From the Departments of Cell Biology and
¶ Medicine, § Howard Hughes Medical Institute
Laboratories, Duke University Medical Center,
Durham, North Carolina 27710
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ABSTRACT |
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Protein kinase C (PKC) links various
extracellular signals to intracellular responses and is activated by
diverse intracellular factors including diacylglycerol,
Ca2+, and arachidonic acid. In this study, using a
fully functional green fluorescent protein conjugated PKCII
(GFP-PKC
II), we demonstrate a novel approach to study the dynamic
redistribution of PKC in live cells in response to G protein-coupled
receptor activation. Agonist-induced PKC translocation was rapid,
transient, and selectively mediated by the activation of
Gq
- but not Gs
- or
Gi
-coupled receptors. Interestingly, although the
stimuli were continuously present, only one brief peak of PKC membrane
translocation was observed, consistent with rapid desensitization of
the signaling pathway. Moreover, when GFP-PKC
II was used to examine
cross-talk between two Gq
-coupled receptors, angiotensin
II type 1A receptor (AT1AR) and endothelin A receptor
(ETAR), activation of ETARs resulted in a
subsequent loss of AT1AR responsiveness, whereas stimulation of AT1ARs did not cause desensitization of the
ETAR signaling. The development of GFP-PKC
II has allowed
not only the real time visualization of the dynamic PKC trafficking in live cells in response to physiological stimuli but has also provided a
direct and sensitive means in the assessment of activation and desensitization of receptors implicated in the phospholipase C signaling pathway.
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INTRODUCTION |
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The protein kinase C
(PKC)1 family of
phospholipid-dependent serine/threonine kinases plays key
roles in the transduction and regulation of many cellular signaling
processes by catalyzing specific substrate phosphorylation (1, 2).
Activation of protein kinase C can be triggered by stimulating a wide
variety of plasma membrane hormone, neurotransmitter, and growth factor receptors, among which seven-transmembrane G protein-coupled receptors (GPCRs) relay extracellular signals to PKC by activating heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) (3). G
proteins are composed of - and
-subunits, both of which serve as signal moieties and mediate cellular responses by modulating the
activity of different effector systems and levels of various second
messengers (4). Gq
is one of the major G protein
-subunits and is coupled to a large number of receptors including
angiotensin receptors and endothelin receptors. The activation of
Gq
by these receptors triggers the hydrolysis of
membrane inositol phospholipids by phospholipase C
to form two
important second messengers, inositol 1,4,5-triphosphate
(IP3) and diacylglycerol (DAG) (1, 5, 6). The binding of
IP3 to its intracellular receptor results in a rise in
intracellular Ca2+ (7). The increased membrane DAG and
intracellular Ca2+ lead to the mobilization of PKC to the
plasma membrane and its subsequent activation (1). The activation of
PKC by DAG, Ca2+, and many other lipid mediators such as
arachidonic acid has been associated with many important biological
functions including cell proliferation, differentiation, and gene
expression (1, 2, 8).
In Gq-coupled receptor signal transduction, PKC
activation not only serves to relay the signal from effector
phospholipase C
to various PKC substrates for initiating downstream
cellular responses, but it also exerts feedback effects on the system
and is involved in the turning off of signaling from the receptors, an
important regulatory process termed desensitization (9, 10). The
ability of PKC to phosphorylate and desensitize a receptor relies on
the existence of consensus PKC phosphorylation site(s) at the
intracellular domains of the receptors (11-14). Since PKC does not
discriminate between agonist-occupied and -unoccupied receptors (15),
activation of PKC is believed to be associated with both homologous and
heterologous desensitization of G protein-coupled receptors.
Consequently, exposure of cells to a Gq
-coupled receptor agonist that causes PKC activation potentially results not only in the
loss of cellular response to that specific agonist but also the
diminution of cellular responses to various other agonists.
The current methods of assessing Gq-coupled receptor
signaling and desensitization are based mainly on the generation of IP3 or the increase of intracellular Ca2+ as a
result of IP3 generation (16-18). However, neither
approach assesses the equally important and independent branch of
phospholipase C
signaling reflected by the production of DAG and the
subsequent membrane translocation and activation of PKC. Although as
important as IP3, DAG is rarely used as a measurement in
assessing Gq
activation because of its quick turnover
(1) and the lack of techniques to follow its real time cellular
distribution or behavior. Moreover, biochemical studies have indicated
that the activation of PKC by DAG is also transient and is mainly
associated with many cellular events mediated by short term activation
of PKC such as hormone secretion and muscle contraction (1, 2). Phorbol
esters mimic the action of DAG, but they are more potent, and their
effects last longer in cells due to their persistence in the cell
membrane (1). This has made feasible the study of PKC subcellular
localizations before and after activation by phorbol esters by
immunofluorescent microscopy in fixed cells (19, 20). However, the same
method is not applicable in following the more physiologically relevant redistribution of PKC in response to receptor activation in live cells.
Furthermore, it is not clear whether the short term of PKC activation
in response to receptor activation is related to the signaling
desensitization at the level of receptors.
In the present study, we report the development of a green fluorescent
protein conjugated PKCII (GFP-PKC
II) to study PKC mobilization in
response to agonist stimulation of G protein-coupled receptors and to
assess the activation and desensitization of these receptors. GFP,
originally identified in the jellyfish Aequorea victoria,
displays an inherent green bioluminescence and has been used as a
fluorescent reporter molecule in the localization of membrane
receptors, cytoplasmic proteins, and secretory proteins (21-28). When
fused to the amino terminus of PKC
II, the resulting fusion protein,
GFP-PKC
II, was found to be fully functional in terms of its
phospholipid-dependent kinase activity and its ability to
translocate from cytoplasm to the plasma membrane in response to
phorbol ester (PMA) stimulation, similar to that reported for a
PKC
/GFP conjugate (28). Interestingly, while in PMA-treated cells
GFP-PKC
II remained on the plasma membrane, in cells stimulated with
physiological signals that activate Gq
-coupled
receptors, the translocation of GFP-PKC
II to the plasma membrane was
found to be transient, reaching a peak and being reversed within
minutes. This provided a real time visual demonstration of the cellular trafficking of a PKC isoenzyme in live cells, revealing a dynamic nature of the interaction of PKC
II with lipid and/or protein molecules on the plasma membrane. Furthermore, since GFP-PKC
II selectively responded to only signals activating Gq
- but
not Gs
- and Gi
-coupled receptors and such
responses were transitory, our results also demonstrate an important
analytical role of GFP-PKC
II as a reporter in the study of
Gq
-coupled receptor activation and desensitization.
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EXPERIMENTAL PROCEDURES |
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Materials--
[-32P]ATP was purchased from NEN
Life Science Products. Monoclonal antibody against GFP was from
CLONTECH. Polyclonal rabbit antibody against
PKC
II was prepared and extensively characterized as described
previously (29). Mammalian expression vector pBK-CMV and GFP plasmid
pEGFP-N1 was from Stratagene and CLONTECH,
respectively. Restriction enzymes were from Promega or New England
Biolabs. Ampli-Taq DNA polymerase was obtained from
Perkin-Elmer. Protein A-Sepharose CL-4B was from Amersham Pharmacia
Biotech. Phosphatidylserine and sn-dioctanoyl-glycerol were
purchased from Avanti Polar Lipids Inc. Eagle's minimum essential
medium, phosphate-buffered saline (PBS), and 1 M HEPES
buffer were from Life Technologies, Inc. Fetal bovine serum was
obtained from Summit Biotechnology Inc. Fluorescein
isothiocyanate-conjugated donkey anti-rabbit IgG was from Jackson
ImmunoResearch Laboratories, Inc. All other chemicals were from
Sigma.
Plasmid Constructs--
The human PKCII cDNA was inserted
between BamHI and KpnI sites of pBK-CMV. A unique
BssHII restriction site was located three base pairs prior
to the start codon (ATG) of PKC
II cDNA. To construct GFP-PKC
II, EGFP cDNA without the stop codon was first amplified from plasmid pEGFP-N1 by polymerase chain reaction using
5'-oligonucleotide primer 5'-GTGAACCGTCAGATCCGCTAG-3' (based on the
sequence of pEGFP-N1 from 575 to 595) and 3'-primer
5'-CCATCTTGCGCGCCTTGTACAGCTCGTCCATGC-3' (with the native
sequence of pEGFP-N1 from 1376 to 1396 underlined). The polymerase
chain reaction fragment containing EGFP cassette was gel-purified,
digested with BamHI and BssHII, and directly inserted between the BamHI and BssHII sites of
plasmid pBK-CMV-PKC
II prior to the 5'-end of the PKC
II cDNA
(Fig. 1). The sequence of the construct was confirmed by DNA
sequencing. The cDNAs of AT1AR, ETAR,
2AR, and D2R were subcloned in pcDNA I
or pcDNA I/amp mammalian expression vectors (Invitrogen).
Cell Culture and Transfection-- HEK 293 cells from the American Type Culture Collection (ATCC) were maintained in Eagle's minimum essential medium supplemented with 10% (v/v) fetal bovine serum in a 5% CO2 incubator at 37 °C. Cells were seeded at a density of 2.0 × 106 cells/100-mm dish and transfected using a modified calcium phosphate method with 1-10 µg of plasmid (30).
Immunoprecipitation--
HEK 293 cells were transfected with
EGFP, PKCII, or GFP-PKC
II. Immunoprecipitation was performed
48 h after transfection as follows. The cells were washed with
cold PBS and solubilized in 0.5 ml of lysis buffer with protease
inhibitors (50 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 0.5% Nonidet P-40, 10 mM NaF, 1 mM
sodium orthovanadate, 1 mM dithiothreitol, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 10 µg/ml trypsin/chymotrypsin
inhibitor, 5 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl
fluoride) for 1 h, and the lysates of EGFP, PKC
II, or
GFP-PKC
II transfected cells were immunoprecipitated with PKC
II
antibody. Protein A-Sepharose beads were used to absorb immunoprecipitates and were then washed four times with lysis buffer
followed by one wash with kinase buffer (50 mM Tris-HCl, pH
7.4, 10 mM NaF, 1 mM
Na3VO4, 0.5 mM EDTA, 0.5 mM EGTA, 2 mM MgCl2). The kinase
activity of immunoprecipitated proteins was analyzed by protein kinase
C assay (see below).
Immunoblot--
Cell lysates from HEK 293 cells transfected with
EGFP, PKCII, or GFP-PKC
II were separated by SDS-polyacrylamide
gel electrophoresis, and electrophoretically transferred onto
nitrocellulose membranes (31). The membranes were blocked in PBS with
0.1% Tween 20 and 5% dried milk, probed with anti-GFP antibody
(1:2500 dilution) or rabbit anti-PKC
II antibody (1:2000 dilution),
and exposed using the enhanced chemiluminescence (ECL) Western blotting
detection system (Amersham Pharmacia Biotech).
Protein Kinase C Assay--
PKC activity was measured using the
vesicle assay for PKC as described previously (32). Standard assay
conditions were as follows: 20 mM Tris-HCl, pH 7.4, 100 µM MgCl2, 1 mM CaCl2,
10 µM ATP, 10-15 µCi/ml [-32P]ATP, 40 µg/ml phosphatidylserine/sn-dioctanoyl-glycerol vesicles, and 200 µg/ml histone IIIS as substrate in a final volume of 250 µl
at 30 °C for 10 min. 10 mM EGTA was used in determining
basal kinase activity. The phosphorylated proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.
Indirect Immunofluorescence--
Transfected cells were seeded
on glass coverslips placed in six-well culture dishes at a density of
5 × 105 cells/well. For experiments involving phorbol
ester treatment, cells were treated with 100 nM PMA for 5 min. The cells were then rinsed briefly in PBS and fixed in 3.7%
paraformaldehyde for 10 min. The fixed cells were permeabilized in PBS
containing 0.2% Triton X-100 for 10 min and blocked in PBS containing
0.2% bovine serum albumin for 10 min. After 1 h of incubation
with anti-PKCII polyclonal antibody (1:100 dilution), the cells were
washed and incubated with fluorescein isothiocyanate-conjugated donkey
anti-rabbit IgG (1:100 dilution) for 1 h. The coverslips were
mounted onto the slides, and PKC
II immunofluorescence was observed
with a Zeiss LSM-410 laser scanning microscope at 488-nm
excitation.
Confocal Microscopy--
HEK 293 cells were transfected with
GFP-PKCII and one or two of the G protein-coupled receptors as
described in figure legends. 24 h after transfection, the cells
were plated onto 35-mm glass-bottomed culture dishes (MatTek) at a
density of 1 × 105 and incubated for another 24 h for the cells to attach to glass. The cells expressing GFP-PKC
II
(25-40% of the total cell population) were observed under confocal
microscopy. Confocal microscopy was performed on a Zeiss LSM-410 laser
scanning microscope using a Zeiss 40 × 1.3 NA oil immersion lens.
The cells were kept warm during microscopy at 30 °C in culture
medium containing 20 mM HEPES on a heated microscope stage.
GFP-PKC
II fluorescent signals were collected sequentially using the
Zeiss LSM software time series function with single line excitation
(488 nm) with a time interval of 20 s between two scannings.
Various drugs were applied to the cells during the scanning of
GFP-PKC
II transfected cells.
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RESULTS |
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Comparison of Wild-type PKCII and GFP-PKC
II--
Due to its
inherent fluorescence and unique compact structure, GFP has been
reported in many studies to serve as a valuable reporter molecule in
the localization of various proteins without interfering with their
biological activity (21, 33). GFP-PKC
II was constructed by fusing
EGFP to the 5'-end of PKC
II (Fig. 1). When examined using SDS-polyacrylamide gel electrophoresis followed by
immunoblotting, GFP-PKC
II was found to express to the same extent in
HEK 293 cells as wild-type PKC
II but migrated more slowly due to the
added mass of GFP (Fig. 2A).
To compare the biological activity of GFP-PKC
II with that of
wild-type PKC
II, both proteins were immunoprecipitated from HEK 293 cells transiently expressing GFP-PKC
II or PKC
II with an antibody
against PKC
II and analyzed for their relative kinase activity using
a recently developed PKC assay with histone IIIS as the substrate (32). As shown in Fig. 2B, the cells transfected with EGFP
contained very low kinase activity, but transfection of either
GFP-PKC
II or PKC
II significantly increased the phosphorylation of
histone IIIS. The ability of GFP-PKC
II to phosphorylate histone IIIS in a Ca2+- and phospholipid-dependent manner
was comparable with that of wild-type PKC
II, indicating that the
fusion of the GFP molecule to the N terminus of PKC
II has no
significant effect on PKC activity.
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Dynamic and Selective Trafficking of GFP-PKCII in Response to G
Protein-coupled Receptor Activation--
Although the redistribution
of PKC upon phorbol ester stimulation has been extensively studied with
indirect immunofluorescence microscopy (19, 20), the kinetics of PKC
membrane translocation in response to physiological signals have not
been well characterized. One such physiological signal that activates
PKC is through activation of G protein-coupled receptors. Therefore,
initial studies using confocal microscopy examined the real time
cellular distribution of GFP-PKC
II in response to the activation of
the Gq
-coupled AT1AR by its physiological
ligand angiotensin II (Fig.
3A). This was done at 30 °C
in live HEK 293 cells, which had been transiently transfected to
overexpress the AT1AR and GFP-PKC
II. In the absence of
receptor activation, confocal microscopy revealed that GFP-PKC
II was
evenly distributed throughout the cytoplasm. However, upon agonist
activation of the AT1AR, a redistribution of GFP-PKC
II to the plasma membrane and clearance of cytosolic fluorescence occurred
and peaked within 40 s. This angiotensin II-induced GFP-PKC
II membrane trafficking was rapid compared with the translocation caused
by PMA stimulation, which did not peak until after 2-5 min. More
interestingly, unlike PMA-induced translocation in which PKC remained
persistently localized on the plasma membrane, the mobilization of
GFP-PKC
II to the plasma membrane following AT1AR activation was transient, and the redistributed GFP-PKC
II rapidly returned to the cytoplasm within 1 min after translocation. The time
frame for GFP-PKC
II recovery in the cytoplasm in the majority of the
cells observed ranged from 20 s to 1 min after GFP-PKC
II membrane translocation. Moreover, even in the continuous presence of
angiotensin II for 30 min, only an initial brief peak of PKC membrane
translocation was observed (data not shown). These visual results are
consistent with biochemical studies indicating a dynamic nature of
PKC
II in interacting with molecules on the plasma membrane (1,
34).
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Desensitization of Gq-coupled Receptor Signaling
Revealed by GFP-PKC
II Trafficking--
The observation that there
was only one brief peak of PKC membrane translocation even in the
continuous presence of angiotensin II or endothelin suggests that the
transitory nature of PKC trafficking in response to these receptor
agonists might be associated with the rapid desensitization of
AT1AR or ETAR signaling. To further investigate
this phenomenon, HEK 293 cells transfected with GFP-PKC
II and
AT1AR were sequentially exposed to two pulses of
stimulation by angiotensin II separated by 10 min (Fig.
4A). Whereas the first exposure resulted in a transient GFP-PKC
II trafficking between the
cytoplasm and plasma membrane and GFP-PKC
II returned to the cytoplasm within 1 min following its membrane translocation, there was
no apparent PKC mobilization in response to the second angiotensin II
stimulation, indicating the AT1AR signaling was turned off as a result of initial receptor activation at a step prior to PKC
translocation or at the level of PKC. The possibility that PKC itself
might be desensitized and loses its ability to respond to further
receptor activation was examined by exposing AT1AR and
GFP-PKC
II-containing cells to PMA following agonist activation of
the AT1AR. As shown in Fig. 4B, PMA triggered a
second peak of stable GFP-PKC
II redistribution to the plasma
membrane in cells preexposed to angiotensin II. The time profile and
extent of this PKC translocation were indistinguishable from
PMA-induced PKC redistribution in cells untreated with receptor
agonist. Similar results were obtained when cells were transfected with
GFP-PKC
II and ETAR and pretreated with endothelin (Fig.
4, C and D). These results strongly suggest that
the transient nature of PKC trafficking in response to
Gq
-coupled receptor activation is a direct consequence of the rapid desensitization of receptor signaling at a step prior to
PKC translocation.
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Cross-talk of Gq-coupled Receptor Signaling Revealed
by GFP-PKC
II Trafficking--
The AT1AR and
ETAR share the same Gq
-mediated signaling
pathway in which Gq
activates phospholipase C
and
results in the hydrolysis of phosphoinositol lipid to generate
IP3 and DAG, the latter serving as a second messenger for
activation of PKC. To further study the rapid receptor desensitization
as well as the relationship between signaling pathways of different
receptors, AT1AR and ETAR were cotransfected
with GFP-PKC
II into HEK 293 cells, and the effect of their potential
cross-talk on the trafficking of GFP-PKC
II was examined and
visualized by confocal microscopy. In initial experiments, the cells
were prestimulated with angiotensin II to activate the
AT1AR and induce PKC response. As described above, the
resulting GFP-PKC
II trafficking was transient and GFP-PKC
II
returned to the cytoplasm within 1 min following its membrane
translocation. No second peak of PKC translocation was observed in the
continuous presence of agonist for as long as 30 min. However, when
endothelin was subsequently added to the angiotensin II-pretreated
cells to activate the ETAR, a second peak of GFP-PKC
II
translocation was apparent within 1 min, the time profile and extent of
which were indistinguishable from the first peak (Fig.
5A). As both AT1AR
and ETAR induce PKC response by activating
Gq
, which increases the activity of phospholipase C, the
ability of endothelin to mobilize PKC in cells prestimulated with
AT1AR agonist further indicates that the activation of
AT1AR did not desensitize Gq
-mediated
signaling pathways, but instead the desensitization occurred at the
level of the AT1AR receptor itself. Moreover, these results
also demonstrate that the activation of AT1ARs does not
contribute to the desensitization of the ETAR.
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DISCUSSION |
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In the present work, the development of a fully functional green
fluorescent protein conjugated PKCII has allowed visualization in
live cells of the real time interaction of PKC with the plasma membrane
in response to extracellular signals. Whereas stimulation by phorbol
esters (e.g. PMA) causes a persistent localization of PKC to
the plasma membrane, our results reveal a dynamic nature of PKC
II
trafficking between the cytoplasm and plasma membrane in response to
physiological signals such as those activating G protein-coupled
receptors. PKC
II responds selectively only to specific signals that
activate receptors coupled to Gq
but not
Gs
and Gi
proteins. The membrane
translocation of PKC
II triggered by the activation of
Gq
-coupled receptors is rapid and transient and is
followed immediately by the returning of PKC to the cytoplasm within
minutes, indicating a desensitization of the signaling pathway.
Moreover, when GFP-PKC
II trafficking in response to the sequential
activation of two distinct Gq
-coupled receptors
(i.e. AT1AR and ETAR) was studied,
the results indicated that the signaling desensitization occurs at the
level of receptors and that there is cross-talk between the two
receptors. Thus, GFP conjugated PKC fusion proteins serve as a novel
useful tool not only for studying the dynamic localization of PKCs in
signal transduction in live cells but also for detecting the activation and desensitization of receptors coupled to phospholipase C such as
Gq
-coupled receptors.
PKC cellular localization has been extensively studied in culture cells
using antibody staining and immunofluorescent microscopy (19, 20).
However, many signal transduction events involving PKC are rapid,
transient, and difficult to follow in fixed cells. Green fluorescent
protein, because of its inherent bioluminescence and stoichiometric
labeling, represents a sensitive optical reporter to follow the real
time localization of many proteins in live cells and is thus well
suited for the study of transitory and dynamic distribution of
molecules in the process of signal transduction (26). Recently, the GFP
technique has been used in the study of cellular distribution of PKC
(28). In our study, when EGFP was fused to the N terminus of PKC
II,
GFP-PKC
II conjugate displayed Ca2+ and
phospholipid-dependent kinase activity comparable with that of wild-type PKC
II and was localized mainly in the cytoplasm and
excluded from the nuclei. Moreover, in cells stimulated with PMA, like
wild-type PKC
II, GFP-PKC
II redistributed to the plasma membrane.
The stable association of GFP-PKC
II with the plasma membrane is a
true reflection of the persistence of PMA in the plasma membrane as
well as the stable interaction of PKC
II to PMA. Therefore, although
GFP is about one-third the size of PKC
II (Fig. 1), as reported for
many other proteins, our data demonstrate that GFP-PKC
II conjugate
retains the function of native PKC
II in terms of biochemical
behavior and cellular localization, although the possibility cannot be
ruled out that the coupling of GFP to PKC
II may alter the affinity
of the enzyme to its interacting molecules.
The activation of PKC is triggered by a large number of extracellular
signals including hormones, neurotransmitters, and growth factors that
act through cell surface receptors. The activation of these receptors
regulates the intracellular level of various PKC activators including
DAG, Ca2+, and many other lipid mediators. G
protein-coupled receptors were used in this work as an example to
address PKC cellular trafficking and distribution in response to
various physiological stimuli. Four receptors were examined for their
ability to stimulate GFP-PKCII redistribution, including
Gs
-coupled
2AR, Gi
-coupled
D2R, and Gq
-coupled AT1AR and
ETAR. The physiological relevance of PKC activation
mediated by these receptors is apparent since PKC responded selectively
only to signals activating Gq
-coupled receptors
(i.e. AT1AR and ETAR) but not
Gs
- and Gi
-coupled receptors,
corresponding to the fact that among the three major G proteins only
Gq
mediates the production of DAG at the plasma membrane
and a rise of intracellular Ca2+. More importantly, unlike
PMA-stimulated PKC translocation, physiological signals activating
AT1ARs and ETARs induce a redistribution of GFP-PKC
II to the plasma membrane, which appears to be transient, consistent with the biochemical studies indicating that DAG is one of
the plasma membrane stimuli for PKC and that the interaction between
DAG and PKC is rapid and reversible (1, 34).
However, it is somewhat unexpected that only one rapid cycle of PKC translocation from the cytoplasm to plasma membrane and back was observed although receptor agonists were present continuously. This lack of further PKC responsiveness is probably the result of a rapid desensitization of the agonist-mediated signaling pathway upstream of PKC, since PKC itself still retains the ability to respond normally when subsequently exposed to PMA. In addition, sequential stimulation of the AT1AR and then the ETAR with their corresponding agonists induced consecutive transient cycles of PKC translocation, one after each stimulation. As the AT1AR and ETARs presumably share the same signaling components downstream of the receptors, this suggests that the desensitization of AT1AR signaling occur at the level of the receptor itself. More interestingly, although activation of the AT1AR does not affect the activity of the ETAR, activation of the ETAR not only shuts off its own signaling but also causes the desensitization of the AT1AR, indicating differential regulation of the two receptors.
The rapid desensitization of G protein-coupled receptors is achieved
mainly through phosphorylation of the receptors by two classes of
serine/threonine protein kinases: the second messenger activated
protein kinases, PKA and PKC; and the G protein-coupled receptor
kinases that specifically phosphorylate agonist-activated receptors
(15). Although the role of PKC in regulating AT1AR desensitization in different tissues is still variable depending on
experimental conditions, recently it was reported that the AT1AR was phosphorylated by both G protein-coupled receptor
kinases and PKC in response to short term angiotensin II stimulation in HEK 293, a cell line widely used in the study of receptor
desensitization (16). In contrast, in the same cell line, the
ETAR was mainly phosphorylated by G protein-coupled
receptor kinases but not by activated PKC in response to agonist (36).
These studies suggest that while both receptors serve as substrates for
G protein-coupled receptor kinases, only the AT1AR (but not
ETAR) has the unique biochemical property to undergo
agonist-dependent phosphorylation by PKC. The homologous
desensitization of both receptors probably involves G protein-coupled
receptor kinase or possibly PKC in the case of the AT1AR.
However, our results suggest that cross-talk (i.e.
heterologous desensitization) between the two receptors is mainly
mediated by PKC, and the distinct ability to be phosphorylated by PKC
might underlie the observed differential desensitization properties of
the two receptors. For instance, the inability of the ETAR
to be phosphorylated by PKC might account for its lack of heterologous
desensitization by activation of the AT1AR. In addition,
despite the finding that the AT1AR was phosphorylated by
PKA in intact aortic vascular smooth muscle cells (RASM) (37), we
demonstrate that activation of PKA by stimulating the
2AR does not lead to desensitization of the
AT1AR, consistent with the lack of PKA phosphorylation of
the AT1AR found in HEK 293 cells (16).
A large variety of signaling pathways are known to culminate in the
activation of PKC, including those mediated by Gq
protein-coupled receptors (1, 34). The numbers of such receptors are
expected to expand rapidly with the progress of genomic sequencing and challenge the conventional biochemical measurements that assess individual receptor-specific properties for defining their
corresponding ligands, detecting signaling activation and measuring
change of second messenger levels. In this study, by combining the
inherent fluorescence of GFP with the translocation property of PKC, we have developed a potential live cell biosensor that may provide simple,
sensitive, and rapid assessment of the involvement of PKC activation in
the signaling pathways of these receptors. In addition, since GFP-PKC
redistribution can serve as a sensitive indicator for receptor
activation, it may provide a simple and universal tool for screening
new ligands for receptors coupled to PKC as well as for associating
newly discovered receptors with their cognate ligands and physiological
functions. Moreover, by monitoring inhibition of PKC translocation, it
may be potentially also applicable to the identification of the
inhibitors of PKC itself as well as inhibitors that block the signaling
components leading to activation of PKC. In the case of GPCRs, GFP-PKC
translocation has been shown in this study to be a specific measure of
Gq
-coupled receptor activation. The transitory nature of
the translocation makes GFP-PKC a useful tool for studying
Gq
-coupled receptor desensitization. Compared with
detecting Ca2+ signals, measuring PKC trafficking
represents a more direct and accurate assessment of the properties of
the plasma membrane receptors without concerns from the permeability,
solubility, and compartmentalization of Ca2+ indicators and
interference from plasma membrane and intracellular Ca2+
channels (e.g. the IP3 receptor) (7, 18, 38).
Furthermore, with the discovery of numerous novel G protein-coupled
receptors by genomic sequencing, GFP-PKC should also be extremely
useful in quickly identifying those receptors coupled to
Gq
and their ligands and functions.
The visualization of GFP-PKCII dynamic translocation in this study
provides a direct real time assessment of the distribution of a PKC in
live cells in response to changes of intracellular PKC activators
triggered by physiological stimuli. To date, 12 members are identified
as belonging to the PKC superfamily, associated with a wide variety of
cellular signaling events, such as mitogenesis and tumorigenesis (2,
8). The use of GFP conjugates as optical reporters should provide
valuable information concerning not only the specific cellular
distribution of different PKC isoenzymes but also their dynamic
trafficking in response to various physiological stimuli. Therefore,
GFP-PKC conjugates may represent ideal optical tools in the study of
specific functions and kinetics of each PKC isoenzyme in different
signal transduction systems. Furthermore, when employed as a biosensor,
GFP-PKC fusion proteins may also provide a unique and sensitive means
for studying the kinetics and components of signal transduction
pathways in which PKCs are involved.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL-43707.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: Dept. of
Biochemistry, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425.
1
The abbreviations used are: PKC, protein kinase
C; PKA, cAMP-dependent protein kinase; GFP, green
fluorescent protein; EGFP, enhanced GFP; AT1AR, angiotensin
II type 1A receptor; ETAR, endothelin A receptor;
2AR,
2-adrenergic receptor;
D2R, dopamine D2 receptor; HEK 293 cells, human
embryonic kidney 293 cells; PMA, phorbol 12-myristoyl 13-acetate;
IP3, inositol 1,4,5-triphosphate; DAG, diacylglycerol; PBS,
phosphate-buffered saline; CMV, cytomegalovirus; GPCR, G
protein-coupled receptor.
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