Real-Time Optical Monitoring of Ligand-Mediated Internalization of
1b-Adrenoceptor with Green Fluorescent Protein
Takeo Awaji,
Akira Hirasawa,
Masakazu Kataoka,
Hitomi Shinoura,
Yasuhisa Nakayama,
Tatsuo Sugawara,
Shun-ichiro Izumi and
Gozoh Tsujimoto
Department of Molecular and Cell Pharmacology National
Childrens Medical Research Center Setagaya-ku, Tokyo, 154
Japan
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ABSTRACT
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The study of G protein-coupled receptor
signal transduction and behavior in living cells is technically
difficult because of a lack of useful biological reagents. We show here
that a fully functional
1b-adrenoceptor
tagged with the green fluorescent protein
(
1bAR/GFP) can be used to determine the
molecular mechanism of internalization of
1bAR/GFP in living cells. In mouse
T3
cells,
1bAR/GFP demonstrates strong, diffuse
fluorescence along the plasma membrane when observed by confocal laser
scanning microscope. The fluorescent receptor binds agonist and
antagonist and stimulates
phosphatidylinositol/Ca2+ signaling in a
similar fashion to the wild receptor. In addition,
1bAR/GFP can be internalized within minutes
when exposed to agonist, and the subcellular redistribution of this
receptor can be determined by measurement of endogenous fluorescence.
The phospholipase C inhibitor U73,122, the protein kinase C activator
PMA, and inhibitor staurosporine, and the
Ca2+-ATPase inhibitor thapsigargin were used to
examine the mechanism of agonist-promoted
1bAR/GFP redistribution. Agonist-promoted
internalization of
1bAR/GFP was closely
linked to phospholipase C activation and was dependent on protein
kinase C activation, but was independent of the increase in
intracellular free Ca2+ concentration. This
study demonstrated that real-time optical monitoring of the subcellular
localization of
1bAR (as well as other G
protein-coupled receptors) in living cells is feasible, and that this
may provide a valuable system for further study of the biochemical
mechanism(s) of agonist-induced receptor endocytosis.
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INTRODUCTION
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1-Adrenoceptors (
1ARs) play critical
roles in the regulation of a variety of physiological processes (1).
Considerable progress has been made toward a molecular description of
the structures and signal transduction mechanisms of
1ARs (2). The primary structure of cloned
1ARs corresponds to the predicted topographic model of
the superfamily of G protein-coupled receptors (GPCRs), and
substantial evidence indicates the importance of agonist and G
protein-regulated phospholipase C (PLC) for the generation of
phosphoinositide (PI)-derived second messengers for Ca2+
signaling in response to
1AR activation (2, 3, 4). The
convergence of recent pharmacological and molecular cloning studies has
revealed the presence of at least three subtypes of
1ARs, among which the
1bAR subtype was
the first to have its primary structure and is apparently prototypic of
the large family of Ca2+-mobilizing GPCRs.
Despite these important advances, much still remains unclear in our
understanding of regulation of
1AR function,
particularly regarding the disposition of the receptor in the cell
membrane and the influence of agonist on receptor distribution,
responsiveness, and metabolism. Although desensitization of
1AR responses by agonist has been reported, the kinetics
of these processes vary among different systems (5, 6, 7), and controversy
also exists over whether agonists cause sequestration of the receptor
from the extracellular surface (7, 8, 9). Furthermore, prolonged agonist
exposure has been reported to decrease total receptor number in some
(10, 11) but not all systems (5, 12). Attempts to study receptor
distribution at the subcellular level have been limited by the lack of
specific structural probes. In other systems, immunological approaches
have provided powerful tools for studying receptor localization and
organization at the cellular level (13, 14, 15). However, even this
technique has major limitations, including application in living cells,
nonstoichiometric labeling of receptors, the eventual dissociation of
the antibody from the receptor, and an inability to label intracellular
receptors in nonpermeabilized cells.
Green fluorescent protein (GFP) from the jellyfish Aequorea
victoria has been used as a reporter of gene expression and a
fusion tag to monitor protein localization within living cells
(16, 17, 18, 19). It has an inherent green bioluminescence that can be excited
optically by blue light or by nonradiative energy transfer (19, 20, 21),
and it stoichiometrically labels when integrated into cDNA as either an
amino- or a carboxyl-terminal fusion protein. Here, we report the
characterization of what seems to be a fully functional
carboxyl-terminal
1bAR/GFP fusion protein.
1bAR/GFP stably expressed in mouse
T3 cells has
normal antagonist and agonist binding and activation of
Ca2+-mobilization and is sequestered (internalized) in
response to agonist stimulation. Furthermore, agonist-promoted
1bAR/GFP internalization can be readily monitored in
living cells and pharmacologically characterized. This study suggests
that
1bAR/GFP and other similarly conjugated GPCRs
should prove to be important tools for the optical measurement of
biochemical and biophysical processes that are relevant to GPCR signal
transduction.
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RESULTS
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Pharmacological Comparison of Wild-Type
1bAR and
1bAR/GFP
Expression constructs used in the present study are shown in Fig. 1
. Mouse
T3 cells did not contain any
detectable
2-[ß-[4-hydroxy-3-[125I]iodo-4-hydroxyphenyl]-ethyl-aminomethyl]
tetralone ([125I]HEAT)-binding sites, and norepinephrine
(NE) (100 nM) did not elicit a response of intracellular
free Ca2+ concentration either before or after transfection
with the expression vector alone (data not shown). In contrast,
membrane preparations from
T3 cells stably transfected with the
wild-type
1bAR genes or
1bAR/GFP genes
showed saturable bindings of [125I]HEAT (Table
1A). The saturation isotherms for both
receptors are nearly identical, as summarized in Table 1
. The agonist-
and antagonist-binding characteristics of each receptor were likewise
similar (Table 1B). Pretreatment of the membrane preparation in
hypotonic buffer with 10 µM chloroethylclonidine (CEC)
for 30 min inactivated more than 95% of [125I]HEAT
binding sites in both cell lines.

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Figure 1. Constructs for Wild-Type and GFP-Fused
1bARs
As described in Materials and Methods, we constructed
GFP-fused 1bAR. Dashed boxes are the putative
membrane-spanning domains. Black boxes are the epitope regions for
antipeptide antibodies. The amino acid compositions of the epitope
regions for antipeptide antibodies and those across the fusion
point are also shown in the figure.
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The coupling to G protein of the wild-type
1bAR and
1bAR/GFP was investigated by measuring their ability to
stimulate whole cell PLC (Fig. 2
). Basal
levels of inositol triphosphate (IP3) production
were unaffected by the presence of the GFP, and the conjugated receptor
activated PLC as well. The EC50 values for PLC stimulation
by NE were similar: 53.4 ± 8.5 nM (n = 3) for
wild-type and 55.6 ± 5.3 nM (n = 3) for the
conjugate.
The effects of NE on the elevation of [Ca2+]i
were also compared. NE (100 nM) caused a rapid increase in
[Ca2+]i in a single
T3 cell stably
expressing wild-type
1bAR (Fig. 3B
) and
1bAR/GFP (Fig. 3C
); however, NE (100 nM) did not increase
[Ca2+]i in
T3 cells transfected with only
GFP (Fig. 3A
). Activation of endogenous PI-linked receptor GnRH
receptor by GnRH (100 nM) caused a rapid rise in
[Ca2+]i either in untransfected
T3 cells
(data not shown) or in cells stably expressing
1bAR/GFP
(Fig. 3D
). The effect of the PLC inhibitor U73,122 was also examined.
U73,122 (10 µM) did not cause any change in the basal
[Ca2+]i level. In both cell lines,
pretreatment with U73,122 (10 min) almost abolished the NE-induced
[Ca2+]i response, whereas an inactive analog,
U73,343, had no effect on the NE-induced
[Ca2+]i response (data not shown).
Localization of
1bAR and
1bAR/GFP
The similar pharmacological and biochemical properties exhibited
by wild-type
1bAR and
1bAR/GFP suggest
that their cellular distribution and trafficking might be similar. The
series of micrographs shown in Fig. 4
demonstrate that they have similar cellular distribution. We first
examined the cellular distribution of receptor using fluorescent
anti-
1bAR antibody 1B-N1-C (Fig. 4
, AD) and also the
endogenous receptor GFP fluorescence (Fig. 4E
) by the fluorescent
confocal microscopy. The immunocytochemical analysis with 1B-N1-C
showed that the fluorescence distribution of
1bAR is
typical of a plasma membrane-labeling pattern in
T3 cells stably
transfected either with the wild-type
1bAR genes (fixed
cells, Fig. 4A
; living cells, Fig. 4B
) or
1bAR/GFP genes
(fixed cells, Fig. 4C
; living cells, Fig. 4D
). Furthermore, as shown in
Fig. 4F
, the endogenous receptor fluorescence (Fig. 4E
) was well
correlated with immunostaining (Fig. 4D
) in living
T3 cells stably
transfected with
1bAR/GFP genes, confirming a plasma
membrane-labeling pattern. No fluorescent signal was detected in
untransfected
T3 cells, and the fluorescent signals were distributed
uniformly throughout whole cell in
T3 cells transfected only with
the GFP gene (data not shown).
NE-Stimulated Redistribution of
1bAR/GFP
We next investigated whether we could monitor the changes in the
subcellular localization of
1bAR/GFP using this
experimental system. Figure 5
showed a
consecutive X-Y scan of a single
T3 cell after the application of NE
(100 nM). The redistribution of GFP-associated fluorescent
signal became apparent about 8 min after the application of NE.
Redistribution reached a steady state at about 15 min, which lasted
unchanged until 60 min after application of NE. The time courses for
NE-induced subcellular distribution of the endogenous receptor GFP
fluorescence signal and immunofluorescent signal (expressed by a cell
surface localization ratio) are shown in Fig. 6
. The internalization kinetics of
GFP-associated fluorescent signal and the immunofluorescent signal were
similar; however, consecutive monitoring could not be performed for
immunocytochemical analysis, since fluorescent dye became quickly
photobleached. On the other hand, GFP-associated fluorescent signal was
found to be bleach-resistant and could be consecutively monitored at
1-min intervals.

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Figure 5. Real-Time Monitoring of NE-promoted Internalization
of 1bAR/GFP
Internalization of 1bAR/GFP was monitored at 1-min
intervals after NE (100 nM) stimulation using a confocal
laser scanning microscope. Shown are images obtained at 5, 10, 15, and
30 min after NE stimulation. The internalization of GFP-associated
fluorescent signal became apparent about 8 min after the application of
NE. The internalization reached a steady state after about 15 min. The
results presented are representative of at least three similar
experiments.
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Internalization of many receptors and integral membrane proteins occurs
by endocytosis to a common endosomal compartment from which further
intracellular sorting or recycling to the cell surface proceeds (22).
To determine whether
1bAR trafficking occurs by a
similar pathway, we further examined fluorescence colocalization of the
internalized receptors with Cy3-conjugated transferrin, a classic
endosomal marker. Confocal images obtained from control cells showed
that
1bAR/GFP is mainly localized to the cell surface
while transferrin receptors reside in internal vesicles (Fig. 7A
). After 30 min of NE exposure, the
internalized
1bAR/GFP colocalizes with Cy3-conjugated
transferrin in endosomes (Fig. 7B
). This result indicates that receptor
translocates to endosomes during agonist exposure. In addition,
agonist-induced receptor internalization was completely prevented by
pretreatment of cells with hyperosmotic sucrose solutions (0.45
M), a procedure that has been shown previously to inhibit
receptor-mediated endocytosis (23) (data not shown). In sum, these
results are consistent with an endocytic pathway of receptor
internalization.
In the following series of experiments, we therefore examined effects
of various pharmacological treatments on the NE (100
nM)-induced redistribution of GFP-associated fluorescent
signal at 30 min after application of NE.
Characterization of the Internalization of
1bAR/GFP
Without any stimulation (control), most of the GFP-associated
fluorescent signal was localized on plasma membrane (cell-surface
localization ratio = 0.98 ± 0.02, n = 6) (Fig. 8A
). After 30 min of NE stimulation, this
ratio had decreased to 0.65 ± 0.12 (n = 6, Fig. 8B
).
Pretreatment with
-AR antagonist phentolamine (10 µM)
completely inhibited both the NE (100 nM)-induced
internalization of
1bAR/GFP (Fig. 8
, C and D) and the
NE-stimulated increase in [Ca2+]i (data not
shown). In addition, the PLC inhibitor U73,122 (10 µM, 10
min) was found to inhibit both the NE-promoted internalization (Fig. 8
, E and F) and NE-stimulated increase in
[Ca2+]i (data not shown).

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Figure 8. Effects of Various Agents on Redistribution of
1bAR/GFP
1bAR/GFP distribution was recorded by a confocal laser
scanning microscope. A and B, The effect of NE on
1bAR/GFP distribution. Before NE stimulation,
1bAR/GFP was distributed over the cell surface (A).
Exposure to NE (100 nM) for 30 min resulted in the
internalization of 1bAR/GFP (B). C and D, the effect of
pretreatment with phentolamine (10 µM, 10 min) on
NE-promoted internalization of 1bAR/GFP. Before NE
stimulation, 1bAR/GFP was distributed over the cell
surface (C). Pretreatment with phentolamine inhibited NE (100
nM, 30 min)-promoted internalization of
1bAR/GFP (D). E and F, The effect of pretreatment with U73,122 (10 µM, 10 min) on
NE-promoted internalization. Before NE stimulation,
1bAR/GFP was distributed over the cell surface (E).
Pretreatment with U73,122 inhibited NE (100 nM, 30
min)-promoted internalization of 1bAR/GFP (F). G and H,
The effect of thapsigargin on 1bAR/GFP distribution.
Before application of thapsigargin, 1bAR/GFP was
distributed over the cell surface (G). Application of thapsigargin (1
µM) for 30 min did not cause any internalization of
1bAR/GFP (H). I and J, The effect of PMA on
1bAR/GFP distribution. Before application of PMA,
1bAR/GFP was distributed over the cell surface (I).
Application of PMA (1 µM, 30 min) caused an
internalization of 1bAR/GFP (J). K and L, The effect of
pretreatment with staurosporine (10 µM, 10 min) on
NE-promoted internalization. Before NE stimulation,
1bAR/GFP was distributed over the cell surface (K).
Pretreatment with staurosporine inhibited NE (100 nM, 30
min)-promoted internalization of 1bAR/GFP (L). M and N,
The effect of GnRH on 1bAR/GFP distribution. Before GnRH
stimulation, 1bAR/GFP was distributed over the cell
surface (M). Application of GnRH (100 nM, 30 min) caused
the internalization of 1bAR/GFP (N). Each result
presented is representative of at least six experiments.
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Thapsigargin, which blocks ATP-dependent Ca2+ uptake into
endoplasmic reticulum and elicits an increase in
[Ca2+]i without activating PLC, did not cause
any change in subcellular localization of
1bAR/GFP (Fig. 8
, G and H). The protein kinase C (PKC) activator PMA (1
µM, 30 min) did not increase
[Ca2+]i, but did promote a redistribution of
1bAR/GFP in a similar fashion to NE stimulation (Fig. 8
, I and J), although to a lesser extent. Staurosporine (10
µM, 10 min), which inhibits PKC activity, suppressed the
NE-mediated internalization of
1bAR/GFP (Fig. 8
, K and
L). Furthermore, stimulation of endogenous PI-linked GnRH receptor by
GnRH (100 nM) was also found to cause internalization of
1bAR/GFP (Fig. 8
, M and N). The internalization of
GFP-associated fluorescent intensity promoted by the various
pharmacological treatments is summarized in Fig. 9
.

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Figure 9. Effects of Various Pharmacological Agents on
1bAR/GFP Redistribution
Internalized GFP-associated fluorescent intensity as shown in Fig. 8
was summarized in a quantitative fashion as described in
Materials and Methods. A, Effects of pretreatment with
phentolamine (Phe), U73,122, or staurosporine (Stauro) on NE-promoted
1bAR/GFP internalization. T3 cells stably expressing
1bAR/GFP were treated with NE alone (100 nM)
for 30 min, or pretreated for 10 min with phentolamine (10
µM) or U73,122 (10 µM) or with
staurosporine (10 µM) and then stimulated by NE (100
nM) for 30 min. *, P < 0.05
vs. NE alone. B, Effects of NE, GnRH, PMA, or
thapsigargin (Thap) on 1bAR/GFP internalization. T3
cells stably expressing 1bAR/GFP were treated for 30 min
by NE (100 nM), GnRH (100 nM), PMA (1
µM), or thapsigargin (1 µM). *,
P < 0.05 vs. without stimulation.
Values are given as mean ± SD of at least six
independent experiments.
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DISCUSSION
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The
1bAR/GFP fusion protein has enabled us to
visualize
1bAR with subcellular resolution in live cells
and to pharmacologically characterize mechanisms of agonist-induced
receptor internalization. Our data demonstrate that the ligand binding,
signal-coupling properties, cellular distribution, and trafficking
behavior of
1bAR/GFP closely resemble those of the
wild-type
1bAR. In mouse
T3 cells, the fluorescence
distribution of
1bAR/GFP is characteristic of a plasma
membrane-labeling pattern, and the application of agonist promoted the
internalization of
1bAR/GFP. The second messenger
mechanism for this internalization was pharmacologically determined by
using this optical experimental system. NE-promoted internalization of
1bAR/GFP was blocked by the
1AR
antagonist phentolamine and by the PLC inhibitor U73,122. The
agonist-induced internalization was mimicked either by activation of
PLC through endogenous PI-linked GnRH receptor (24) or by stimulation
of PKC with PMA, but not by a simple rise in
[Ca2+]i with thapsigargin. Furthermore, the
PKC inhibitor staurosporine blocked the NE-induced internalization.
Taken together, NE-promoted internalization of
1bAR/GFP
appears to be closely linked to PLC activation and dependent on PKC
activation in particular. Our present results, which were obtained by
real-time optical monitoring of subcellular localization in living
cells, are in good agreement with previous observations made by
cell-free biochemical assay (9) or by immunohistochemical analysis of
fixed cells (14).
GFP is now widely used to monitor intracellular localization of
proteins in intact cells. However, its size (238 amino acids) (25) in
comparison with the overall size of the
1bAR protein
(515 amino acids) (3) and other GPCRs (26, 27) makes it an unlikely
candidate for the formation of a functional GPCR/GFP fusion protein.
Both the present study and recent work with ß2-AR (28),
however, suggest that the GFP adduct does not significantly change the
inherent physical or biochemical behavior of GPCR and that optical
methods can be generally useful even for GPCRs. Optical studies in
cultured cells, of
1bAR in particular and of GPCRs in
general, are difficult due to the small number of membrane receptors
expressed. Thus, GPCRs produce only marginal signals when tagged with
fluorophores or labeled with fluorescent agonists or antagonists, a
procedure that often modifies the behavior of these compounds (29). In
addition, the introduction of foreign epitopes into receptor cDNA is
now a standard technique used to enhance detection, permitting antibody
recognition of
1AR (30) and other GPCRs in flow
cytometry or fluorescence microscopy (31). However, even this technique
has major limitations, including its applicability to living cells,
nonstoichiometric labeling of receptors, the eventual dissociation of
the antibody from the receptor, and an inability to label intracellular
receptors in nonpermeabilized cells. An ideally labeled receptor should
be relatively unperturbed by its fluorescent tag, exhibit little or no
change in its biochemical or biophysical behavior, have a large
fluorescence signal above background when excited by visible light in
addition to being photostable, and be stoichiometrically labeled. As
shown in this report, the observed behavior of
1bAR/GFP indicates that
1bAR/GFP and
other similarly conjugated GPCRs should be important tools both
in vitro and in vivo for the optical measurement
of biochemical and biophysical processes that are relevant to GPCR
signal transduction.
We observed a relatively minor degree (
35%) of internalization of
the
1bAR by the fluorescence detection method using GFP
and the fluorescent anti-
1bAR antibody, compared with
the internalization observed by the use of other GPCRs such as
ß-adrenoceptor (32). However, the internalization of
1bAR/GFP, which was observed by optical monitoring of
the fluorescence signal in living cells, is in good agreement with
previous observations that were made using radioligand-binding assay on
membrane preparations (32, 33). Thus, both the time course and the
extent of
1bAR internalization after exposure to high
concentrations of an
1AR agonist are similar to that
found in previous studies that employed DDT1 MF-2 cells,
which express
1bAR naturally (33). Therefore, we
considered that, in general, the agonist-promoted internalization of
the
1bAR may not be as marked compared with that
promoted by other GPCRs. The reason for this relatively minor degree of
internalization of the
1bAR is not clear.
Furthermore, our data showed that agonist-promoted internalization of
1bAR/GFP appears to be closely linked to PLC activation
and is dependent on PKC activation, but independent of
[Ca2+]i increase. At present, the signals
controlling
1bAR internalization are not well known
because of the lack of specific structural probes for the receptor
in vivo. Using measurement of radioligand binding to assess
receptor redistribution in Chinese hamster ovary cells transfected with
receptor cDNA, Toews (12) reported that agonist or PMA
stimulation causes receptor internalization, which is blocked by
staurosporine. In addition, a recent immunohistochemical study by
Fonseca et al. (14) suggested that PKC-dependent
phosphorylation resulting from
1AR stimulation induces
receptor internalization. Our data obtained from real-time optical
monitoring of
1bAR/GFP are generally in good agreement
with these studies; moreover, we observed that not only homologous
stimulation (by NE) but also heterologous stimulation (by GnRH) of PLC,
and eventually PKC, resulted in the internalization of
1bAR, although the latter process appeared to be less
potent. Thus, phosphorylation of
1bAR by PKC clearly
plays an important role in the desensitization (7, 14) and
internalization of
1bAR.
In conclusion, this study demonstrates that real-time optical
monitoring of subcellular localization of
1bAR (as well
as other GPCRs) on living cells are feasible, and that this approach
combined with appropriate pharmacological tools would provide a
valuable system to further study the biochemical mechanism(s) of
agonist-induced receptor endocytosis.
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MATERIALS AND METHODS
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cDNA Construction
Expression vectors (Fig. 1
) were constructed on SR
promoter-based mammalian expression vector pME18S (34). The cDNA
for the hamster
1b-adrenoceptor (3) was the kind gift of
Dr. Susanna Cotecchia (Institute de Pharmacologie et Toxicologie,
Lausanne, Switzerland). To generate the
1bAR/GFP
construct, the coding region of GFP mutant S65T (20) (the kind gift of
Dr. H. Takahashi, Mitsubishi Kasei Inst. Life Sciences) was
amplified by primer 1(aaagggcccatgagtaaaggagaagaacttttc) and primer 2
(aaaactagttttgtatagttcatccatggc), which produced a 5'-ApaI
site for ligation. The
1bAR expression vector
pME-
1b (35) was digested by ApaI and
XbaI. Both enzyme-digested products were ligated to obtain a
construct designated pME-
1bAR/GFP. The modified region
of these construct was confirmed by sequencing with an ABI 373A DNA
sequencer (Applied Biosystems Inc., Foster City, CA). We chose the
ApaI site in the carboxyl terminus for the GFP to be
integrated because the region distal to the ApaI site varies
in
1aAR splice variants with similar pharmacological
properties (36), and deletion of the region in
1bAR was
shown not to affect the binding and signal transduction properties
(37). All experiments were performed with wild-type receptors in
parallel whenever possible.
Transfection and Selection of Stably Expressing Cells
T3 cells were maintained in DMEM with 10% FBS. The
constructs, pME18S-
1b and pME-
1bAR/GFP,
were transfected into
T3 cells by Lipofectin (GIBCO, Life
Technologies, Gaithersburg, MD) according to manufacturers
instructions. Using a cell sorter (FACsort, Becton Dickinson & Co.,
Mountain View, CA), we selected and enriched anti-N terminus
antibody-positive and/or GFP-positive cells at 72 h, 1 week, and 2
months after transfection.
[125I]HEAT Binding Assay
Crude particulate membrane fractions were collected from
T3
stable cells as described previously (38). Briefly, the harvested cells
were pelleted by centrifugation at 500 x g for 5 min
and washed, and the pellet was homogenized in 2 ml ice-cold buffer A
(250 mM sucrose, 5 mM Tris-HCl, 1
mM MgCl2, pH 7.4) and centrifuged at 1,000
x g at 4 C for 10 min to remove nuclei. The supernatant was
then centrifuged at 35,000 x g for 20 min at 4 C, the
pellet was homogenized, and the homogenates were resuspended in buffer
B (50 mM Tris-HCl, 10 mM MgCl2, 10
mM EGTA, pH 7.4) to a final protein concentration of 0.1
mg/ml. The protein concentration was measured using the bicinchoninic
acid protein assay kit (Pierce Chemical Co., Rockford, IL).
Radioligand binding with [125I]HEAT studies was performed
as described previously (35, 38, 39). Briefly, measurement of specific
[125I]HEAT binding was performed by incubating 0.1 ml of
membrane preparation (
30 µg of protein) with
[125I]HEAT (2,200 Ci/mmol) in a final volume of 0.15 ml
buffer B for 60 min at 25 C in the presence or absence of competing
drugs. The incubation was terminated by adding ice-cold buffer B and
immediately filtering through Whatmann GF/C glass-fiber filters with a
Brandel cell harvester (model 30, Gaithersburg, MD). Each filter
was collected, and the radioactivity was measured. Binding assays were
always performed in duplicate. For competition curve analysis, each
assay contained about 70 pM [125I]HEAT. At
this concentration, nonspecific binding, defined as binding displaced
by 10 mM phentolamine, represented less than 40% of the
total binding. Data were analyzed by computer with an iterative
nonlinear regression program LIGAND (40).
In some experiments with CEC treatment, the membrane preparation was
incubated in a 1 ml volume of hypotonic buffer (5 mM
Tris-HCl, 5 mM EDTA, pH 7.6) with CEC (100
µM) for 30 min at 37 C, after which the reactions were
stopped by adding 16 ml ice-cold buffer, and centrifuged at 35,000
x g for 20 min at 4 C. The membrane was washed two times
and resuspended in buffer B and used for the binding assay.
Measurement of Inositol-1,4,5-Triphosphate
[Ins(1, 4, 5)P3]
A portion (106) of the suspended
T3 cells
treated with NE (1 nM-10 µM) for 5 sec
were immediately added by 0.2 volume of ice-cold 20% perchloric acid.
After centrifugation, the supernatant was adjusted to pH 7.0 using
HEPES-KOH solution, and the sediment was eliminated by centrifugation.
Amounts of Ins(1, 4, 5)P3 in a sample were measured by a RRA
with a D-myo-inositol 1,4,5-triphosphate [3H]
assay kit, TRK 1000 (Amersham, Buckinghamshire, U.K.). Values of
Ins(1, 4, 5)P3 were expressed as picomoles/106 of
T3 cells.
Antibody Preparation
Generation of an antipeptide antibody (designated as 1B-N1-C)
was described previously (41). Briefly, peptide was synthesized
corresponding to amino acids 1227 (peptide: 1B-N1;
(C)SAPAQWGELKDANFTG) of the published hamster
1bAR
sequence (3), conjugated to the carrier protein keyhole limpet
hemocyanin and injected to rabbits. Antisera were screened against the
peptides by using cross-dot systems (Sebia, Moulineaux, France) and
visualized by ABC system (Vector Laboratories, Burlingame, CA). By
immunoblotting and immunoprecipitation studies, we confirmed that the
antibodies detect the
1bAR (41).
Antiserum was purified on 1 ml of protein A-Sepharose CL-4B column
(Pharmacia Biotech, Tokyo, Japan) equilibrated with 20 mM
phosphate buffer, pH 7.5, and eluted with glycine-HCl buffer (100
mM, pH 2.2), into 1-ml fractions, which were immediately
neutralized with 1 M Tris-HCl buffer, pH 8.5. The resulting
antibody fractions were concentrated by a Centricon 30
microconcentrator (Amicon, Danvers, MA) and stored at -20 C. Antibody
was labeled by Cy3 (Amersham) according to the protocol of Southwick
et al. (42) and used for immunocytochemical analysis.
Confocal Laser Scanning Microscope Analysis
Immunofluorescence Detection (Fixed Cells)
T3 cells stably expressing wild-type
1bAR and
1bAR/GFP were seeded at 1 x 105 per
well of the eight-well Lab-Tek chamber slide (Nunc, Napervile, IL) in
0.5 ml medium. Fixation was performed in 80% acetone for 5 min. Cells
were then incubated with 0.05% Triton X-100 in PBS. Cy3-conjugated,
affinity-purified anti-
1bAR antibody [5 µg/ml,
1B-N1-C, (41)], was brought in PBS containing 10% goat serum
and 0.05% Triton X-100, and applied to cells, which were subsequently
kept in a humidified chamber for 1 h at room temperature. Cells
were then washed twice with PBS, and coverslips were applied using
Gel/Mount (Biomeda, Foster City, CA).
Immunofluorescence Detection (Living Cells)
For immunocytochemical staining of living cells,
T3 cells stably
expressing wild-type
1bAR and
1bAR/GFP
were washed three times with Tyrode solution (135.0 mM
NaCl, 5.4 mM KCl, 0.33 mM
NaH2PO4, 5.0 mM HEPES, 0.5
mM MgCl2, 5.55 mM glucose, 1.25
mM CaCl2, pH 7.4), and incubated with ice-cold
Tyrode solution containing 1 µg/ml of the Cy3-labeled antibody for 30
min at 4 C, after which the cells were washed three times with ice-cold
Tyrode solution.
After immunocytochemical staining, cells were examined by using
LSM-GB200 laser scanning microscope (Olympus, Tokyo, Japan) with
argon-ion laser set at 514 nm for excitation of Cy3.
GFP Detection
T3 cells stably expressing wild-type
1bAR and
1bAR/GFP were seeded at 1 x 105 per
well of the cover glass-bottom culture dish (MatTek Corp., Ashland, MA)
in 2.0 ml of medium and examined using LSM-GB200 within 30 min at room
temperature.
Labeling of Cells with Cy3-Transferrin
Transferrin was labeled by Cy3 (Amersham) according to manufacturers
instructions.
T3 cells stably expressing
1bAR/GFP in
35-mm dishes were rinsed three times with serum-free DMEM and incubated
for 12 h with Cy3-transferrin. At the termination of labeling, the
cells were again rinsed three times with warm serum-free DMEM and
examined by using LSM-GB200 laser scanning microscope (Olympus) with
argon-ion laser set at 514 nm for excitation of Cy3.
Monitoring of
[Ca2+]i
and Subcellular Distribution of
1bAR/GFP
Changes in [Ca2+]i as well as the
distribution of fluorescent signal were monitored by GB-200 confocal
laser scanning microscope (Olympus). The fluorescence intensity
change of intracellularly loaded Fura Red was used to estimate the
[Ca2+]i change upon stimulation.
T3 cells
stably expressing wild-type
1bAR and
1bAR/GFP cultured in a cover glass-bottom culture dish
(MatTek Corp.) were incubated with 0.5 µM Fura Red
tetrakisacetoxymethyl ester (Fura Red/AM) dissolved in Tyrode solution
containing 0.1% BSA for 30 min at 37 C. After the cells were washed
twice with Tyrode solution, changes in
[Ca2+]i were monitored with a sample interval
of 10 sec in Tyrode solution containing yohimbine 100 nM
and propranolol 100 nM. The argon laser beam (wavelength
488 nm) was focused with a water-immersion objective lens (Olympus UV
ApoLSM 40x). Fluorescent signals were split with a dichroic mirror
(550 nm), and change in fluorescence was measured through interference
filters of 590-nm highcut filter and 500- to 530-nm bandpass filter for
monitoring of [Ca2+]i and subcellular
distribution of
1bAR/GFP, respectively. Three frames
before the stimulation were averaged pixel by pixel to obtain resting
fluorescence (F0). The resting frame was then divided by
each frame in a pixel-by-pixel basis, and the normalized fluorescence
intensity value (Ft) was used to estimate the
[Ca2+]i change upon stimulation. We did not
convert the fluorescence intensity change to absolute values of
[Ca2+]i because it was difficult to
unambiguously determine the background fluorescence intensity and the
resting [Ca2+]i, both of which are required
for [Ca2+]i determination using
nonratiometric dyes.
Fluorescence Measurements
Confocal images were digitally acquired into two-dimensional arrays of
picture elements (pixels). Each pixel is a square with a width of 0.1
µm and assigned an intensity value ranging from 0 (black) to 255
(white). Cellular edges in each image were outlined manually, and the
intensity value of each pixel within the outlined area was measured.
The sum of the intensity value within the outlined area was considered
as the total cellular fluorescence intensity, while the sum of the
intensity value within 0.5 µm (5 pixel) depth from the cellular edge
was considered as fluorescence signal localized on plasma membrane.
Image analysis were performed using IPLab software (Signal Analytics
Corp., Vienna, VA). The subcellular distribution of Cy3- or
GFP-associated fluorescence was expressed by a cell surface
localization ratio: the fluorescent signal localized on plasma membrane
divided by the total cellular fluorescence intensity. To quantify the
effect that various pharmacological agents have on
1bAR/GFP redistribution, cells were chosen randomly in
each experiment, and one image was obtained from each dish of each
individual experiments (Fig. 9
). Each image contains, on average, three
to seven cells, and all cells in the images were analyzed for
quantification. At least six independent experiments were performed for
each treatment.
Materials
Hamster
1bAR cDNA was a kind gift of Drs.
S. Cotecchia and R. J. Lefkowitz (Duke University Medical Center,
Durham, NC) (3). The following drugs were used:
[125I]HEAT (specific activity 2,200 Ci/mmol) (New England
Nuclear, Boston, MA); KMD-3213 dihydrobromide,
((-)-(R)-1-(3-hydroxypropyl)-5-[2-[2-[2-(2, 2, 2-trifluoroethoxy)
phenoxy]ethylamino]propyl] indoline-7-carboxamide dihydrobromide)
(Kissei Pharmaceutical Co., Matsumoto, Japan); phentolamine
hydrochloride (ClBA-Geigy, Summit, NJ); GnRH (Tanabe, Osaka, Japan);
prazosin hydrochloride (Pfizer, Groton, CT); yohimbine HCl (Wako Pure
Chemical Industries, Ltd., Osaka, Japan); CEC and 5-methylurapidil
(Research Biochemicals, Natick, MA); (-)-norepinephrine bitartrate
(Sigma, St. Louis, MO); lipofectin (GIBCO, Life Technologies,
Gaithersburg, MD); Fura Red/acetoxymethyl ester (Fura Red/AM)
(Molecular Probes, Eugene, OR); Triton X-100 (Wako Pure Chemical
Industries, Osaka, Japan); U73,122,
1-[6-[[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione;
U73,343,
1-[6-[[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione;
Cy3 (Amersham); transferrin (Sigma). All other chemicals were of
reagent grade.
Statistics
ANOVA was performed and when a statistical difference was
detected, a Dunnetts multiple comparison test was used to determine
the difference between groups. All data are presented as the mean
± SD, and the statistically significant difference was
determined at the P < 0.05 level unless otherwise
stated.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. H. Takahashi (Mitsubishi Kasei Institute of
Life Sciences) for the kind gift of the cDNA-encoding GFP mutant S65T.
We are also grateful to Dr. Susanna Cotecchia (Institute de
Pharmacologie et Toxicologie, Lausanne, Switzerland) for the generous
gift of the cDNA-encoding hamster
1bAR and to Dr.
Stojilkovic (National Institutes of Health, Bethesda, MD) for mouse
T3 cells. We also thank Dr. Thomson (International Medical
Information Center, Tokyo, Japan) for the language editing.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Gozoh Tsujimoto, Department of Molecular and Cell Pharmacology, National Childrens Medical Research Center, 335-31 Taishido, Setagaya-ku, Tokyo, 154 Japan. E-mail:
gtsujimoto{at}nch.go.jp
Received for publication May 1, 1997.
Revision received March 6, 1998.
Accepted for publication April 20, 1998.
 |
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