From the Departments of Ophthalmology and Visual
Sciences and the
Department of Cellular and Molecular
Physiology, Yale University School of Medicine, New Haven, Connecticut
06520-8026 and the § Department of Physiology and
Biophysics, Mount Sinai School of Medicine, New York University,
New York, New York 10029
Received for publication, August 21, 2000, and in revised form, October 16, 2000
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ABSTRACT |
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To investigate how G protein There is good evidence that the in vivo localization
and organization of G proteins and the proteins they interact with are important factors that regulate the specificity, kinetics, and magnitude of signaling pathways (1). Recent studies using fusions of
GFP1 with G protein-coupled
receptors, a G protein G protein For Varying effects of receptor-mediated activation on the localization of
To elucidate the factors responsible for targeting of Construction of GFP and RFP Fusion
Proteins--
Cell Fractionation--
HEK-293 cells (ATCC, CRL-1573)
(12.5 × 106 per 150-mm dish) were transfected with
37.5 µg of plasmid using DEAE-dextran (31). 48 h after
transfection, the cells were lysed by 10 passages through a 27-gauge
needle in 0.5 ml of an ice-cold buffer containing 50 mM
Tris (pH 8.0), 2.5 mM MgCl2, 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, and 1 mM dithiothreitol, and 10% glycerol. Nuclei were pelleted (750 × g, 5 min, 4 °C) and the
postnuclear supernatant was then fractionated (18,000 × g, 30 min, 4 °C) into membrane pellets and supernatants.
The pellets were washed once and resuspended in 0.5 ml of the same
buffer. Membrane proteins were quantified using the Lowry assay (32).
Samples (10 µg of membrane proteins and normalized volumes of the
supernatant fractions) were resolved by SDS-polyacrylamide
electrophoresis (10%), transferred to nitrocellulose, and probed with
the anti-EE monoclonal antibody as described (33). The antigen-antibody
complexes were detected using an anti-mouse horseradish
peroxidase-linked antibody according to the ECL Western blotting protocol.
Inositol Phosphate Formation Assay--
Recombinant Measurement of
Xenopus oocytes were surgically extracted, dissociated, and
defolliculated by collagenase treatment, and microinjected with 50 nl
of a water solution containing 1 ng of the desired cRNA. Oocytes were
incubated for 3 days at 19 °C. Whole oocyte currents were then
measured by conventional two-microelectrode voltage clamp with a
GeneClamp 500 amplifier (Axon Instruments). Agarose-cushion microelectrodes were used with resistance between 0.1 and 1.0 M Imaging of GFP and RFP Fusion Proteins--
0.5 × 106 HEK-293 cells were plated onto 35-mm tissue culture
dishes containing a glass coverslip (MatTek Corp., Ashland, MA) and
transiently transfected using LipofectAMINE 2000 Reagent (Life Technologies). Cells were imaged 48-72 h after transfection. The cells
were imaged using an inverted Zeiss microscope or a confocal microscope
(Bio-Rad MRC600). The inverted microscope was fitted with computer
controlled (IPLabs, Scanalytics) filter wheels (Ludl electronics) on
the excitation and emission paths to sequentially image the GFP and RFP
signals (excitation and emission filters from HQ fluorescein
isothiocyanate and Texas Red filter sets, dichroic mirror from
fluorescein isothiocyanate/Texas Red Pinkel filter set; Chroma,
Brattelboro, VT). For the confocal microscopy, factory supplied
fluorescein isothiocyanate and Texas Red excitation/emission optics
were used to image the GFP and RFP signals separately. The pixel
densities across the membrane were fit with IGOR software (Wavemetrics,
Oswego, OR).
Immunohistochemistry--
The cells were fixed in ice-cold,
PBS-buffered paraformaldehyde. The cells were subsequently rinsed with
PBS, and then incubated with 10% normal goat serum in PBS with 0.05%
Triton X-100 for 30 min. The primary antibodies (a rabbit anti-GFP
antibody from CLONTECH, a rabbit anti-peptide
antibody to a COOH-terminal sequence of
Cell fractionation and immunoblotting with an anti-EE monoclonal
antibody showed that
As for
Imaging of
Imaging of
To more clearly visualize the plasma membrane-associated populations of
the Imaging of
The substantial amount of cytosolic A Change in the Cellular Localization of
Stimulation of these cells with 10 µM UK-14,304 caused
PKC
There was no obvious translocation of the A Change in the Cellular Localization of
Therefore, to prevent rapid deactivation of
AlF4
As with hormonal stimulation, treatment of cells with
AlF4
A major difference between the mutations (RC and C9S/C10S) and
stimulation with hormone or AlF4 This is the first report of a functional G protein Our studies with The effects of mutational activation and removal of the palmitoylation
sites of It is initially surprising that the RC mutation, which has an
incomplete inhibitory effect on GTPase activity, decreases the association of There are several potential reasons why membrane-associated
An alternative explanation for why activation does not cause release of
membrane-associated With respect to interactions between The studies presented here provide the starting point for further
investigations of how G protein subunit
localization is regulated under basal and activated conditions, we
inserted green fluorescent protein (GFP) into an internal loop of
G
q.
q-GFP stimulates phospholipase
C in response to activated receptors and inhibits
-dependent activation of basal G protein-gated
inwardly rectifying K+ currents as effectively as
q does. Association of
q-GFP with the
plasma membrane is reduced by mutational activation and eliminated by
mutation of the
q palmitoylation sites, suggesting that
q must be in the inactive, palmitoylated state to be
targeted to this location. We tested the effects of activation by
receptors and by AlF4
on the
localization of
q-GFP in cells expressing both
q-GFP and a protein kinase C
-red fluorescent protein
fusion that translocates to the plasma membrane in response to
activation of Gq. In cells that clearly exhibit protein
kinase C
-red fluorescent protein translocation responses,
relocalization of
q-GFP is not observed. Thus, under
conditions associated with palmitate turnover and
dissociation,
q-GFP remains associated with the plasma
membrane. These results suggest that upon reaching the plasma membrane
q receives an anchoring signal in addition to
palmitoylation and association with
, or that during activation,
one or both of these factors continues to retain
q in
this location.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit, and other proteins in the G protein
signaling pathway, have provided new insights into how these proteins
behave under basal and activated conditions (2-5). However, an
important player in this pathway, the G protein
subunit, has
not yet been visualized in this way, presumably because the importance
of both the amino and carboxyl termini for localization and function
has precluded the fusion of GFP to these sites. Understanding the
mechanism by which
subunit localization is regulated requires tools
such as GFP-tagged
subunits that allow real-time visualization of
subunits in living cells. Studies using conventional procedures
such as cell fractionation and immunohistochemistry provide only a
limited view of the process, are subject to artifacts, and have yielded conflicting results.
subunits are peripheral membrane proteins that attach to
the plasma membrane as a result of amino-terminal myristoylation and/or
palmitoylation, and association with the
subunits. Whereas myristoylation occurs co-translationally and is stable throughout the
lifetime of the
subunit, palmitate is added post-translationally and turns over upon stimulation by activated receptors (6-10). Members
of the
i family (
i1,
i2,
i3,
o,
z, and
t) are myristoylated and all except
t are
also palmitoylated.
q and
s are
palmitoylated, but not myristoylated. However, the existence of
additional membrane-targetting signals associated with the amino
termini of
q and
s has been proposed (11,
12).
q and
s, the roles of palmitoylation
and association with
in regulating localization are
controversial. Mutations of the palmitoylation sites have been reported
either to cause these
subunits to partition exclusively in the
soluble rather than the particulate fraction (13) or to have little or
no effect on the fractionation of these
subunits (7, 11, 14-16).
Mutation of
q residues important for
interaction
has been reported to decrease palmitoylation and disrupt association
with the plasma membrane (17), but mutational activation of
s, which should decrease association with
, has
been reported to be associated with both decreased (6, 18) and normal
(19) association with the plasma membrane.
q and
s have been observed.
-Adrenergic receptor stimulation has been reported to cause
s to move from the particulate to the soluble fraction
(18, 20) and from the plasma membrane to the cytoplasm (18). However,
in other studies,
-adrenergic receptor-dependent
relocation of
s was not observed (19, 21). Thyrotropin-releasing hormone induced patching of
11 has
been reported to occur within 10-60 min of hormone exposure, followed by internalization within 2-4 h, time scales that are slow compared with that of the activation cycle (22).
q/11 has also
been reported to transiently translocate to the plasma membrane of adrenal glomerulosa cells in response to stimulation by angiotensin II
for 1-5 min (23).
q
to the plasma membrane and to determine whether the cellular
localization of
q changes during the activation cycle,
we have followed
q localization in vivo under
basal and activated conditions using a functional
q-GFP
fusion protein in which GFP is inserted into an internal loop of
q. The effects of mutational activation on targeting of
q-GFP have been tested by substituting cysteine for
Arg183 of
q, which inhibits GTPase activity
(24). We have also tested the effects of mutating the
q
palmitoylation sites, Cys9 and Cys10 (13), on
q-GFP localization. To determine whether
membrane-associated
q-GFP relocalizes upon activation,
the effects of stimulation by hormone-activated receptors or
AlF4
have been tested. Our results
indicate that although the basal activation state and palmitoylation
are important for initially targeting
q-GFP to the
plasma membrane, activation of membrane-associated
q-GFP, which is thought to cause palmitate turnover and
dissociation from
, does not cause relocalization. These results
suggest that upon reaching the plasma membrane
q
receives an anchoring signal in addition to palmitoylation and
association with
, or that during activation, one or both of
these factors continues to retain
q in this location.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q-GFP was generated from murine
q (25) and "humanized" GFP with the S65T mutation
(26) using polymerase chain reactions that produced DNA fragments with
overlapping ends that were combined subsequently in a fusion polymerase
chain reaction (27). GFP was inserted in between
q
residues 124 and 125. A 6-residue linker sequence (SGGGGS) was inserted
at both of the junctions between
q and GFP using
oligonucleotide-directed in vitro mutagenesis (28) using the
Bio-Rad Muta-Gene kit.
qRC-GFP and
q-C9S/C10S-GFP were generated from
q-GFP
using oligonucleotide-directed in vitro mutagenesis. All
q and
q-GFP constructs contain an
epitope, referred to as the EE epitope (29) that was generated by
mutating
q residues SYLPTQ (171) to EYMPTE.
PKC
-RFP was produced by replacing a KpnI/NotI
fragment encoding eGFP in pPKC
-EGFP (CLONTECH)
with a KpnI/NotI fragment encoding RFP
(CLONTECH). For expression in Xenopus
oocytes,
q and
q-GFP were subcloned as
NotI fragments into pGEM-HE (30). Subcloning and mutagenesis
procedures were confirmed by restriction enzyme analysis and DNA sequencing.
subunits
were transiently expressed in HEK-293 cells using DEAE-dextran (31).
106 cells per 60-mm dish were transfected with 1 µg of
vector containing the porcine
2a-adrenergic receptor
(34) and varying amounts of vector alone or vector containing an
subunit construct as indicated in the figure legends. 24 h after
transfection, the cells were replated in 24-well plates and labeled
with [3H]inositol (5 µCi/ml for 24 h). The assay
for intracellular inositol phosphates was performed as described
(33).
-Dependent Activation of Basal G
Protein-gated Inwardly Rectifying K+ Currents in
Xenopus Oocytes--
A human homolog of GIRK4, referred to as GIRK4*,
in which threonine is substituted for Ser143, was used as
described previously (35). Briefly, plasmids expressing GIRK4*,
q, or
q-GFP were linearized with Nhel and
cRNAs were transcribed in vitro using the "message
machine" kit (Ambion). RNAs were electrophoresed on formaldehyde gels
and concentrations were estimated from two dilutions using RNA marker
(Life Technologies, Inc.) as a standard.
.
Oocytes were constantly superfused with a high potassium solution having (in mM): 91 KCl, 1 NaCl, 1 MgCl2, and 5 KOH/HEPES (pH 7.4). To block currents, the oocyte chamber was perfused
with solutions of the same composition with 3 mM
BaCl2. Typically oocytes were held at 0 mV (EK)
and currents were constantly monitored by 500-ms pulses to a command
potential of
80 mV for 200 ms followed by a step to +80 mV for
another 200 ms and the cycle was repeated every 2 s. Current
amplitudes were measured at the end of the 200-ms pulse at each potential.
q/
11 from PerkinElmer Life Sciences, or a
rabbit anti-peptide antibody to an internal sequence of
q/
11 corresponding to residues 277-294
of
q from Sigma) were diluted with PBS and 10% normal goat serum with 0.05% Triton X-100 to a final concentration of 1:1000,
1:250, or 1:250, respectively, and added for an overnight incubation. The following day, the cells were washed with three changes of PBS, incubated for 60 min in a 1:500 dilution of a Texas
Red-conjugated goat anti-rabbit secondary serum (Jackson ImmunoResearch) and then washed three times in PBS. All experiments involved examining plates processed in parallel that were not exposed
to the primary antibody. The signal to noise in each experiment was
determined by comparing the signals produced in the presence and
absence of the primary antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q and
q-GFP Exhibit Similar
Functional Properties--
To produce a functional
q-GFP fusion protein, GFP was inserted in between
q residues 124 and 125 in the
B/
C loop of the helical domain (Fig. 1). The rationale
for this fusion strategy was that the helical domain of
q does not specify activation of PLC (33), and the
insertion site corresponds to a region in GPA1, a G protein
subunit
in Saccharomyces cerevisae, in which there is an insertion
of ~100 residues relative to other
subunits (36, 37). This design
avoided modifying the amino or carboxyl termini of
q, which are important for interaction with receptors,
PLC, and
, and for membrane attachment (11, 38). A 6-residue
linker sequence (Ser-Gly-Gly-Gly-Gly-Ser) was placed at each of the
junctions between
q and GFP. To enable comparison of the
expression levels of
q and
q-GFP, both
constructs contain an epitope, referred to as the EE-epitope (29),
located in the
E/
F loop of the helical domain. This epitope does
not interfere with receptor-mediated stimulation of PLC by
q (39).
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Fig. 1.
Model of
q-GFP. The structure of GFP
(54) is green. The
subunit structure is that of
t·GTP
S (55). The helical domain is pink
and the GTPase domain is light blue. GTP
S is yellow. The
SGGGGS linkers between GFP and the
subunit are shown schematically
in dark blue. This figure was drawn using MidasPlus,
developed by the Computer Graphics Laboratory at University of
California, San Francisco.
q and
q-GFP were
expressed in transiently transfected HEK-293 cells at similar levels
and exhibited similar distribution patterns (Fig.
2). In addition to associating with the
membrane pellet, both
q and
q-GFP were
isolated in the cytoplasmic fractions. To investigate whether the
activation state of
q affects targeting to the plasma
membrane, GFP was inserted into
qRC, in which
substitution of cysteine for Arg183 causes constitutive
activation of
q by reducing its GTPase activity (24).
For both
q and
q-GFP, the RC mutation
caused a minor increase in the relative amount of protein that
fractionated in the supernatant compared with the amount in the
membrane pellet. To test the role of palmitoylation in membrane
targeting, GFP was also inserted into
q-C9S/C10S, in
which serines were substituted for the
q palmitoylation
sites (13). The C9S/C10S mutations also increased the relative amount
of both
q and
q-GFP in the supernatant.
The effect of the C9S/C10S mutations was more pronounced than that of
the RC mutation, but neither
q-C9S/C10S nor
q-C9S/C10S-GFP was completely soluble.
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Fig. 2.
Expression of
q and
q-GFP constructs in HEK-293 cells.
HEK-293 cells were transfected with 3 µg/106 cells of
plasmid encoding the indicated constructs. The cells were fractionated
into membrane pellets (P), and supernatants (S),
which were separated using SDS-polyacrylamide gel electophoresis and
immunoblotted as described under "Experimental Procedures." The
fractionation patterns of
q,
q-R183C, and
q-C9S/C10S are similar or identical to those of the
corresponding GFP-fusion proteins. The R183C mutation modestly
increases the relative amount of protein in the supernatant, while the
C9S/C10S mutations have a more pronounced effect. Similar results were
obtained in two additional experiments.
q-GFP and
q exhibited indistinguishable
abilities to stimulate PLC in response to stimulation by the
2a-adrenergic receptor in transiently transfected
HEK-293 cells (Fig. 3). Although these cells express
q endogenously, inositol phosphate
production in response to stimulation with the
2-adrenergic agonist, UK-14,304, was minimal in cells
that were transfected solely with plasmid encoding the
2a-adrenergic receptor. Co-transfection of these cells
with plasmid encoding either
q or
q-GFP
resulted in agonist-dependent increases in inositol
phosphate production. The relationship between the magnitude of the
response and the amount of transfected plasmid was the same for the
q- and
q-GFP-expressing plasmids. Thus, the ability of
q to interact with receptors and
effectors is not impaired by the GFP insertion.
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Fig. 3.
q and
q-GFP exhibit identical abilities to
stimulate PLC in response to receptor stimulation. For each data
point, 106 HEK-293 cells were transfected with 1 µg of
plasmid encoding the
2a-adrenergic receptor and the
indicated amounts of plasmid encoding
q
(circles) or
q-GFP (squares).
Inositol phosphate (IP) formation was measured in the
presence (filled symbols) or absence (open
symbols) of 10 µM UK-14,304. The relationship
between the amount of IP formation and the amount of transfected
plasmid is the same for the
q- and
q-GFP-encoding plasmids. Values represent the means of
triplicate determinations ± S.D. from a single experiment, which
is representative of 2 such experiments.
q, the RC mutation caused constitutive activation
of
q-GFP (Fig. 4).
Stimulation of cells co-transfected with plasmid encoding the
2a-adrenergic receptor and plasmid encoding either
qRC or
qRC-GFP with UK-14,304 resulted in
further elevation of inositol phosphate levels, indicating that the RC
mutation inhibits, but does not abolish GTP hydrolysis. In contrast to
q-GFP, which stimulated PLC to the same extent as
q did (Figs. 3 and 4),
qRC-GFP was less
effective at PLC stimulation than
qRC was. Since the RC
mutation has been reported to destabilize
s (20), it is
possible that combining this mutation with the insertion of GFP results
in decreased stability of
qRC-GFP compared with that of
q-GFP or
qRC.
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Fig. 4.
Substitution of Arg183 in
q with cysteine (the RC mutation)
causes constitutive activation of
q-GFP. 106 HEK-293
cells were transfected with 3 µg of vector containing the indicated
construct and 1 µg of vector containing the
2a-adrenergic receptor. Inositol phosphate
(IP) formation was measured in the presence (light
gray bars) or absence (dark gray bars) of 10 µM UK-14,304. The RC mutation increases the basal
activity of both
q and
q-GFP. Further
increases in activity in response to UK-14,304 indicate that this
mutation inhibits, but does not abolish GTPase activity. Values
represent the means of triplicate determinations ± S.D. from a
single experiment, which is representative of three such
experiments.
q-GFP and
q also exhibited identical
abilities to interact with
, as demonstrated by inhibition of
-dependent activity of the G protein-gated inwardly
rectifying K+ channel, GIRK4 (Fig.
5). Agonist-independent (basal) potassium currents through GIRK4 homotetrameric channels expressed in
Xenopus oocytes can be inhibited by coexpression of
i subunits (35, 40). Since GIRK channels bind to and are
activated by only the
subunits of G proteins (41, 42), this
inhibition serves as a functional assay of
subunit binding to
subunits. Coexpression of
q displayed the same
inhibitory effect on
-stimulated basal GIRK4 currents.
Coexpression of
q-GFP with GIRK4 channels reduced K+ currents to the same extent as
q did,
indicating that GFP-tagged
q can bind to
and
prevent
-mediated activation of the channel as efficiently as
q can.
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Fig. 5.
q and
q-GFP exhibit identical abilities to
inhibit
-dependent
basal activity of GIRK4. K+ currents were recorded
from a GIRK4 mutant, GIRK4(S143T), referred to as GIRK4*, that conducts
large currents through homotetrameric channels expressed in
Xenopus oocytes (40). The addition of Ba2+
selectively blocks K+ currents resulting from heterologous
expression of GIRK4* currents (A-C). Coexpression of
q or
q-GFP with GIRK4* significantly and
similarly reduces agonist-independent (basal) currents compared with
those in oocytes expressing only GIRK4*. Summary data in D
represent averages in each group from five oocytes isolated from the
same frog. Similar results were obtained from experiments in oocytes
isolated from a different frog.
q-GFP,
qRC-GFP, and
qC9S/C10S in Living Cells and After Extraction with 1%
Triton X-100--
Imaging of transiently expressed
q-GFP in live HEK-293 cells with confocal microscopy
demonstrated signal in the plasma membrane and the cytoplasm (Fig.
6A). In some cells there were
distinct patches of
q-GFP in the plasma membrane. There
was some variability in the ratio of signal at the membrane compared
with that in the cytoplasm, but there was no correlation with the level
of expression. Bright cells had the same variability as did the dim
ones.
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Fig. 6.
Confocal imaging of
q-GFP and
q-GFP mutants in living cells.
HEK-293 cells were transiently transfected with: A,
q-GFP; B,
qRC-GFP; or
C,
qC9S/C10S-GFP. The cells were imaged with
a ×100 lens, numerical aperture of 1.2.
q-GFP exhibits
distinct signal in the plasma membrane, as well as in the cytoplasm and
the nucleus. The RC mutation greatly reduces the relative amount of
signal in the plasma membrane compared with that in the cytoplasm,
while the C9S/C10S mutations eliminate all detectable plasma membrane
signal. Bar = 10 µm.
qRC-GFP-expressing HEK-293 cells (Fig.
6B) demonstrated a significantly higher ratio of cytoplasmic
to plasma membrane signal than in
q-GFP-expressing cells
(Fig. 6A). As with
q-GFP, there was
expression level independent cell-to-cell variability in the
localization pattern. The C9S/C10S mutations had a more dramatic effect
on localization than the RC mutation in that they completely eliminated
detectable plasma membrane labeling by
q-GFP (Fig.
6C). These results suggest that targeting of
q-GFP to the plasma membrane requires both association
with
, which would be impaired by the RC mutation, and
palmitoylation of Cys9 and Cys10.
q-GFP constructs, we treated cells expressing these
constructs with 1% Triton X-100 to extract the soluble proteins. Surprisingly, although
qRC-GFP and
q-C9S/C10S-GFP exhibited quite similar distribution
patterns in living cells (Fig. 6), treatment of cells expressing one or
the other of these constructs with 1% Triton X-100 produced very
different results. Treatment of
qRC-GFP-expressing
HEK-293 cells (Fig. 7C) with
1% Triton X-100 revealed a signal in the plasma membrane (Fig.
7D) that was similar to that seen in Triton-treated
q-GFP-expressing cells (Fig. 7B). Therefore,
although the RC mutation decreases the percentage of
q-GFP that is associated with the plasma membrane, there
is residual
qRC-GFP signal at this location that is
masked in living cells because it is of similar intensity to the
cytoplasmic signal. However, in contrast to the results obtained with
cells expressing
q-GFP or
qRC-GFP, after
treatment of
q-C9S/C10S-GFP-expressing cells (Fig.
7E) with 1% Triton X-100, no staining of the plasma membrane was seen (Fig. 7F). Thus, Cys9 and
Cys10, the
q palmitoylation sites, are
essential for plasma membrane association.
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Fig. 7.
Imaging of
q-GFP constructs in living cells and
after extraction with 1% Triton X-100. Transiently transfected
HEK-293 cells were imaged on an inverted Zeiss microscope before and
after the addition of ice-cold 1% Triton X-100. The cells were
transfected with plasmid encoding
q-GFP (A
and B),
qRC-GFP (C and
D), or
q-C9S/C10S-GFP (E and
F). A, C, and E are images of the
living cells, while B, D, and F are
images derived from the same cells 2.5 min after the addition of
detergent. Although there is much less detectable plasma membrane
signal due to
qRC-GFP than to
q-GFP in
living cells, the signals in the Triton-treated cells are similar.
However, although the signals due to
qRC-GFP and
q-C9S/C10S-GFP are quite similar in living cells, there
is no detectable signal after Triton treatment of cells transfected
with
q-C9S/C10S-GFP. 2-s acquisition times were used for
all images, but considerable variation in signal strength required
adjusting the brightness/contrast of the final images by plotting
restricted ranges of the pixel values as follows: A,
400-2000; B, 400-1000; C, 400-3000;
D, 400-1500; E, 300-2500; F,
300-1250. Bar = 30 µm.
q-GFP in Living and Fixed Cells--
In
previous immunohistochemistry studies,
q was found
either predominantly in the plasma membrane (22), in the plasma and Golgi membranes (43), or associated with the cytoskeleton (23, 44).
Reasoning that preparation of specimens for immunohistochemistry might
affect the apparent localization pattern of
q and
therefore contribute to these diverse observations, we investigated
whether the
q-GFP signal was altered during fixation and
immunostaining (Fig. 8). The
q-GFP signal observed in living cells (Fig.
8A) did not change after fixation with 2% paraformaldehyde,
extraction with 0.05% Triton X-100, and treatment with an
antibody to GFP followed by a Texas Red-conjugated secondary antibody
(Fig. 8B). Furthermore, the Texas Red signal (Fig.
8C) was virtually identical to the intrinsic GFP signal.
Therefore, standard fixation procedures and detergent permeabilization
do not appear to selectively mask the
q-GFP signal or
extract it from particular cellular compartments.
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Fig. 8.
Imaging of
q-GFP in living cells and after
fixation. Transiently transfected HEK-293 cells were imaged on an
inverted Zeiss microscope, and the immunohistochemical labeling was
done on the microscope such that the same cells could be imaged after
each step. A, GFP signal before fixation. B, GFP
signal after fixation with paraformaldehyde, Triton X-100 extraction,
and antibody treatments. C, Texas Red anti-GFP antibody
signal. The GFP signal is the same before and after immunohistochemical
labeling and is accurately represented by the anti-GFP antibody signal.
Bar = 25 µm.
q-GFP potentially
could have been caused by the insertion of GFP. However, based on a comparison of the immunolocalization patterns of
q-GFP
and
q using an antibody to a COOH-terminal
q/
11 peptide, this does not appear to be
the case. The cytosolic signal seen with GFP in
q-GFP-expressing cells (Fig.
9A) was clearly labeled by the anti-
q antibody in the same cells (Fig. 9B)
and in cells expressing
q (Fig. 9C). The
q-GFP seen at the membrane with GFP (Fig. 9A) was under-represented by the Texas Red anti-
q antibody
signal (Fig. 9B). It is possible that association of
q with receptors limits access of the anti-peptide
antibody at the membrane, since the carboxyl termini of
subunits
are involved in receptor interaction. A second antibody to an internal
q epitope gave similar results (data not shown).
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Fig. 9.
Imaging of
q-GFP and
q after fixation. Transiently
transfected HEK-293 cells were imaged on an inverted Zeiss microscope
after fixation and immunohistochemical labeling. A, GFP
signal in HEK-293 cells transfected with plasmid encoding
q-GFP, and B, Texas Red anti-
q
peptide antibody signal in the same cells. The signals in A
and B are similar, but plasma-membrane-associated
q-GFP is less apparent in B. C,
Texas Red anti-
q peptide antibody signal in HEK-293
cells transfected with plasmid encoding
q. The
substantial amount of cytosolic signal in both B and
C indicates that the insertion of GFP is not responsible for
the presence of cytosolic
q-GFP. Variation in signal
strength required adjusting the brightness/contrast of the final images
by plotting restricted ranges of the pixel values as follows:
A, 0-3200; B, 0-2600; C, 0-800.
Bar = 10 µm.
q-GFP Is
Not Observed upon Hormonal Stimulation--
The effects of the RC and
C9S/C10S mutations on the localization of
q-GFP
suggested that targeting of
q to the plasma membrane requires association with
and palmitoylation of Cys9
and Cys10. Therefore, we investigated whether
receptor-mediated activation, predicted to cause dissociation of
q from
and palmitate turnover, leads to release
of
q-GFP from the plasma membrane. To be able to monitor
activation of
q-GFP, we co-transfected HEK-293 cells with plasmids encoding
q-GFP, the
2a-adrenergic receptor, and PKC
-RFP. PKC-GFP fusion
proteins transiently associate with the plasma membrane in response to
stimulation of Gq-coupled receptors (3). Thus, the
PKC
-RFP signal served to confirm signaling through Gq on
a cell by cell basis.
-RFP to transiently associate with the plasma membrane (Fig.
10, A and B). In
any given field, not all of the cells exhibited a PKC translocation
response, but in those that did, no change in the localization of
q-GFP was observed (Fig. 10, C and
D). Simultaneous images of PKC
-RFP and
q-GFP were collected every 2 s for 800 s,
which produced a three-dimensional stack of images with time in the Z
dimension. The PKC
-RFP and
q-GFP signals at the
membrane can be followed more carefully over time by looking at a
90o rotation of a small region of the image stack
(boxes, Fig. 10, A and C). Such
projections are shown in Fig. 10, B and D. Each projection extends 400 frames from top to bottom and in Fig.
10B the dramatic transient movement of PKC
-RFP to the
plasma membrane is clear. Fig. 10D shows that the
q-GFP signal remained in the plasma membrane throughout
the duration of the experiment. Video 1 is a movie showing the entire
sequence of PKC
-RFP and
q-GFP images in this
experiment.
View larger version (57K):
[in a new window]
Fig. 10.
A change in the cellular localization
of q-GFP is not observed upon
hormonal stimulation. HEK-293 cells were co-transfected with
vectors encoding
q-GFP, PKC
-RFP, and the
2a-adrenergic receptor. The cells were heated to
30 °C and imaged with confocal microscopy. 400 images were collected
at 2-s intervals, and the cells were stimulated with 10 µM UK-14,304 in between frames 3 and 5. This produced a
three-dimensional stack of images with time in the Z dimension.
A, images of PKC
-RFP at selected time points indicating
hormone-dependent transient association with the plasma
membrane. B, 90o rotation of PKC
-RFP images
within the box in A over the whole time course such that
time is now the y axis. C, images of
q-GFP in the same cells and time points as in
A demonstrating that stimulation with hormone does not
result in detectable relocalization. D, 90° rotation of
q-GFP images within the boxed region in
C over the whole time course. The images in A and
C are 31 × 42 µm. Video is available (Video 1). In
the video as in the figure, the images on the left are of
PKC
-RFP and those on the right are of
q-GFP.
q-GFP signal
following stimulation, but it is possible that the movement of
q-GFP is more rapid and/or less pronounced than the
movement of PKC
-RFP. To explore the spatial limits within which a
change in the localization of
q-GFP could have been
detected, we quantified the membrane staining pattern of
q-GFP within the boxed region shown in Fig. 10C for the first 400 s of the experiment. For each
frame, the signal across the membrane was fit to a Gaussian
distribution (Fig. 11A) and
the S.D. of the distribution was used to estimate the width of the
signal at the membrane. Fig. 11B shows these standard deviations plotted versus time in the stimulated cell from
Fig. 10 (left) and in an unstimulated control cell. In the
stimulated cell, the standard deviations varied between 0.04 and 0.08 µm with no clear increase preceding the PKC response. For comparison, the membrane signal of a control cell measured in the same manner showed a variability in thickness that exceeded any measured in the
responding cell. If
q-GFP translocates from the membrane in response to stimulation, it must do so more quickly than the frame
rate (1 image/2 s) can capture and/or move only short distances that
would not change the membrane profile by more than 0.04 µm.
View larger version (30K):
[in a new window]
Fig. 11.
Width of the
q-GFP membrane signal over time in
stimulated and unstimulated cells. To quantify the
q-GFP signal at the membrane, the signal across the
membrane was fit to a Gaussian distribution and the S.D. of the fit was
used to estimate the width of the signal. A, fit of the
q-GFP signal across the membrane from the boxed
region of frame 100 in Fig. 10C. B, plot of the
standard deviations of the Gaussian fits of the
q-GFP
signals in the boxed region of the first 200 frames in Fig.
10C (left) versus time and from a
similar region of
q-GFP membrane signal in a cell
stimulated with vehicle (right). In the stimulated cell, the
width of the membrane signal did not change during the interval between
the stimulus and the PKC response. Variability in membrane thickness
was greater in the control cell than in the stimulated cell.
q-GFP Is
Not Observed Upon Stimulation with Aluminum Fluoride--
One
potential reason why release of
q-GFP from the plasma
membrane was not observed in response to receptor stimulation is that
the activation/deactivation cycle of
q is too fast to be measured on the time scale over which we collected images. It has been
proposed that rapid GTP hydrolysis may allow receptors and G proteins
to remain bound throughout the GTPase cycle (45). In the presence of
PLC or RGS4, Gq can hydrolyze GTP with a deactivation half-time of 25-75 ms at 30 °C (46).
q-GFP by GTP
hydrolysis, we stimulated with AlF4
.
AlF4
activates
subunits by binding
to the GDP-bound form and mimicking the
-phosphate of GTP in the
transition state intermediate of the GTPase reaction (47, 48).
Stimulation with AlF4
thus provided a
means to test whether prolonged activation of
q would
result in its release from the plasma membrane.
stimulation of HEK-293 cells
transiently co-transfected with
q-GFP and PKC
-RFP
produced oscillating PKC translocation responses. Fig.
12A shows a PKC
-RFP image
before the first translocation response. Fig. 12B shows a
PKC
-RFP image in the same field during one of the membrane
translocation responses. Fig. 12C, a projection of the
PKC
-RFP image stack in the boxed region in Fig.
12B rotated such that time is the Y axis, shows
the oscillating PKC
-RFP responses. Although
AlF4
activates all G proteins, its
effect on PKC localization involves activation of
q,
because the number and magnitude of translocation responses was greater
in cells that were co-transfected with
q-GFP than in
cells transfected only with PKC
-RFP. Of 82 cells co-transfected with
q-GFP and PKC
-RFP (from two experiments), 46 exhibited at least one response, with a total of 216 oscillations. In
contrast, of 61 cells transfected with PKC
-RFP alone (from two
experiments), only 18 responded, with a total of 63 oscillations.
Therefore, although HEK-293 cells express
q
endogeneously, the level of expression appears to be limiting with
respect to mediating PKC responses to
AlF4
.
View larger version (106K):
[in a new window]
Fig. 12.
A change in the cellular localization
of q-GFP is not observed upon
stimulation with AlF4
.
HEK-293 cells were co-transfected with plasmids encoding
q-GFP and PKC
-RFP. Cells were stimulated with 30 µM AlCl3, 10 mM NaF at 37 °C
and images were collected on the inverted microscope in sets of 10 in
which 1
q-GFP image was followed by 9 PKC
-RFP images.
AlF4
was added after frame 1 (first
PKC
-RFP image). The interval between frames was ~1 min for the
first 2 sets of images. 20 sets of images were then collected with
~10-s intervals between frames. Images were collected out to 46 min
after application of the stimulus. The first response in the field
shown occurred in frame 13 (12.41 min after addition of
AlF4
; A) PKC
-RFP, before
the first response (frame 9, 8.27 min after application of the
stimulus). This is used as the "before" image, because cell
movement in the initial frames made it impossible to make direct
comparisons with later frames. B, PKC
-RFP during
oscillation response (frame 69, 26.86 min after application of the
stimulus). C, 90o rotation of PKC
-RFP images
within the box in B over the whole time course such that
time is now the y axis. Increases in signal in the plasma
membrane (right) correspond to decreases in signal in the
cytoplasm (left) and vice versa. In this experiment, 8 transient translocations of PKC
-RFP occurred. D,
q-GFP before the first PKC
-RFP response (frame 10, 10.33 min after application of the stimulus); E,
q-GFP during PKC
-RFP oscillation response (frame 70, 27.08 min after application of the stimulus). F,
90o rotation of
q-GFP images within the
boxed region in E over the whole time course,
indicating that stimulation with AlF4
does not cause detectable relocalization. Bar = 20 µm. Video is available (Video 2). In the video, as in the figure, the
images on top are of PKC
-RFP and those on the bottom are
of
q-GFP. Since 1
q-GFP image was
collected for every 9 PKC
-RFP images, the single
q-GFP image that corresponds to each set of 9 PKC
-RFP
images is shown below the PKC
-RFP images.
did not produce a detectable
change in the localization of
q-GFP in cells that
clearly gave PKC
-RFP translocation responses. Images of the
q-GFP signal before (Fig. 12D) and during
(Fig. 12E) the PKC
-RFP responses showed the same
distribution pattern. The consistency of the
q-GFP
signal after stimulation with AlF4
is
further demonstrated in Fig. 12F, which shows a projection of the
q-GFP image stack in the boxed region
in Fig. 12E, rotated as in Fig. 12C. Even when
activation of
q-GFP is irreversible, there is no
movement out of the membrane within the time frame of many cellular
responses. Video 2 is a movie showing the entire sequence of PKC
-RFP
and
q-GFP images in this experiment.
is
that the mutations affect
q-GFP from the moment that it
is synthesized, while stimulation occurs subsequent to the initial targeting of
q-GFP to its cellular locations. The
mechanistic implications of the different effects of the mutations
versus the stimulations are discussed below.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit-GFP
fusion protein.
q-GFP exhibits normal interactions with a G protein-coupled receptor, with
, and with PLC, and has made it possible to track the behavior of
q in living cells
without the potential artifacts that can arise from antibody staining, and from studying fixed or fractionated cells. We have tested the roles
of activation state and palmitoylation in targeting of newly
synthesized
q to the plasma membrane. We have also
investigated the effects of activation by receptors and by
AlF4
on the localization of
q. These studies reveal that although activation and
lack of palmitoylation can disrupt targeting of
q to the
plasma membrane, membrane-associated
q does not relocate upon activation.
q-GFP provide new insights into the
cellular location and behavior of
q in resting cells.
The observed inhomogeneity of the
q-GFP membrane
population may indicate compartmentalization with other signaling
proteins, which could contribute to the efficiency and specificity of
signaling. For instance, previously reported clusters of
2A-adrenergic receptors (49) resemble the
q-GFP patches seen here. Such compartmentalization has
been observed for the
-adrenergic receptor-Gs-adenylyl
cyclase system, which has been localized to low density plasma membrane
fractions that can be separated from the bulk of the plasma membrane
(50). In addition, although
subunits have generally been thought to be primarily associated with the plasma membrane, our results with
q-GFP indicate that a significant amount of
q is also in the cytoplasm. The functional significance
of this cytoplasmic
q population remains to be determined.
q-GFP are consistent with the "two-signal model" for plasma membrane binding (17). According to this model, the
initial localization signal for
subunits such as
q
that can be palmitoylated, but not myristoylated is association with
, which is prenylated on the
subunit. After localization of the heterotrimer to the plasma membrane, in which palmitoyl transferase is proposed to be located (51), palmitoylation takes place, which leads
to stable association with the plasma membrane. In support of this
model, mutation of
q residues important for
interaction decreases both palmitoylation and association with the
plasma membrane (17). Mutational activation of
q-GFP
would thus decrease plasma membrane binding by impairing association with
, which binds preferentially to the GDP-bound form of
subunits. Although both mutational activation and removal of the palmitoylation sites reduce association of
q-GFP with
the plasma membrane, the effect of removing the palmitoylation sites is
stronger. The partial effect of the RC mutation is consistent with the
fact that it inhibits but does not abolish GTPase activity. The
fraction of
qRC-GFP that is in the GDP-bound form will
be able to associate with the plasma membrane.
q-GFP with the plasma membrane, while
treatment with AlF4
, which causes
irreversible activation, has no such effect. However, there is a
significant difference between these two manipulations of
q-GFP. Whereas the mutation is present from the moment
that the protein is synthesized, AlF4
is applied to cells in which there is already a membrane-associated pool of
q-GFP. The simplest explanation for the apparent
discrepancy is that the activated state can prevent targeting of newly
synthesized
q-GFP to the plasma membrane, but after
q-GFP associates with the membrane, this association is
resistant to subsequent activation.
q-GFP does not relocate to the cytoplasm upon
activation. Although release from the membrane might be predicted if
palmitoylation and association with
were the only signals
important for membrane localization, one scenario is that upon membrane
association an additional localization signal is acquired that ensures
that
q-GFP remains there throughout the activation
cycle. This hypothetical signal could be a protein-protein interaction
or an additional modification that occurs once
q-GFP
reaches the plasma membrane. For instance, a feature of the amino
terminus of
q other than palmitoylation of
Cys9 and Cys10 is proposed to contribute to
hydrophobicity and membrane association (11).
q-GFP is that upon activation the
palmitates may not be completely removed and/or
q-GFP
may not completely dissociate from
. It is also possible that
only one of these two localization signals is required to retain as opposed to initially target
q-GFP in the membrane. With
regard to the level of palmitate that remains after activation,
although receptor stimulation increases turnover of palmitate on
s, the stoichiometry of palmitoylation on
s isolated from basal and stimulated cells was found to
be the same (21). In the case of
q, receptor stimulation
causes turnover of palmitate (9, 10), but the effect on the
stoichiometry of palmitoylation has not been determined.
and
during the GTPase
cycle, the generally accepted model for G protein activation assumes
that
and
dissociate from each other upon activation. However, it is possible that
and
merely shift relative to each other, rather than completely separate. The contact between the
subunit and switch II is likely to be severed during activation, since switch II residues are required for the interactions of most if
not all
subunits with their effectors (52). However, it is possible
that the connection of the
subunit to the amino terminus of the
subunit is preserved throughout the cycle. Given that
q
and
q-GFP inhibit
-dependent basal
activity of GIRK4 with equal effectiveness, they are likely to possess
similar affinities for
.
subunits are organized in
vivo under basal and activated conditions. Given the overall structural similarity of the
subunits, the GFP insertion site that
worked for
q is likely to be useful for other
subunits as well. For instance, residues in this region of
s (53) and
i2 (33) can be replaced with
homologs from other
subunits without disrupting function.
GFP-tagged
subunits will also make it possible to follow
subunits over time during development in specialized cells such as
neurons and polarized epithelia in tissue culture and intact animals.
Additionally, these reagents will be useful for colocalization studies
with RFP-tagged
subunits and receptors. Fluorescence resonance
energy transfer between GFP-tagged
subunits,
subunits, and
receptors may also be possible, which would enable monitoring of the
interactions of these proteins during activation cycles.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Galina Grishina and Rolando Medina
for producing the q-GFP construct, and Thomas Hynes for
the computer graphics, helpful discussions, and critical reading of the text.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants GM50369 (to C. H. B.), EY08362 (to T. E. H.), and HL54185 (to D. E. L.).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.
The on-line version of this article (available at
http://www.jbc.org) contains Video 1 (Fig. 10) and Video 2 (Fig. 12).
¶ Established Investigator of the American Heart Association.
** Established Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 203-785-3202; Fax: 203-785-4951; E-mail: catherine.berlot@yale.edu.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M007608200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GFP, green
fluorescent protein;
RFP, red fluorescent protein;
PKC, protein kinase
C;
GTPS, guanosine 5'-O-(3-thiotriphosphate);
PLC, phosphoinositide phospholipase C;
PBS, phosphate-buffered saline.
![]() |
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