From the Departments of ¶ Anesthesiology, ** Biochemistry,
§ Cell Biology, §§ Medicine,
The substance P receptor (SPR) is a G
protein-coupled receptor (GPCR) that plays a key role in pain
regulation. The SPR desensitizes in the continued presence of agonist,
presumably via mechanisms that implicate G protein-coupled receptor
kinases (GRKs) and The substance P or neurokinin 1 receptor is a member of the G
protein-coupled receptor superfamily that regulates signaling for pain
perception in both the peripheral and central nervous systems (1). Its
role in pain transmission has been convincingly demonstrated in animal
models using genetic techniques. Disruption of either the gene for the
SP1 precursor
preprotachykinin A or the gene for the SPR in the mouse dramatically
reduces the animal's response to painful stimuli (2, 3). Therefore, a
pharmacological blockade of SPR activation or an enhancement of
mechanisms that attenuate SPR signaling may ultimately lead to novel
therapies for pain.
The SPR is coupled to a Gq-mediated signaling cascade. Thus,
the binding of SP to the receptor results in the hydrolysis of membrane
phosphoinositides, the formation of inositol trisphosphate and
diacylglycerol, the mobilization of intracellular calcium, and the
activation of protein kinase C (PKC) and translocation to the plasma
membrane (4-6). Biochemical and electrophysiological studies on cells
expressing either endogenous or transfected SPR indicate that SPR
signaling diminishes within seconds of agonist exposure (7-11). The
molecular events that lead to this agonist-dependent desensitization in cells remain ill-defined. Studies performed over the
last several years on GPCRs such as The goal of the present study was to ascertain the extent and kinetics
of GRK and Materials--
Cell culture media and physiological buffers were
obtained from Life Technologies, Inc. Substance P was obtained from
Sigma. H332PO4 (specific activity,
8500-9120 Ci/mmol) was from New England Nuclear (Boston, MA). Plasmid
constructs of green fluorescent protein conjugated to PKC Cell Culture and Transfection--
HEK293 cells were maintained
in minimum essential medium with 10% fetal bovine serum and 1:100
(v/v) penicillin/streptomycin in a 5% CO2 incubator at
37 °C. Cells were transfected with 5-10 µg of plasmid
pcDNA1.1 containing human SPR cDNA and 1-2.5 µg of plasmid
containing cDNA for either PKC Whole Cell Phosphorylation--
Human SPR-transfected HEK293
cells (106 cells/well) were labeled with 0.3 mCi of
32Pi in 1 ml of HEPES/Krebs buffer (10 mM HEPES, pH 7.4, 118 mM KCl, 1.17 mM MgSO4, 1.3 mM CaCl2,
0.34 mM NaHCO3, and 11.7 mM glucose) for 90 min at 37 °C. The cells were then stimulated with 1 µM SP for various times. After this treatment, cells were
washed with cold buffer and then lysed with 1 ml of ice-cold lysis
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.1 mM
Na3VO4, 10 mM
Na4P2O7, 1 mM EGTA, 10 mM NaF, 0.5% Nonidet P-40, 1 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, and 0.1 mM phenylmethylsulfonyl fluoride). The lysate was
centrifuged, precleared by incubating with protein A-Sepharose, and
incubated for 1 h with 5 µl of a polyclonal antibody raised
against the C terminus of human SPR (16). The mixture was then
incubated for 1 h at 4 °C. The protein A-Sepharose was
collected by centrifugation, washed with lysis buffer containing 1 M NaCl, resuspended in 50 µl of SDS-polyacrylamide gel
electrophoresis sample buffer (2% SDS, 60 mM Tris-HCl, pH
6.8, 10% glycerol, 10% Confocal Microscopy--
Transfected cells were split into 35-mm
plastic dishes containing a centered, 1-cm, glass-bottomed well (MaTEK,
Inc., Ashland, MA). Cells were placed in 1 ml of minimum essential
medium buffered with 20 mM HEPES and treated with 100 µl
of 1 µM SP in medium. Images were collected with a Zeiss
laser scanning confocal microscope (LSM-410), and relative intensities
were calculated from 8-bit pixel values using Scion Image for the PC
(Release 2). Net translocation was calculated by subtracting the
average background intensity in the cytoplasm from the membrane
intensity. Images were printed using Adobe Photoshop 5.0.
PKC Translocation after Human SPR Exposure to SP--
The
translocation of PKC to the plasma membrane occurs upon agonist
stimulation of Gq-coupled GPCRs (19). Fig.
1 shows how the temporal distribution of
PKC Translocation of GRK2 to the Plasma with SP Treatment--
The
cell cytoplasm is a major reservoir of GRK2 (18). If GRK2 plays a major
role in initiating human SPR desensitization, then its physical
translocation from the cytoplasm to the plasma membrane should be
apparent within the temporal window defined by the reversible
translocation of SP-activated PKC. To investigate the kinetics of GRK2
behavior during SP stimulation, GRK2-GFP was coexpressed along with the
human SPR in HEK293 cells (Fig. 3).
Previous studies have documented that GRK2-GFP is a functional kinase
because it phosphorylates rhodopsin in both a G Agonist Stimulation of Human SPR Results in Its
Phosphorylation--
Because GRK2 translocates to the plasma membrane
after SPR stimulation, we examined whether this event leads to receptor
phosphorylation. HEK293 cells expressing human SPR were labeled with
32Pi, and the receptor was immunoprecipitated
from control and SP-treated cells. The results, which are shown in Fig.
4, indicate that SP causes a rapid
phosphorylation of the receptor within 30 s of SP exposure. Thus,
SP-induced translocation of GRK2 correlates with SP-induced
phosphorylation of the SPR. A similarly rapid (t1/2 < 1 min) SP-dependent phosphorylation of the human SPR
also occurs in Chinese hamster ovary cells that stably express the
receptor.3
Translocation of We visually document in real time a rapid activation and
desensitization of the human substance P receptor. The results indicate that SPR desensitization coincides with the active recruitment of GRK2
to the plasma membrane and phosphorylation of the receptor, followed by
the recruitment of Studies on the mechanism of homologous desensitization of GPCRs using
the An interesting observation made here is that SP causes a profound
change in cell shape. This membrane remodeling appears to be more
pronounced at the free surfaces of the cells, as shown in Fig.
7. Although the underlying mechanism of
this phenomenon remains obscure, it may involve direct interactions of
SP or GRK2 and tubulin (18, 29). It is interesting to speculate that the observed SP-induced changes in cell morphology may be due to the
SPR's high affinity for GRK2 (16), which would keep it localized on
the membrane, where it can phosphorylate tubulin (18). Furthermore,
whereas a role of the SPR in chemotaxis has been reported previously
(30, 31), additional studies are needed to delineate the mechanism by
which SP induces changes in cell shape.
Pharmacology,
Howard
Hughes Medical Institute, Duke University Medical Center, Durham, North
Carolina 27710
ABSTRACT
Top
Abstract
Introduction
References
-arrestins. The temporal relationship of these
proposed biochemical events has never been established for any GPCR
other than rhodopsin beyond the resolution provided by biochemical
assays. We investigate the real-time activation and desensitization of
the human SPR in live HEK293 cells using green fluorescent protein
conjugates of protein kinase C, GRK2, and
-arrestin 2. The
translocation of protein kinase C
II-green fluorescent protein to
and from the plasma membrane in response to substance P indicates that the human SPR becomes activated within seconds of agonist exposure, and
the response desensitizes within 30 s. This desensitization process coincides with a redistribution of GRK2 from the cytosol to the
plasma membrane, followed by a robust redistribution of
-arrestin 2 and a profound change in cell morphology that occurs after 1 min of SPR
stimulation. These data establish a role for GRKs and
-arrestins in
homologous desensitization of the SPR and provide the first visual and
temporal resolution of the sequence of events underlying homologous
desensitization of a GPCR in living cells.
INTRODUCTION
Top
Abstract
Introduction
References
2-adrenergic
receptor (
2AR) and rhodopsin have proposed that
agonist-dependent desensitization of GPCRs requires
phosphorylation by G protein-coupled receptor kinases (GRKs) followed
by the binding of
-arrestin proteins (12-14). The applicability of
this homologous desensitization paradigm to the SPR is suggested by
recent in vitro studies demonstrating that GRKs efficiently
phosphorylate the human and rat SPR (15, 16).
-arrestin involvement in SPR signaling in live cells. To
this end, HEK293 cells were transfected with human SPR and green
fluorescent protein conjugates of PKC
II, GRK2, or
-arrestin 2. The effects of human SPR activation on the cellular distribution of
these proteins were then visualized in real time by confocal
microscopy. Our data demonstrate that during SP-mediated homologous
desensitization of the human SPR, the translocations of GRK2 and
-arrestin 2 from the cytosol to the plasma membrane closely follow
the activation of PKC. Interestingly, whereas redistribution of
-arrestin 2 to activated GPCRs has previously been observed (17),
the SPR is the first GPCR whose activation led to the observable
translocation of GRK2 to the plasma membrane in real time. Hence, these
observations in live cells represent the first visual confirmation that
the temporal sequence of biochemical events underlying homologous GPCR
desensitization does indeed occur.
EXPERIMENTAL PROCEDURES
II, GRK2,
and
-arrestin 2 were made and characterized as described previously
(17-19).
II-GFP, GRK2-GFP, or
-arrestin
2-GFP using coprecipitation with calcium phosphate as described
previously (17).
-mercaptoethanol, and 0.025% bromphenol
blue), and the phosphorylated proteins were visualized by
SDS-polyacrylamide gel electrophoresis followed by autoradiography.
RESULTS
II-GFP changes in HEK293 cells upon activation of SPRs by 100 nM SP. As seen in Fig. 1, the increase in PKC
II-GFP
fluorescence at the plasma membrane, which separates the two cells,
becomes evident within 3 s. This redistribution of PKC reflects
SPR activation, which probably occurs even more rapidly. However, our
ability to measure shorter time intervals is limited because there is
image degradation upon employing more rapid scanning rates. As can be
observed in Fig. 1, PKC translocation remains stable for 20-30 s,
begins to wane after 30 s, and disappears by 60-90 s. To
demonstrate that the reversibility of PKC translocation upon SPR
activation is consistent with receptor desensitization and is not
simply a generalized waning of the PKC response, cells were also
exposed to phorbol 12-myristate 13-acetate. This compound persistently
activates PKC by a receptor-independent mechanism (20, 21). Fig.
2 shows that the exposure of the cells to
100 nM phorbol 12-myristate 13-acetate results in a
translocation of PKC to the plasma membrane that shows no sign of
decreasing even after 5 min. These data indicate that the waning of PKC
upon SPR activation is due to an upstream event which probably occurs at the level of the receptor. A similar conclusion was reached in a
recent study by Feng et al. (19). It was found that
stimulation of Gq-linked angiotensin or endothelin receptors
also produces a single transient pulse of PKC translocation. However,
the sequential activation of angiotensin receptors followed after a few
minutes by the activation of endothelin receptors produced a second
independent pulse of PKC translocation. This indicated that the
desensitization occurred at the level of the receptor itself and was
not due to the desensitization of downstream Gq-mediated
signaling. The 60-s time period after SPR activation thus defines a
window in which changes in the distributions of the cellular proteins
responsible for homologous SPR desensitization should become
apparent.
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Fig. 1.
Activation and desensitization of the human
substance P receptor as visualized by the redistribution of
PKC II. HEK293 cells expressing both PKC
II-GFP and the human
SPR were exposed to 100 nM substance P at time 0. The
redistribution of PKC
II was followed by confocal microscopy using
repetitive 3-s scanning intervals. The activation of the receptor and
its subsequent desensitization coincide with the increased plasma
membrane fluorescence that occurs by 3 s and the subsequent loss
of membrane fluorescence that is evident after 30 s.
Bar = 10 µm.
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Fig. 2.
Activation of PKC II by the phorbol ester
phorbol 12-myristate 13-acetate. Phorbol 12-myristate 13-acetate
was added to the cell media at time 0 (left panel), and the
redistribution of PKC
II-GFP was followed by confocal microscopy. The
redistribution of PKC
II to the plasma membrane shows no signs of
reversal up to 5 min (right panel). Bar = 10 µm.
-independent and
-dependent manner (18), facilitates
2AR
internalization (18), and phosphorylates
2AR in intact
cells.2 As seen in Fig. 3,
the increase in fluorescence from the plasma membrane after SP
treatment reflects the agonist-dependent translocation of
GRK2-GFP. Using 3-s scanning intervals, it is seen that GRK2 accumulates at the membrane interface between two cells within 6-9 s
of agonist treatment. This translocation peaks at between 15 and
21 s and wanes thereafter but does not entirely disappear, even
after 1 min. These results visually demonstrate that human SPR
activation recruits GRK2 to the plasma membrane during the period in
which the receptor desensitizes. These data represent the first
demonstration in living cells of a physical translocation of GRK2 to
the plasma membrane after GPCR activation.
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Fig. 3.
Redistribution of GRK2 to the plasma membrane
after SPR activation. HEK293 cells containing GRK2-GFP and human
SPR were treated with SP at time 0 (top left panel). The
kinetics of GRK2 translocation are followed using 3-s scanning
intervals on the confocal microscope. GRK2 translocation occurs within
6 s (top panels), rapidly peaks, and then begins to
wane (bottom panels) during the first minute after SPR
activation. Bar = 10 µm.
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Fig. 4.
Phosphorylation of the human SPR. HEK293
cells (106 cells/well) containing the human SPR were
incubated with 32Pi for 90 min before exposure
to 100 nM SP for 0.5-2 min. Control cells were not exposed
to agonist. The SPR was immunoprecipitated, and the receptor
phosphorylation was analyzed by SDS-polyacrylamide gel
electrophoresis/autoradiography as described under "Experimental
Procedures." A near maximal increase in SPR phosphorylation occurs
within 30 s of exposure to SP. These data confirm that the
physical translocation of GRK2 coincides with receptor
phosphorylation.
-Arrestin 2 to the Plasma with SP
Treatment--
Fig. 5 shows the
redistribution of
-arrestin in a representative cell containing both
-arrestin 2-GFP and the human SPR in response to SP. The cell
cytoplasm acts as a reservoir for
-arrestins (17). This can be
observed in the cell shown in the top left panel of Fig. 5
as a homogenous distribution of
-arrestin 2-GFP fluorescence. The
addition of SP at time 0 to the surrounding media results in an
enhancement of fluorescence at the cell margins from
-arrestin 2-GFP
translocation that is apparent by 30 s and is delayed relative to
PKC
II and GRK2 translocations.
-Arrestin 2 translocation does not
peak for 60-120 s after SP exposure and remains localized at the
plasma membrane. Interestingly, the data for Fig. 5 show that after a
minute of SPR stimulation, the cell morphology begins to change
dramatically.
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Fig. 5.
Redistribution of -arrestin 2 to the
plasma membrane after SPR activation. HEK293 cells containing
human SPR and
-arrestin 2-GFP were exposed to 100 nM SP.
The distribution of
-arrestin 2 was observed using repetitive 30-s
confocal scans. Translocation of
-arrestin 2 occurs within 15-30 s
and reaches a steady state after about 60-90 s. Note the striking
change in cell morphology that begins after 1 min of SP stimulation.
Bar = 10 µm.
DISCUSSION
-arrestin 2 to the plasma membrane. Thus, these
data establish a role for GRKs and
-arrestins in the homologous
desensitization of SPRs. Moreover, these results represent the first
direct confirmation in whole cells that the putative sequence of
biochemical events underlying homologous GPCR desensitization (receptor
activation and GRK-mediated phosphorylation followed by the binding of
-arrestins) actually occurs as postulated from a variety of in
vitro and whole cell studies. Fig. 6
summarizes the temporal relationship of SPR activation and the
redistribution of GRKs and
-arrestins.
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Fig. 6.
Temporal redistribution of PKC, GRK2, and
-arrestin 2 upon SPR activation. The relative increases in the
intensity of green fluorescent protein fluorescence at the cell
membrane were determined as described under "Experimental
Procedures," and smooth curves were drawn to represent their temporal
redistributions.
2AR as a model have shown that increases in plasma membrane GRK activity occur after agonist treatment (22, 23). These
findings did not necessarily require that a simultaneous recruitment of
GRK from the cytosol also occurs. A significant finding of the present
study is that a sustained physical translocation of GRK2 occurs with
agonist stimulation of the human SPR. This type of GRK behavior in the
presence of the substance P receptor has been suggested by
immunochemical studies using fixed and extracted neuronal cells (24).
Similar studies using antibodies to assess GRK2 distribution in fixed
HEK293 cells also seem to indicate that GRK2 is recruited to membranes
during the early stages of desensitization and
2AR
endocytosis (25). However, we have been unable to demonstrate any
persistent observable physical translocation of GRK2-GFP to activated
2AR, despite results demonstrating that overexpressed
GRK2-GFP enhances
2AR phosphorylation and sequestration.
Similarly, we have been unable to document a physical translocation of
GRK2-GFP to other types of GPCRs including the angiotensin II type IA
receptor.4 Thus, our finding
of an observable physical GRK2-GFP translocation upon SPR stimulation
is intriguing. A potential explanation for this may follow from the
observation that the human SPR is an excellent substrate of GRK2. It
can be nearly maximally phosphorylated in the absence of G
with a
Km of 6 nM (16). In contrast, GRK2-mediated phosphorylation of the
2AR occurs with a
Km of 49 nM and requires G
(26).
The affinity of GRK2 for the
2AR is over 100-fold lower
than the 0.3 nM Kd observed in
vitro for
-arrestin 2 binding to the receptor (27). Therefore, for some GPCRs,
-arrestin may normally prevent persistent
GRK/receptor interactions by displacing the kinase from the receptor,
but GRK may remain at the membrane upon activation of GPCRs for which it has high affinity. This interpretation of the results also support
previous observations that GRKs facilitate but are not absolutely
necessary for
2AR endocytosis (28).
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Fig. 7.
Interference contrast and fluorescence
imaging of substance P-induced cell shape changes in HEK293 cells.
Before SP exposure, transfected cells exhibit smooth boundaries in both
interference contrast (top left panel) and by imaging of
fluorescent -arrestin 2-GFP (top right panel). Upon
treatment with 100 nM SP for 5 min, the free boundaries of
the cells exhibit multiple membrane protrusions visible in both types
of imaging (bottom panels).
In summary, the present study visually illustrates the kinetic behavior
of GRK2 and -arrestin 2 during homologous desensitization of the
human substance P receptor. The data provide direct evidence that the
sequence of biochemical events proposed for homologous GPCR
desensitization actually occurs in live cells. Moreover, the rapid but
persistent membrane recruitment of GRK2 by the SPR suggests that the
kinase may be involved in cell regulatory processes other than receptor desensitization.
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ACKNOWLEDGEMENTS |
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We thank Drs. Eric D. Roush and Jie Zhang for helpful discussions and Susan R. S. Tumey for editorial assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants NS 33405 (to M. M. K.), HL03422 (to L. S. B.), and NS19576 (to M. G. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Depts. of
Anesthesiology and Pharmacology, Box 3094, Duke University Medical
Center, Durham, NC 27710. Tel.: 919-681-4775; Fax: 919-681-8089;
E-mail: kwatr001{at}mc.duke.edu.
2 L. S. Barak, S. S. G. Ferguson, and M. G. Caron, unpublished results.
3 E. D. Roush and M. M. Kwatra, unpublished results.
4 L. S. Barak, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
2AR,
2 adrenergic receptor;
-arrestin 2-GFP,
-arrestin
2 tagged with green fluorescent protein;
GPCR, G protein-coupled
receptor;
GRK, G protein-coupled receptor kinase;
GRK2-GFP, GRK2 tagged
with green fluorescent protein;
PKC, protein kinase C;
PKC
II-GFP, PKC
II tagged with green fluorescent protein;
SP, substance P;
SPR, SP receptor.
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REFERENCES |
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