding ARR-GFP. Clone 23 was selected because a large proportion of cells coexpressed both NK1R and ARR-GFP at high and
uniform levels, assessed by flow cytometry (Fig. 1A) and
microscopy (Fig. 5). Cells were studied at <passage 15 when they
retained NK1R-GFP and NK1R plus ARR-GFP.
-arrestins determines the
subsequent cellular responsiveness to agonists and is important for
signal transduction. We examined substance P (SP)-induced trafficking
of
-arrestin1 and the neurokinin-1 receptor (NK1R) in KNRK cells in
real time using green fluorescent protein. Green fluorescent protein
did not alter function or localization of the NK1R or
-arrestin1. SP
induced (a) striking and rapid (<1 min) translocation of
-arrestin1 from the cytosol to the plasma membrane, which preceded
NK1R endocytosis; (b) redistribution of the NK1R and
-arrestin1 into the same endosomes containing SP and the transferrin
receptor (2-10 min); (c) prolonged colocalization of the
NK1R and
-arrestin1 in endosomes (>60 min); (d) gradual resumption of the steady state distribution of the NK1R at the plasma
membrane and
-arrestin1 in the cytosol (4-6 h). SP stimulated a
similar redistribution of immunoreactive
-arrestin1 and
-arrestin2. In contrast, SP did not affect G
q/11
distribution, which remained at the plasma membrane. Expression of the
dominant negative
-arrestin319-418 inhibited SP-induced
endocytosis of the NK1R. Thus, SP induces rapid translocation of
-arrestins to the plasma membrane, where they participate in NK1R
endocytosis.
-Arrestins colocalize with the NK1R in endosomes until
the NK1R recycles and
-arrestins return to the cytosol.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestins participate in uncoupling and endocytosis of many
GPCRs. For example, GRK2/3 phosphorylate the
2-adrenergic receptor (
2AR) (2, 3), and
-arrestin1/2 interact with GRK-phosphorylated
2ARs to
uncouple them from heterotrimeric G-proteins and terminate signal
transduction (4-6).
-Arrestins also serve as adaptor proteins for
clathrin-mediated endocytosis of the
2AR since
-arrestins interact with clathrin with high affinity (7), and
overexpression of dominant negative mutants inhibits receptor
endocytosis (8-10). GRK-mediated phosphorylation is also necessary for
endocytosis of certain GPCRs, presumably by enhancing their
interactions with
-arrestins (11-13). However, expression of
dominant negative
-arrestin mutants has no effect on endocytosis of
the angiotensin II type 1A receptor or the m1, m3, and m4 muscarinic
cholinergic receptors (9, 14). Thus,
-arrestins do not mediate
endocytosis of all GPCRs.
-arrestins determine cellular responsiveness to agonists and may be
important for signal transduction. Receptor endocytosis contributes to
desensitization by depleting the cell surface of receptors available to
interact with agonists in the extracellular fluid (1), and endocytosis
of
2ARs is required for stimulation of mitogen-activated
protein kinases (15). Agonists of several GPCRs induce translocation of
GRK2/3 and
-arrestins from the cytosol to the plasma membrane where
they interact with cell-surface receptors to participate in uncoupling
and endocytosis (16-18). However, the duration of these interactions
and the fate of GRK2/3 and
-arrestins during endocytosis and
recycling of GPCRs have not been examined. These are important issues
because resensitization requires endocytosis and processing of
receptors, which may entail dissociation of ligand and
-arrestin,
receptor dephosphorylation, and recycling (19-25).
-arrestins and to determine the role of
-arrestins in endocytosis of the SP or neurokinin-1 receptor (NK1R).
Regulation of cellular responses to SP are of interest in view of the
important role of the NK1R in pain transmission in the spinal cord,
regulation of intestinal motility and secretion, mediation of
neurogenic inflammation, and in depression in humans (26, 27).
-Arrestins and GRKs may mediate NK1R uncoupling since GRK2/3
phosphorylate the NK1R in reconstituted systems and membrane assays
(28, 29), and disruption of
-arrestins abrogates NK1R
desensitization (30). The role of
-arrestins in SP-induced endocytosis of the NK1R has not been examined. We and others (23, 25,
31-33) have shown that SP induces endocytosis and recycling of the
NK1R. By using fluorescent SP to detect the NK1R in neurons, we showed
that SP causes translocation of
-arrestins to the plasma membrane
and to endosomes containing the NK1R (34). However, limitations of
using fluorescent SP include inability to detect receptors at later
times when SP is degraded (23) and difficulty in colocalization with
other proteins by immunofluorescence, since incompletely fixed peptides
are washed out during staining (35).
-arrestin1/2, and G
q/11, which couples to the NK1R (28, 36). This approach requires transfected cells but enables direct observation of
trafficking in real time for prolonged periods. We expressed dominant
negative
-arrestin to determine the role of
-arrestins in NK1R
endocytosis. Our aims were as follows: (a) generate cell
lines expressing NK1R-GFP or wild type NK1R plus
-arrestin1-GFP
(ARR-GFP) and investigate whether GFP affects the function of these
proteins; (b) examine SP-induced trafficking of NK1R-GFP and
ARR-GFP in real time and determine their precise subcellular
distribution for 0-6 h after stimulation with SP; (c)
determine if SP induces redistribution of endogenous
-arrestin1,
-arrestin2, and G
q/11 in cells expressing wild type
NK1R; and (d) examine the effect of a dominant
negative mutant of
-arrestins on SP-induced endocytosis of the
NK1R.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Sources and dilutions of primary antibodies used for immunofluorescence
-arrestin1, and dominant
negative rat
-arrestin1319-418 were generated by
polymerase chain reaction using the following primers and
Pfu DNA polymerase. For NK1R, the forward primer was 5'-GGCCGGGAATTCGCCACCATGGATAACGTCCTTCCTATG-3'
(EcoRI site underlined, followed by the Kozak translation
initiation site, and the N terminus of NK1R cDNA in
bold), and the reverse primer was
5'-GGCCGGGGATCCCCGGCCAGCATGTTAGAGTAGAA-3'
(BamHI site underlined, C terminus of the NK1R cDNA in
bold). A 1253-base pair fragment amplified from rat NK1R cDNA was
separated on an agarose gel and purified using QiaEx extraction kit.
For
-arrestin1, the forward primer was
5'-GGCCGGAAGCTTGCCACCATGGGCGACAAAGGGACACGA-3' (HindIII restriction site underlined, N terminus of
-arrestin1 cDNA in bold), and the reverse primer was
5'-GGCCGGCCGCGGTCTGTTGTTGAGGTGTGGAGA-3' (SacII site underlined, C terminus of
-arrestin1 in
bold). A 1284-base pair fragment amplified from rat
-arrestin1 cDNA (Dr. R. Lefkowitz, Duke University) was
purified. A fragment of bovine
-arrestin1319-418 acts
as a dominant negative mutant (10). A construct of rat
-arrestin1319-418 was generated using the forward
primer
5'-GGCCGGAAGCTTGCCACCATGGTTTCCTACAAAGTCAAAGTG-3' (HindIII site underlined, sequence corresponding to rat
-arrestin1319-325 in bold), and the same reverse primer
as for wild type
-arrestin1. All constructs were ligated into
pEGFP-N1 vector and used to transform JM109 E. coli in Luria
broth containing 30 µg/ml kanamycin. The sequences of the chimeras
were verified using a reverse primer (5'-CGTCGCCGTCCAGCTCGACCAG-3')
corresponding to the 46-67 region of the GFP.
-arrestin (below). Highly expressing
clones were further enriched by fluorescence-activated cell sorting.
Cells were maintained and prepared for experiments as described (23,
32, 38). To express GFP constructs transiently, KNRK-NK1R cells (stably expressing FlagNK1R) were transfected by overnight incubation with 5 µg/ml cDNA encoding control vector pEGFP-N1, ARR-GFP, or ARR319-418-GFP and Lipofectin. Cells were plated on glass
coverslips 48-72 h before experiments.
-arrestin1 and
-arrestin2 are expressed in KNRK cells, and to assess specificity of
the antibodies (37). Cells (~107) were pelleted and lysed
in 1 ml of Laemmli or RIPA buffer containing a protease inhibitor
mixture. Lysates were fractionated by SDS-PAGE on 4-15 or 12%
polyacrylamide gels and transferred to nitrocellulose. Membranes were
incubated in 3% non-fat milk in PBS for 60 min or 3% BSA in PBS for
60 min and then with antibodies to GFP (1:10,000 overnight, 4 °C),
-arrestin1 (1:250, 1 h, room temperature),
-arrestin2
(1:10,000, 1 h, room temperature),
-arrestin1 + 2 (1:10,000,
1 h, room temperature), or G
q/11 (0.5 µg/ml,
1 h, room temperature). Membranes were washed and incubated with
goat anti-rabbit or mouse IgG conjugated to horseradish peroxidase (1:5,000) for 60 min at room temperature. Proteins were detected as
described (37). In controls, primary antibodies were preincubated with
1-2 µg/ml antigens overnight at 4 °C.
-arrestin1,
-arrestin2,
-arrestin1 + 2, G
q/11, and markers of
endosomes and lysosomes were localized by immunofluorescence (23, 34).
In some experiments, cells were incubated with Cell Tracker CM-DiI to
identify the plasma membrane (37). All observations were in duplicate
in n > 3 experiments. Cells were observed by confocal
microscopy (25). For observation of live cells, care was taken to
minimize laser exposure by limiting the number of optical sections
(2-3 per time point) and the laser intensity (<3%, aperture of 3-4
mm), so as to maintain fluorescence intensity.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of NK1R-GFP and ARR-GFP in KNRK
cells. To generate cells expressing NK1R-GFP, KNRK cells were
transfected with NK1R-GFP (KNRK-NK1R-GFP cells). To generate cells
expressing both the FlagNK1R plus ARR-GFP, KNRK-NK1R cells were
transfected with cDNA encoding ARR-GFP (KNRK-NK1R + ARR-GFP cells). A, analysis by flow cytometry with the
GFP signal (vertical axis) to detect GFP-tagged proteins and
the phycoerythrin (PE) signal (horizontal axis)
to detect the FlagNK1R using a Flag M2 antibody and a
phycoerythrin-conjugated secondary antibody. Note the low
autofluorescence of untransfected KNRK cells and that a high proportion
of cells express NK1R-GFP and FlagNK1R plus ARR-GFP. B,
Western blots of untransfected KNRK cells, KNRK cells expressing the
GFP vector without insert (KNRK-GFP), KNRK-NK1R-GFP cells,
and KNRK-NK1R + ARR-GFP cells (4-15% SDS-PAGE, with 2 µl of cell
lysate/lane). In the left panel the blot was probed with the
GFP antibody. In the right panel the blot was probed with
the GFP antibody preabsorbed with 1 µg/ml GFP-GST that was used for
immunization. Note the specific detection of proteins of the predicted
masses in transfected cell lines. C, localization of GFP in
KNRK-NK1R-GFP cells (upper panels) and KNRK-NK1R + ARR-GFP
cells (lower panels). The left panels show the
GFP signal. The right panels show GFP immunoreactivity that
was detected in the same cells using the GFP antibody and a secondary
antibody coupled to Texas Red. The NK1R was present at the plasma
membrane (arrowheads) and in some intracellular stores
(arrows). In contrast, -arrestin1 is in the cytosol
(arrows). Scale bar = 10 µm.
Western blotting verified expression of NK1R-GFP and ARR-GFP (Fig.
1B). In extracts of KNRK cells transfected with GFP vector without insert, the GFP antibody recognized a major protein with an
apparent molecular mass of ~27 kDa, corresponding to the predicted size of GFP. A second minor protein was also detected, but its identity
is unknown. In extracts of KNRK-NK1R-GFP cells, the GFP antibody
recognized a broad band with an apparent molecular mass of ~90 kDa.
The molecular mass of the rat NK1R is predicted to be ~46 kDa, but
there are two potential N-linked glycosylation sites, and
the glycosylated receptor has a larger mass (38). The broad protein
band probably represents glycosylated NK1R-GFP. In extracts of
KNRK-NK1R + ARR-GFP cells, the GFP antibody recognized a major protein
with an apparent molecular mass of ~75 kDa. This protein corresponds
to the known mass of -arrestin1 (~48 kDa) plus GFP (~27 kDa).
The GFP antibody did not interact with proteins in extracts of
untransfected KNRK cells, and signals in the transfected cells were
abolished by preabsorption of the antibody with 1 µg/ml GFP-GST
fusion protein. These results confirm expression NK1R-GFP and ARR-GFP
of the predicted sizes.
To determine the subcellular distribution of NK1R-GFP and ARR-GFP in unstimulated cells, we localized these proteins simultaneously using the GFP signal and immunofluorescence with the GFP antibody and a secondary antibody coupled to Texas Red. In KNRK-NK1R-GFP cells, NK1R-GFP was principally localized to the plasma membrane and also detected in an intracellular compartment (Fig. 1C, upper panels). In KNRK-NK1R + ARR-GFP cells, ARR-GFP was uniformly distributed throughout the cytosol and was not found at the plasma membrane in vicinity of the NK1R (Fig. 1C, lower panels, arrows). Staining was similar by both methods, validating the specificity of the GFP antibody.
Functional Characterization of Cells Expressing NK1R-GFP and ARR-GFP
SP-induced Ca2+ Mobilization--
GFP is a compact
protein, but its molecular mass is almost half that of the NK1R and
-arrestin1. To determine whether GFP interferes with signal
transduction of the NK1R, we measured SP-induced Ca2+
mobilization. SP stimulated a prompt but transient increase in [Ca2+]i in both KNRK-NK1R-GFP cells and KNRK-NK1R + ARR-GFP cells with a similar potency and efficacy (EC50
~0.5 nM) (Fig. 2A). This potency is similar
to that we have previously reported in KNRK cells expressing wild type
NK1R and
-arrestins (~0.6 nM) (38). Thus, GFP does not
interfere with signal transduction of the NK1R. To determine if GFP
interferes with either the ability of the NK1R to undergo
desensitization or with the role of
-arrestin1 in desensitization,
we compared desensitization of SP-induced Ca2+ mobilization
in cells expressing NK1R-GFP or FlagNK1R plus ARR-GFP. Exposure of
KNRK-NK1R-GFP cells to 1 nM SP for 2 min caused a prompt
increase in [Ca2+]i, which rapidly declined to
basal values even in the continued presence of SP (Fig. 2B).
When cells were washed and exposed again to 1 nM SP 5 min
after the first challenge, the response was strongly desensitized.
Identical results were obtained in KNRK-NK1R + ARR-GFP cells (Fig.
2C). This desensitization is similar to that we have
reported in cells expressing wild type NK1R and
-arrestin (24).
Thus, GFP does not interfere with NK1R desensitization or with the
capacity of
-arrestin1 to participate in this process.
|
Internalization of 125I-SP--
To determine if
endocytosis of the NK1R was affected by GFP, and whether GFP altered
the ability of -arrestin1 to participate in endocytosis, we
quantified internalization of 125I-SP in cells expressing
NK1R-GFP or FlagNK1R plus ARR-GFP. In KNRK-NK1R-GFP cells, 93.6 ± 0.4% of specifically bound SP was at the surface and 5.0 ± 0.1%
was internalized after 60 min at 4 °C (Fig. 2D). Warming
to 37 °C resulted in a rapid decline in surface label and a
concomitant increase in internalized label, which was almost maximal
after 20 min (18.9 ± 5.9% surface, 79.1 ± 5.3%
internalized). Similar results were obtained using KNRK-NK1R + ARR-GFP
cells. At 4 °C, 96.7 ± 0.6% of specifically bound SP was at
the surface and 5.3 ± 0.8% was internalized (Fig.
2D). After 20 min at 37 °C, 19.0 ± 4.5% of
specifically bound SP was at the surface and 79.4 ± 5.0% was
internalized. These rates of internalization are similar to those
observed in KNRK cells expressing wild type NK1R and
-arrestins
(32). Thus, GFP does not interfere with endocytosis of the NK1R or with
the capacity of
-arrestin1 to participate in this process. Total
specific binding at 4 °C was similar in both cell lines
(KNRK-NK1R-GFP, 16.7 ± 5.4% total counts; KNRK-NK1R + ARR-GFP,
16.7 ± 5.6% total counts), which suggests that these cell lines
expressed similar numbers of NK1Rs.
SP-induced Trafficking of NK1R-GFP and ARR-GFP in Real Time
The effects of SP on the subcellular distribution of the NK1R and
-arrestins have not been examined in real time. Therefore, we
directly observed SP-induced trafficking of NK1R-GFP in KNRK-NK1R-GFP cells and ARR-GFP in KNRK-NK1R + ARR-GFP cells over time.
NK1R-GFP--
Before addition of SP, NK1R-GFP was principally
localized at the plasma membrane (Fig.
3A). After 30 s
incubation with 10 nM SP, NK1R-GFP was still mostly
detected at the plasma membrane (Fig. 3B). After 10 min,
NK1R-GFP was present in numerous endosomes localized beneath the plasma
membrane and in a perinuclear location (Fig. 3C, arrows),
and the intensity of the signal at the cell surface was diminished
(Fig. 3C, arrowheads). After 60 min, NK1R-GFP was localized
in a prominent perinuclear pool of endosomes, and there was diminished
labeling of the plasma membrane (Fig. 3D, arrows). These
results show that SP binds to NK1R-GFP in live cells and rapidly
induces receptor internalization to superficial and then perinuclear
endosomes.
|
ARR-GFP-- Before addition of SP, ARR-GFP was uniformly distributed throughout the cytosol (Fig. 3E). Within 30 s of adding 10 nM SP, ARR-GFP was prominently detected at the plasma membrane, and the intensity of the cytosolic signal declined (Fig. 3F, arrowheads). After 10 min, ARR-GFP was detected in endosomes that were located immediately beneath the plasma membrane and in a perinuclear location (Fig. 3G, arrows), and the intensity of the signal at the plasma membrane was diminished. After 60 min, ARR-GFP was localized in a prominent perinuclear pool of endosomes (Fig. 3H). Thus, when SP binds to the NK1R at the plasma membrane, a large proportion of ARR-GFP rapidly translocates to the plasma membrane. Membrane translocation of ARR-GFP precedes endocytosis of the NK1R but is quickly followed by redistribution of ARR-GFP into endosomes.
Colocalization of Cy3-SP with ARR-GFP
SP induced translocation of NK1R-GFP and ARR-GFP into vesicles
that appeared to be similar in size, shape, and location. To determine
if these proteins were colocalized, we used Cy3-SP to localize the
NK1R. The advantage of using Cy3-SP to detect the NK1R is that it
interacts initially only with functional receptors at the plasma
membrane and, unlike a receptor antibody or a GFP tag, will not detect
the NK1R in the biosynthetic pathway. Therefore, signals are especially
clear. We have previously reported that Cy3-SP colocalizes with the
wild type NK1R in KNRK cells (23). To determine if Cy3-SP similarly
colocalizes with NK1R-GFP and whether GFP alters trafficking of the
NK1R, we examined SP-induced trafficking in KNRK-NK1R-GFP cells. When
incubated with cells at 4 °C, Cy3-SP colocalized with NK1R-GFP at
the cell surface (not shown). After 1-10 min at 37 °C, Cy3-SP and
NK1R colocalized in endosomes (not shown). We have previously shown
that after internalization the NK1R recycles and SP is degraded in KNRK
cells (23). Thus, Cy3-SP can be used to localize the NK1R during early stages of endocytosis. To determine if the NK1R colocalizes with ARR-GFP, we examined SP-induced trafficking in KNRK-NK1R + ARR-GFP cells. After incubation with 100 nM Cy3-SP at 4 °C,
ARR-GFP (Fig. 4A) and Cy3-SP
(Fig. 4B) was colocalized at the plasma membrane (Fig.
4C). After 1-10 min at 37 °C, ARR-GFP and Cy3-SP were
colocalized in the same superficial and perinuclear vesicles (Fig. 4,
D-F). Because Cy3-SP colocalizes with the NK1R
at the plasma membrane and in endosomes, these results indicate that
the NK1R colocalizes with -arrestins during the early stages of
endocytosis.
|
Time Course of Colocalization of ARR-GFP with Immunoreactive NK1R
SP is degraded in lysosomes after endocytosis, and therefore
Cy3-SP cannot be used to localize the NK1R during later stages of
receptor trafficking. To permit simultaneous localization of the NK1R
and -arrestin1 in KNRK-NK1R + ARR-GFP cells for prolonged periods,
we localized the NK1R using a primary antibody to the C-tail and a
Texas Red-conjugated secondary antibody, and we localized
-arrestin1
using GFP. In the absence of SP, NK1R was mainly localized to the
plasma membrane (Fig. 5A,
arrowheads), and ARR-GFP was distributed throughout the cytosol
(Fig. 5B, arrows), with minimal colocalization (Fig.
5C). When cells were incubated with 10 nM SP for
60 min at 4 °C, NK1R was principally localized to the cell surface
(Fig. 5D), and ARR-GFP was redistributed from the cytosol to
the plasma membrane (Fig. 5E), where it colocalized with the NK1R (Fig. 5F). After 5 min at 37 °C, NK1R was detected
in numerous superficial vesicles, and there was diminished NK1R
immunoreactivity at the cell surface (Fig. 5G). ARR-GFP was
no longer detected at the plasma membrane but was observed in vesicles
of the same size, shape, and location as those containing NK1R, as
indicated by superimposition of confocal images (Fig. 5, H
and I). After 30 min, NK1R was localized to a prominent
perinuclear pool of large vesicles (Fig. 5J). ARR-GFP was
colocalized with the NK1R in these centrally located vesicles (Fig. 5,
K and L). After 240 min, NK1R was prominently
detected at the cell surface (Fig. 5M, arrowhead) with diminished localization in vesicles (Fig.
5M, arrow). ARR-GFP was still present in some
centrally located vesicles that also contained the NK1R (Fig. 5,
N and O, arrows). NK1R-GFP and ARR-GFP resumed
their steady state distribution at the cell surface and in the cytosol,
respectively, 6 h after exposure to SP. Thus, SP induces
translocation of
-arrestin1 from the cytosol to the plasma membrane,
where it colocalizes with the NK1R, followed by endocytosis of the NK1R
and
-arrestin1 to the same vesicles. The NK1R and
-arrestin1
remain colocalized until the NK1R returns to the plasma membrane and
-arrestin1 resumes its cytosolic distribution.
|
Identification of Organelles Containing NK1R-GFP and ARR-GFP
To identify organelles containing NK1R-GFP and ARR-GFP, we stained
KNRK-NK1R-GFP cells and KNRK-NK1R + ARR-GFP cells with antibodies to
the transferrin receptor and to lysosomal-associated membrane protein-1
(LAMP-1). To prevent degradation of proteins in lysosomes and loss of
signal, cells were incubated with 10 mM NH4Cl
during the experiment (23). The transferrin receptor and LAMP-1 were
detected by immunofluorescence using a Texas Red-conjugated secondary
antibody. When cells were incubated with 10 nM SP for 60 min at 4 °C, washed, incubated at 37 °C for 30-60 min, ARR-GFP (Fig. 6, A-C) and NK1R-GFP
(not shown) were detected in vesicles in a central and peripheral
location that also contained the transferrin receptor. Thus,
these vesicles are early endosomes. In contrast, vesicles containing
ARR-GFP (Fig. 6, D-F) and NK1R-GFP (not shown) were distinct from lysosomes. We have previously shown that SP induces
redistribution of immunoreactive NK1R into early endosomes, which also
contain the transferrin receptor, but not to lysosomes (23). Even when
cells were incubated with 10 nM for 4 or 6 h at
37 °C, NK1R-GFP and ARR-GFP were principally detected in vesicles that were distinct from lysosomes (not shown). Thus, GFP does not alter
trafficking of the NK1R to endosomes, and SP induces translocation of
both ARR-GFP and NK1R-GFP to early endosomes but not lysosomes.
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SP-induced Trafficking of Immunoreactive NK1R, -Arrestins, and
G
q/11 in KNRK-NK1R Cells
SP-induced trafficking of the NK1R and -arrestin1 could be
affected by GFP or by overexpression of
-arrestin1. To determine whether SP causes a similar redistribution of endogenous
-arrestin1, we simultaneously localized
-arrestin1 with a monoclonal antibody and the NK1R with a polyclonal antibody in KNRK cells expressing FlagNK1R. To examine whether SP caused a similar redistribution of
endogenous
-arrestin2, we localized
-arrestin2 using a polyclonal antibody and the NK1R using a monoclonal M5 Flag antibody. We similarly
colocalized G
q/11 with a polyclonal antibody and NK1R with a monoclonal antibody to determine whether G
q/11
remains at the plasma membrane with the NK1R or is sorted to endosomes. Incubation of cells in SP-free medium at 4 °C and warming to
37 °C had no effect on the subcellular distributions of these proteins.
In cells that were incubated in SP-free medium at 4 °C and
immediately fixed, the NK1R was prominently localized to the plasma membrane, and -arrestin1 was cytosolic (Fig.
7A-C). After incubation with
10 nM SP at 4 °C, the NK1R was localized at the plasma
membrane, and
-arrestin1 was detected at the plasma membrane and
diffusely in the cytosol (Fig. 7, D-F). After
2-60 min at 37 °C,
-arrestin1 colocalized with the NK1R in
superficial and perinuclear endosomes (Fig. 7, G-I). SP had
a similar effect on the subcellular localization of
-arrestin2. In
the absence of SP,
-arrestin2 was principally cytoplasmic. SP
induced translocation of
-arrestin2 to the plasma membrane, where it
colocalized with the NK1R at 4 °C and then to endosomes containing
the NK1R at 37 °C (Fig. 7, J-L). Similar results were
obtained using a well characterized antibody that interacts with both
-arrestin1 + 2 (Fig. 7, M-O). The NK1R and
-arrestins resumed their steady state distribution 6 h after exposure to SP. Thus, SP induces a similar redistribution of endogenous
-arrestin1 and
-arrestin2 and GFP-tagged
-arrestin1.
|
In cells that were incubated with 10 nM SP at 4 °C,
Gq/11 was detected at the plasma membrane where it
colocalized with the NK1R (not shown). After 10 min at 37 °C,
G
q/11 remained at the plasma membrane, and the NK1R was
detected in endosomes (Fig. 8,
A and B). Thus, whereas SP induces membrane
translocation of
-arrestins followed by endocytosis of
-arrestins
and the NK1R into the same vesicles, SP has no effect on the
subcellular distribution of G
q/11.
|
Specificity of Antibodies--
We have previously shown that the
Flag antibody and the C-terminal NK1R antibody specifically interact
with the NK1R in KNRK-NK1R cells (38). Preincubation of antibodies to
Flag, NK1R, and Gq/11 with the peptides used for
immunization abolished the staining of cells (not shown), confirming
specificity. The fusion proteins used to generate antibodies to
-arrestins were not available for preabsorption controls. However,
these antibodies were affinity purified before use and have been
characterized (5). To investigate further antibody specificity, we
examined expression of
-arrestins and G
q/11 by
Western blotting. Antibodies to
-arrestin1 and
-arrestin2
detected single proteins of ~50 and 45 kDa, respectively (Fig.
9). The antibody to
-arrestin1 + 2 detected a broad band that appears to comprise two proteins of
~45-50 kDa. The antibody to G
q/11 detected a single
prominent band of ~42 kDa. Preincubation of G
q/11
antibody overnight at 4 °C with the peptide antigen diminished the
signal (not shown). Thus, the antibodies specifically interact with
proteins of the predicted sizes in KNRK cells. These results also
confirm expression of
-arrestin1 and
-arrestin2 by KNRK
cells.
|
The Role of -Arrestin1 in Endocytosis of the NK1R--
To
determine the role of
-arrestin1 in endocytosis of the NK1R,
KNRK-NK1R cells were transiently transfected with GFP vector (control),
ARR-GFP, or dominant negative ARR319-418-GFP (10). The
transient transfection permitted evaluation of both transfected and
non-transfected control cells in the same experiment.
In cells expressing vector without insert, GFP was uniformly
distributed throughout cells. Binding and endocytosis of Cy3-SP were
unaffected by expression of GFP (Fig.
10A). In cells transfected with wild type -arrestin1-GFP, ARR-GFP was uniformly distributed throughout the cytosol before exposure to Cy3-SP (not shown). After 60 min at 4 °C, Cy3-SP was detected at the plasma membrane of cells
expressing ARR-GFP and in non-transfected cells (Fig. 10B, upper
panel, arrowheads). This binding induced redistribution of ARR-GFP
from the cytosol to the plasma membrane. After 5-30 min at 37 °C,
Cy3-SP was detected in superficial and then perinuclear endosomes in
cells expressing ARR-GFP and in non-transfected cells (Fig.
10B, center and lower panels, arrows).
At all time points, Cy3-SP colocalized with ARR-GFP in endosomes. Thus,
expression of ARR-GFP does not affect binding or endocytosis of
Cy3-SP.
|
In cells transfected with dominant negative
-arrestin319-418GFP, ARR319-418-GFP
was detected in superficial and perinuclear vesicles before exposure to
Cy3-SP. This distribution was unaffected by Cy3-SP. At 4 °C, Cy3-SP
was detected at the plasma membrane of cells expressing ARR319-418-GFP and in non-transfected cells (Fig.
10C, upper panels, arrowheads). After 5-30 min at 37 °C,
Cy3-SP was prominently detected at the plasma membrane in cells
expressing ARR319-418-GFP at high levels (Fig. 10C,
center and lower panels, arrowheads). In marked
contrast, Cy3-SP was detected in endosomes in a superficial and then
perinuclear location in non-transfected cells. Expression of
ARR319-418-GFP strongly inhibits SP-induced endocytosis of
the NK1R. Thus,
-arrestins play an important role in SP-induced
endocytosis of the NK1R.
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DISCUSSION |
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In unstimulated cells, The NK1R and Gq/11
colocalize at the plasma membrane. SP caused the following:
(a) rapid (1 min) and striking translocation of
-arrestins from the cytosol to the plasma membrane, where they are
required for NK1R endocytosis; (b) marked redistribution of
-arrestins and NK1R from the plasma membrane to the same endosomes
(2-10 min), whereas G
q/11 remains at the plasma
membrane; (c) prolonged (>60 min) association of
-arrestins and the NK1R in endosomes; and (d) gradual
(4-6 h) redistribution of
-arrestins to the cytosol and NK1R to the
plasma membrane. To our knowledge, this is the first detailed
examination of SP-induced trafficking of
-arrestins, and the first
direct demonstration that
-arrestins participate in SP-induced
endocytosis of the NK1R.
Expression of Functional NK1R-GFP and ARR-GFP--
We placed GFP
at the intracellular C terminus of the NK1R. Despite the importance of
the NK1R C-tail for desensitization (39, 40) and trafficking (41),
comparisons of SP-induced Ca2+ mobilization, uncoupling and
endocytosis in cells expressing ARR-NK1R, with our previous reports in
KNRK cells expressing wild type NK1R and in neurons that naturally
express the NK1R, suggest that GFP does not affect the function of the
NK1R. First, SP stimulated Ca2+ mobilization in cells
expressing NK1R-GFP with a similar potency to that which we have
previously reported in KNRK cells expressing wild type NK1R (38).
Second, the Ca2+ response in cells expressing NK1R-GFP was
transient in the continued presence of SP and desensitized to repeated
stimulation by SP. We have previously shown that wild type NK1R
similarly desensitizes in transfected cells and neurons (24, 34).
Finally, SP induced endocytosis and trafficking of NK1R-GFP in a
similar manner to that reported for wild type NK1R in KNRK cells and
neurons (23, 25, 32). Thus, NK1R-GFP behaves similarly to wild type
NK1R in transfected cells and in cells that naturally express this receptor (23-25, 34, 38). In support of our results, others have
reported that GFP does not affect signaling or trafficking of
cholecystokinin A and 2-adrenergic receptors
(42-44).
Because GFP has been attached to the C terminus of -arrestin2
without affecting its function (18), we placed GFP at the C terminus of
-arrestin1. The C terminus of
-arrestins interacts with clathrin
(45) to mediate endocytosis, and N-terminal domains interact with
GRK-phosphorylated GPCRs to uncouple them from G-proteins (46).
Comparisons of cells expressing ARR-GFP, with our previous observations
in KNRK cells and neurons expressing wild type
-arrestins, suggest
that GFP does not affect the function of
-arrestin1. SP-induced
endocytosis of the NK1R and desensitization of SP-induced Ca2+ mobilization in cells expressing ARR-GFP in a similar
manner to its effects on endocytosis and desensitization of the NK1R in
cell lines and neurons that express wild type
-arrestins (23-25, 34, 38). Furthermore, SP stimulated a similar redistribution of ARR-GFP
and immunoreactive
-arrestins in KNRK cells and neurons (34). Thus,
ARR-GFP participates as expected in endocytosis and uncoupling of the
NK1R, and ARR-GFP redistributes similarly to wild type
-arrestins in
cell lines and neurons. These observations suggest that GFP does not
alter the function or trafficking of
-arrestin1.
SP-induced Trafficking of the NK1R, Gq/11, and
-Arrestin1/2--
SP caused rapid endocytosis of the NK1R, whereas
G
q/11 remained at the plasma membrane. Thus, the NK1R
may couple to G
q/11 at the plasma membrane and rapidly
dissociate. Studies in reconstituted systems and in membrane assays
also suggest that the NK1R couples to G
q/11 (28, 36).
The
2AR and Gs
similarly colocalize at
the plasma membrane of unstimulated cells, but agonists cause redistribution of Gs
to the cytosol and endocytosis of
the
2-AR (47).
SP stimulated a striking, rapid, and transient redistribution of
-arrestin1 and
-arrestin2 from the cytosol to the plasma membrane, followed by internalization of the NK1R and
-arrestins into the same endosomes. SP similarly stimulates translocation of
-arrestins to the plasma membrane and endosomes of neurons (34).
Remarkably, membrane translocation of
-arrestins occurred even at
4 °C, whereas endocytosis of the NK1R is
temperature-dependent. This rapid membrane translocation
may be due to the cytosolic localization of
-arrestins. In support
of our results, agonists of several GPCRs induce rapid translocation of
-arrestins to the plasma membrane and endosomes (7, 18). Although
the mechanism by which SP induces membrane translocation of
-arrestins is unknown, receptor phosphorylation by GRKs increases
the affinity with which
-arrestins interact with GPCRs (48). It is
likely that SP also induces translocation of GRK2/3 from the cytosol to
the plasma membrane of KNRK cells. In support of this suggestion, SP
stimulates a minor and transient translocation of GRK2/3 to the plasma
membrane of neurons expressing the NK1R (34). Membrane targeting of
GRK2/3 entails their interaction with
subunits of heterotrimeric
G-proteins, a precise mechanism because free
subunits are found
in the plasma membrane at sites of receptor activation (16, 17). GRK2/3
probably phosphorylate the NK1R at the plasma membrane and thereby
facilitate interaction of the NK1R and
-arrestins, which mediate
uncoupling and endocytosis. GRK2/3 phosphorylate the NK1R in a
reconstituted system and in membranes (28, 29). The NK1R possess
numerous potential phosphorylation sites in the C-tail, and truncation
of the C-tail impairs desensitization (39, 40) and endocytosis (41).
GRK2/3 also phosphorylate the
2-adrenergic and m2
muscarinic cholinergic receptors to promote uncoupling and endocytosis
(2, 6, 11-13, 49). Phosphorylation may also regulate the activity of
-arrestins (50).
-Arrestins are constitutively phosphorylated in
the cytosol, and dephosphorylation, which occurs at the plasma
membrane, is required for their participation in receptor endocytosis.
The Role of -Arrestins in SP-induced Trafficking of the
NK1R--
SP-mediated redistribution of
-arrestins to the plasma
membrane preceded endocytosis of the NK1R, and overexpression of
-arrestin319-418 markedly inhibited endocytosis of
Cy3-SP. Thus,
-arrestins participate in SP-stimulated endocytosis of
the NK1R.
-Arrestins serve clathrin adaptors (7, 8). The NK1R
internalizes by a clathrin-dependent mechanism and
colocalizes with clathrin during endocytosis in KNRK cells and neurons
(23, 25). Therefore,
-arrestins probably colocalize with clathrin
during NK1R endocytosis. The localization of
ARR319-418-GFP in superficial and perinuclear vesicles in
unstimulated cells resembles the distribution of clathrin (23). The C
terminus of
-arrestins contains a clathrin binding domain (51), and a C-terminal fragment,
-arrestin319-418, constitutively
interacts with clathrin with high affinity (10). Thus,
-arrestin319-418 probably inhibits NK1R endocytosis by
constitutive interaction with clathrin.
-Arrestins participate in endocytosis of other GPCRs that
internalize by clathrin-mediated mechanisms. Overexpression of dominant
negative mutants
-arrestin1-V53D and
-arrestin319-418 inhibits endocytosis of the
2AR (8-10), whereas overexpression of wild type
-arrestins promotes agonist-induced endocytosis of
2-adrenergic and m2 muscarinic acetylcholine receptors
(8, 52). In contrast, agonist-induced endocytosis of the angiotensin II
type 1A receptor and the m1, m3, and m4 muscarinic cholinergic receptors does not depend on
-arrestins (9, 14). Some GPCRs, such as
the endothelin 1 and cholecystokinin A receptors, internalize in part
by caveolin-dependent pathways (53, 54), but the potential role of
-arrestins in this pathway is unknown. Since the mechanism of endocytosis is distinct for different GPCRs and may also depend on
the cellular environment, it is important to examine receptor regulation in cells that naturally express receptors at physiological levels. Thus, our report that SP induces translocation of
-arrestins to the plasma membrane of neurons suggests that
-arrestins play a
physiological role in regulating the NK1R (34).
An unexpected observation was the prolonged colocalization of the NK1R
and -arrestins in the same endosomes. We do not know whether the
NK1R is physically associated with
-arrestins in endosomes, but the
prolonged colocalization suggests that
-arrestins regulate
internalized receptors. Resensitization of responses to SP in KNRK
cells and neurons is blocked by endocytic inhibitors, phosphatase
inhibitors, and acidotropic agents that prevent receptor recycling,
suggesting that the NK1R must be internalized, dephosphorylated, and
recycled for resensitization to occur (23-25). Similarly, endocytosis and recycling are important for resensitization of the
2AR (19-22). Whether
-arrestins participate in
intracellular trafficking or signaling of GPCRs is unknown, although
endocytosis is required for the
2AR to stimulate
mitogen-activated protein kinases (15). The mechanism by which the NK1R
and
-arrestins redistribute to different compartments is also
unknown. Dephosphorylation of the NK1R in acidified endosomes may be
required for dissociation of
-arrestins. In support of this
possibility, endosomes are enriched in phosphatases that
dephosphorylate GPCRs (55), and endosomal acidification is also
necessary for dephosphorylation of the
2-AR (56).
-Arrestins may also interact with the GRK-phosphorylated NK1R at the
plasma membrane to mediate uncoupling of the NK1R from G
q/11 and thereby terminate signal transduction, since
inositol pentakisphosphate, which disrupts the interactions of
-arrestins with receptors, attenuates desensitization of the NK1R
(30). This possibility could be confirmed by overexpression of the
dominant negative mutant
-arrestin1-V53D, which inhibits interaction
of endogenous
-arrestins with GPCRs.
In summary, SP induces a marked redistribution of -arrestins to the
plasma membrane where they participate in clathrin-mediated endocytosis
of the NK1R.
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ACKNOWLEDGEMENTS |
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We thank Patrick D. Gamp and Mark Thoma for technical assistance; Paul Dazin for assistance with flow cytometry; Ian Trowbridge (Salk Institute, San Diego, CA); and John Hutton (Cambridge, UK) and Robert Lefkowitz (Duke University) for providing reagents. The CURE Center was supported by National Institutes of Health Grant DK41301.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK39957, DK43207, NS21710 (to N. W. B.), DK52388 (to E. F. G.), and DK35740 (to J. H. W.).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.
§ Both authors contributed equally to this paper.
** To whom correspondence should be addressed: University of California San Francisco, 521 Parnassus Ave., San Francisco, CA 94143-0660. Tel.: 415-476-0489; Fax: 415-476-0936; E-mail: nigelb{at}itsa.ucsf.edu.
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ABBREVIATIONS |
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The abbreviations used are:
GPCR, G-protein
coupled receptor;
ARR, arrestin;
2AR,
2-adrenergic receptor;
Cy3-SP, Cyanine 3.18-labeled
substance P;
GFP, green fluorescent protein;
EGFP, enhanced green
fluorescent protein;
GRK, G-coupled protein receptor kinase;
NK1R, neurokinin 1 receptor;
SP, substance P;
KNRK cells, Kirsten murine
sarcoma virus transformed rat kidney epithelial cells;
DMEM, Dulbecco's modified Eagle's Medium;
BSA, bovine serum albumin;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
LAMP-1, lysosomal-associated membrane protein-1;
GST, glutathione
S-transferase.
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REFERENCES |
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