A Functional Enhanced Green
Fluorescent Protein (EGFP)-Tagged
Angiotensin II AT1A Receptor
Recruits the Endogenous G␣q/11
Protein to the Membrane and
Induces Its Specific Internalization
Independently of ReceptorG Protein Coupling in HEK-293 Cells
Stéphanie Miserey-Lenkei, Zsolt Lenkei, Charles Parnot,
Pierre Corvol, and Eric Clauser
Institut national de la santé et de la recherche médicale U36
Collège de France
75005 Paris, France
not G protein coupling and signal transduction
mechanisms, as assessed by pharmacological
data using agonists and antagonists and the characterization of AT1A receptor mutants (D74N and
⌬329) for which the coupling and internalization
functions are modified. (Molecular Endocrinology
15: 294–307, 2001)
The angiotensin II (Ang II) AT1A receptor was
tagged at its C terminus with the enhanced green
fluorescent protein (EGFP), and the corresponding
chimeric cDNA was expressed in HEK-293 cells.
This tagged receptor presents wild-type pharmacological and signaling properties and can be immunodetected by Western blotting and immunoprecipitation using EGFP antibodies. Therefore,
this EGFP-tagged AT1A receptor is the perfect tool
for analyzing in parallel the subcellular distributions of the receptor and its interacting G protein
and their trafficking using confocal microscopy.
Morphological observation of both the fluorescent
receptor and its cognate G␣q/11 protein, identified
by indirect immunofluorescence, and the development of a specific software for digital image analysis together allow examination and quantification
of the cellular distribution of these proteins before
and after the binding of different agonist or antagonist ligands. These observations result in several
conclusions: 1) Expression of increasing amounts
of the AT1A receptor at the cell surface is associated with a progressive recruitment of the cytosolic G␣q/11 protein at the membrane; 2) Internalization of the EGFP-tagged AT1A induced by peptide
ligands but not nonpeptide ligands is accompanied
by a G␣q/11 protein intracellular translocation,
which presents a similar kinetic pattern but occurs
predominantly in a different compartment; and 3)
This G␣q/11 protein cellular translocation is dependent on receptor internalization process, but
INTRODUCTION
The vasoactive peptide angiotensin II (Ang II) acts on
its target tissues via seven- transmembrane domain
receptors of the G protein-coupled receptor (GPCR)
family. The most physiologically relevant of these receptors is called AT1 (AT1A and AT1B in rodents). This
receptor is blocked by specific nonpeptide imidazole
antagonists such as Losartan. The activation of this
receptor results in contraction of vascular smooth
muscle cells, aldosterone secretion in glomerular adrenocortical cells, and in many other physiological actions in target tissues including growth-promoting effects (1).
The transduction machinery for GPCR is described
classically as a three-pieces system comprising 1) a
receptor, which is an integral transmembrane protein
with seven hydrophobic ␣ helices, 2) a transducer,
corresponding to a heterotrimeric GTP-binding protein
and associating a ␤␥-complex, which is anchored in
membranes via a prenylated cystein and an ␣-subunit
that may be associated to the membrane via myristate
or palmitate moities (2), and 3) an effector, which can
be a membrane-bound enzyme or an ion channel.
Several cellular events follow the binding of Ang II to
the AT1A receptor, such as activation of the signaling
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Molecular Endocrinology 15(2): 294–307
Copyright © 2001 by The Endocrine Society
Printed in U.S.A.
294
AT1A Receptor and G␣q/11 Protein Cellular Localizations in Response to Ang II
pathways, receptor-ligand internalization, and receptor phosphorylation and desensitization. These processes involved multiple intracellular receptor-protein
interactions. On one hand, the Ang II signals inside the
cell via receptor, G␣q/11protein, phospholipase C (␤PLC) interactions, which activate inositol phosphate/
calcium signaling (for review see Ref. 1), but perhaps
also via a Jak-STAT pathway that includes AT1/Jak2
interactions (3). On the other hand and like most other
GPCRs, the AT1 receptor is internalized after ligand
binding. The kinetics, pharmacology, and molecular
determinants (third intracellular loop and C-terminal
part of the receptor) of this internalization process
have been extensively investigated by several groups
and our laboratory (4–7). However, the molecular
mechanism of this internalization process is unclear
and may be not only a coated pit-dependent or a lipid
raft/caveolae-dependent mechanism (8).
A clear picture of the parallel subcellular distribution
and of simultaneous trafficking for the Ang II AT1 receptor and its interacting proteins, e.g. G proteins, is
not yet available due to technical limitations in the
immunohistochemical, optical tools, and image analysis softwares.
In the present study, a functional Ang II AT1A receptor tagged with the enhanced green fluorescent protein (EGFP) was stably expressed in HEK-293 cells.
Simultaneous morphological analysis of the fluorescent receptor and its coupled endogenous G␣q/11
protein identified with specific antibodies, in the absence or presence of specific ligands using confocal
microscopy, enabled us to make two new and interesting observations:
1. The cell surface localization of the endogenous
G␣q/11 protein increases with the cell surface expression of the AT1A receptor.
2. After Ang II binding, both the receptor and its
coupled G protein are internalized, but in different
intracellular compartments. This phenomenon is dependent on receptor internalization but not on receptor activation and signal transduction.
RESULTS
The AT1A-EGFP Receptor Expressed in HEK-293
Cells Is Functional and Immunodetectable
An AT1A-EGFP chimera was constructed by linking the
EGFP cDNA in frame to the 3⬘-end of the AT1A coding
sequence, resulting in a receptor carrying EGFP on its
carboxy-terminal tail. The AT1A-EGFP receptor was
stably expressed in HEK-293 cells. The binding properties of agonists and antagonists to the AT1A and
AT1A-EGFP receptors are undistinguishable (Table 1).
Stimulation of the tagged receptor with Ang II induces
a 10-fold increase in total inositol phosphate (IP) production (EC50 ⫽ 2.34 ⫾ 0.54 nM) as reported for the
wild-type receptor expressed in Chinese hamster
ovary (CHO) cells (EC50 ⫽ 0.75 ⫾ 0.09 nM) (9). In
295
Table 1. Pharmacological Characterization of the AT1AEGFP Receptor in HEK-293 Cells
AT1A-EGFP
AT1Aa
125
A. Binding parameters of [ I]Ang II for the AT1A-EGFP
receptor in HEK-293 cells
Kd (nM)
0.53 ⫾ 0.09 0.53 ⫾ 0.02
Bmax (103 sites/cell)
102.8 ⫾ 4.0
172 ⫾ 10
Ki (nM)
AT1A-EGFP
AT1Aa
B. Inhibitory constants (Ki) of Ang II agonists and antagonists for the AT1A-EGFP receptor and the AT1A
receptor
Ang II
0.51 ⫾ 0.01
0.49 ⫾ 0.11
[Sar1,Ile8] Ang II
0.36 ⫾ 0.01
0.66 ⫾ 0.10
Losartan
10.5 ⫾ 1.60
6.51 ⫾ 1.87
CGP42112A
518 ⫾ 14.7 4550 ⫾ 380
A, Binding parameters of [125I]Ang II for the AT1A-EGFP receptor in HEK-293 cells; B, inhibitory constants (Ki) of Ang II
agonists and antagonists for the AT1A-EGFP receptors. The
results are expressed as means ⫾ SEM of three independent
experiments.
a
Data from 7 in CHO cells.
addition, a more integrated assay of the signaling
pathway activation was performed, involving the activation of a reporter gene under the control of a promoter regulated by the PLC-protein kinase C (PKC)
pathway (see Materials and Methods). The wild-type
AT1A and AT1A-EGFP receptors, transiently transfected in HEK-293 cells with the reporter construct,
stimulated the reporter gene expression to similar level
and sensitivity (EC50 ⫽ 0.97 nM for the AT1A receptor;
EC50 ⫽ 0.50 nM for the AT1A-EGFP receptor) (Fig. 1).
In addition, the AT1A-EGFP receptor is specifically
immunodetected using an anti-EGFP antibody both
by immunoprecipitation and Western blot (Fig. 2).
The glycosylated form of AT1A receptor corresponds
to a diffuse band from 70 to 90 kDa (Fig. 2, lane II,
zone 2), a value that is more or less in agreement
with the expected molecular mass of this glycoprotein (41 kDa for AT1A ⫹ 27 kDa for EGFP ⫹ 3
N-linked glycosylated chains) and with the few observations of its molecular mass by SDS-PAGE in
the literature (60–65 kDa for Ref. 10 and 50–90 kDa
for Ref. 11). This broad band is specific since no
signal was observed in the HEK-AT1A cell extracts.
Moreover, and as observed by others in the literature, another band with a higher molecular mass
(120 kDa and more) was visible (Fig. 2, lane II, zone
1) and may correspond to either a homodimer or a
heterocomplex with another protein. After N-deglycosylation with peptide N-glycosidase F (PNGase
F), the receptor presents a reduced molecular mass
to 60–80 kDa (Fig. 2, lane III, zone 3), which confirms
its N-glycosylation. The similar heterogenous pattern of the receptor after deglycosylation probably
reflects the high sensitivity of the receptor to cellular
proteases.
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Fig. 1. Stimulation of a Reporter Gene by the AT1A-EGFP and the AT1A Receptor in HEK-293 Cells
Effect of increasing concentrations of Ang II on the transcription of a reporter gene (luciferase) under the control of a promoter
with TPA-responsive elements (CGTCA). Cells were transiently cotransfected with the AT1A, the AT1A-EGFP receptors, or the
vector alone and the reporter construct. The results are expressed as means ⫾ SEM of three independent experiments.
Fig. 2. Immunoprecipitation of the Glycosylated and Deglycosylated Forms of AT1A-EGFP Receptors
Cells expressing the AT1A (I) or the AT1A-EGFP (II and III)
receptors were subjected to immunoprecipitation, and the
AT1A-EGFP (III) immunoprecipitate was further treated with
PNGase F. The results are representative of four independent
experiments.
Ang II-Induced Internalization of the AT1A-EGFP
Receptor Can Be Observed Directly
and Measured
The subcellular distribution of the AT1A-EGFP receptor
was analyzed by confocal laser scanning microscopy
in the HEK-AT1A-EGFP stably transfected cell line. In
the absence of Ang II, the fluorescence of the AT1AEGFP receptor is located at the plasma membrane as
shown on a large confocal view of the cells (Fig. 3A) or
for a typical HEK-AT1A-EGFP cell (Fig. 3B). No fluorescence was detected in nontransfected HEK-293
cells (data not shown). Internalization was studied by
adding the agonist Ang II (100 nM) at 4 C and incubat-
ing the cells at 37 C for 20 min. In these conditions,
fluorescence at the plasma membrane completely disappeared, and the cytoplasm contained a multitude of
fluorescent vesicles as shown by a large confocal view
(Fig. 3C) or a typical AT1A-EGFP-internalized cell (Fig.
3D). Optical sectioning of the cells by 1 ␮m thick serial
confocal slices confirmed the presence of fluorescent
internalization vesicles since their average size was
less than 1 ␮m and they were not connected to the
plasma membrane (data not shown). This Ang IIinduced internalization is specific because it was abolished by acid wash of Ang II before incubation at 37 C.
To better quantify cell surface and cytosoluble fluorescences, an image analysis software was developed, which allows quantification of both the cell surface expression of the receptor on individual round
cells and also the internalization process on the cell
population after ligand application. This quantification
results in the determination of a S⬘/C⬘ ratio index,
which corresponds to the ratio between the specific
mean density of cell surface fluorescence (S-N) called
S⬘ and the specific mean density of cytoplasmic fluorescence (C-N) called C⬘ (see Materials and Methods
and Fig. 3C).
Within the cell population and in basal conditions,
there is an extensive heterogeneity in the AT1A-EGFP
fluorescence intensity at the cell surface from one cell
to another (see Fig. 3A), which results in large variations of the S⬘/C⬘ ratio from 0.39 (low level) to 20.75
(high level). Further investigations suggested that
these variations were related to the cell cycle and that
this heterogenity is due to an absence of growth synchronization in the cell preparation. Thus, to analyze
receptor internalization more accurately during this
study, we analyzed only cells (representing the large
majority of the cellular population, see Fig. 3A) with
similar levels of receptor expression corresponding to
AT1A Receptor and G␣q/11 Protein Cellular Localizations in Response to Ang II
297
Fig. 3. Ang II-Induced Internalization of the AT1A-EGFP Receptor
Cells were examined by confocal microscopy after incubation for 20 min at 37 C without (A and C) or with (B and D) 100 nM
Ang II. C, S is the mean density of surface fluorescence, C is the mean density of cytoplasmic fluorescence, and N is the mean
density of nuclear fluorescence considered as background. A and B, scale bar ⫽ 10 ␮m; C and D, scale bar ⫽ 5 ␮m. E,
Dose-response curve of the AT1A-EGFP receptor internalization determined using the variations of the S⬘/C⬘ fluorescence ratio
in response to increasing concentrations of Ang II (for details see Materials and Methods). n ⫽ 4 for each point. The results are
expressed as means ⫾ SEM.
a total mean fluorescence of 6 to 12 in the absence of
agonist (total mean fluorescence being a fluorescence
index derived from the addition of S, C, and N values
(for details see Materials and Methods and Table 2).
Internalization observed by confocal microscopy
was quantified on the cell population using the same
S⬘/C⬘ ratio described above. Quantification confirms
clearly the internalization process because cells presenting an S⬘/C⬘ ratio of 5.22 ⫾ 1.11 (expressed as
mean ⫾ SD) in basal conditions presented an S⬘/C⬘
ratio below 1.5 after Ang II exposure (Fig. 3D and Table
2), without any change in total cellular fluorescence. In
addition, this method was used to quantify both the
kinetics and dose dependence of internalization. Ang II
clearly induced a dose-dependent internalization of
the receptor (Fig. 3E). The ED50 for Ang II-induced
internalization was 0.31 ⫾ 0.03 nM, an Ang II concentration similar to the EC50 for IP activation and the
dissociation constant (Kd) of the AT1A receptor for Ang
II. Furthermore, the kinetic profiles of AT1A-EGFP receptor internalization induced by Ang II were similar
when measured either by the S⬘/C⬘ ratio using the
morphological quantification method or by the acid
wash biochemical technique (data not shown). In addition, no difference in the internalization kinetics was
observed between the wild-type AT1A receptor and
the AT1A-EGFP receptor measured by the acid wash
biochemical method (data not shown).
Altogether, these data indicate that the AT1A-EGFP
receptor is internalized like the wild-type receptor after
Ang II binding. Moreover, this internalization can be
clearly and precisely monitored by morphological
analysis on individual cells or a cellular population.
Receptor Internalization Is Dependent on the
Peptide Structure but Not on the Agonist
Properties of the Ligand
The ability of large variety of AT1A receptor ligands to
induce the internalization was analyzed next. These
molecules are either natural metabolites of Ang II (Ang
I, Ang III, Ang IV), or peptide analogs of Ang II with
either agonist ([Sar1] Ang II) or antagonist properties
([Sar1,Ile8] Ang II, [Sar1,Ala8] Ang II, [Sar1,Thr8] Ang II).
Others ligands are pseudopeptide (CGP42112A) or
nonpeptide compounds with either agonist (L162, 313)
or antagonist properties (Losartan). All these ligands
were used at concentrations approximately 100-fold
greater than their reported Kd, and internalization was
analyzed as described in the previous section. Based
on the Ang II dose-dependent internalization curve
(Fig. 3E), when the S⬘/C⬘ ratio is higher than 3 there is
no internalization of the receptor; when the S⬘/C⬘ ratio
is below 1.5 there is internalization of the receptor.
Thus, all the peptide or pseudopeptide (CGP42112A)
ligands induce internalization of the AT1A-EGFP receptor independently of their agonist or antagonist status
(Table 3), and nonpeptide ligands do not induce this
process, again independently of their agonist or an-
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Table 2. Effect of Ang II on Cellular Distribution of the AT1A-EGFP, the D74N-AT1A-EGFP, ⌬329-EGFP Receptors, and the
G␣q/11 Subunit
G␣q/11
EGFP
AT1A-EGFP
Untreated
100 nM Ang II
D74N-AT1A-EGFP
Untreated
100 nM Ang II
⌬329-EGFP
Untreated
100 nM Ang II
n
S⬘/C⬘ ratio
10
10
6.87 ⫾ 0.40
0.71 ⫾ 0.14a
7
9
9
13
Total fluorescence intensity
S⬘/C⬘ ratio
Total fluorescence intensity
6.31 ⫾ 0.92
6.41 ⫾ 1.07
1.31 ⫾ 0.16
0.34 ⫾ 0.08a
12.67 ⫾ 2.33
13.51 ⫾ 3.13
4.94 ⫾ 0.80
0.51 ⫾ 0.08a
9.52 ⫾ 1.52
10.77 ⫾ 1.34
1.74 ⫾ 0.41
0.58 ⫾ 0.05b
16.67 ⫾ 1.82
20.07 ⫾ 2.56
5.47 ⫾ 0.25
2.53 ⫾ 0.07b
6.57 ⫾ 0.26
10.06 ⫾ 0.27
1.70 ⫾ 0.13
1.40 ⫾ 0.03
13.54 ⫾ 0.90
19.21 ⫾ 0.72
The S⬘/C⬘ ratios were calculated for the EGFP-associated and the G␣q/11-associated fluorescence in HEK-AT1A-EGFP, HEKD74N-AT1A-EGFP, HEK-⌬329-EGFP cells untreated or stimulated with 100 nM Ang II. Data represent the means ⫾ SEM obtained
from three different experiments. n ⫽ number of examined cells.
a
P ⬍ 0.001, b P ⬍ 0.05 vs without Ang II.
Table 3. Pharmacology of the AT1A-EGFP Receptor Internalization
HEK-AT1A-EGFP
Ligands
S⬘/C⬘ Ratio EGFP
% Internalization (acid wash)a
Untreated
Ang II (10⫺7 M)
Ang II metabolites
Agonists
Ang I (10⫺5 M)
Ang III (10⫺6 M)
Ang IV (10⫺4 M)
Ang II analogs
Agonist
[Sar1] Ang II (10⫺7 M)
Antagonists
[Sar1,Ile8] Ang II (10⫺7 M)
[Sar1,Ala8] Ang II (10⫺7 M)
[Sar1,Thr8] Ang II (10⫺5 M)
Pseudopeptide analogs
CGP42112A (10⫺4 M)
Nonpeptide analogs
Agonist L162,313 (10⫺5 M)
Antagonist Losartan (10⫺6 M)
4.93 ⫾ 0.40
0.36 ⫾ 0.04
10–20
80
0.68 ⫾ 0.10
0.49 ⫾ 0.13
1.86 ⫾ 0.21
n.d.
70
n.d.
0.69 ⫾ 0.12
80
0.85 ⫾ 0.15
0.74 ⫾ 0.03
0.80 ⫾ 0.12
80
n.d.
n.d.
1.06 ⫾ 0.06
n.d.
3.57 ⫾ 1.05
3.88 ⫾ 1.13
n.d.
20
Cells were preincubated with different ligands at a concentration corresponding to around 100-fold of their described kD.
Confocal images were quantified. S⬘/C⬘ ratio and percent of internalization with the acid wash method are given for different
ligands of the AT1A receptor. Each experiment was carried out in triplicate and the results are expressed as means ⫾ SEM.
a
Data from Refs. 4, 6, 38, and 39). n.d., Not determined.
tagonist status. These observations lead us to conclude that occupancy of the peptide binding site of the
AT1A receptor, but not of the nonpeptide binding site
which is known to be different (12–14), is the key event
that triggers internalization.
The Endogenous G␣q/11 Protein Is Colocalized
with the AT1A-EGFP Receptor at the Cell Surface
Since the AT1A receptor is coupled to the G␣q/11
protein, it was interesting to analyze the distribution
of the endogenous G protein parallel to the recom-
binant AT1A-EGFP receptor in HEK-AT1A-EGFP
cells. The G␣q/11 protein was detected in these
cells in Western blots with commercially available
antibodies as a double band around 47 kDa (data
not shown) or by immunofluorescence. In HEKAT1A-EGFP cells, intense labeling indicates the
presence of endogenous G␣q/11 protein both in the
cytoplasm and at the cell surface (Fig. 4B, panel b).
This immunolabeling is specific since both omission
of the primary antibody and the addition of an excess of immunoreactive peptide abolished fluorescence (data not shown).
AT1A Receptor and G␣q/11 Protein Cellular Localizations in Response to Ang II
299
Fig. 4. Colocalization of the G␣q/11 Protein Subunit and the AT1A-EGFP Receptor at the Cell Surface in the Absence of Ligand
A, Representation of the positive correlation (r ⫽ 0.394, P ⬍ 0.0001) between the amount of membrane AT1A-EGFP
receptor (S⬘/C⬘ ratio of EGFP-associated fluorescence) and the amount of membrane G␣q/11 protein (S⬘/C⬘ ratio of
G␣q/11-associated fluorescence) for 198 cells expressing different amounts of AT1A-EGFP receptor. The 198 cells were
taken from seven independent experiments. B, Large confocal views (a–d) or single cell views (e–h) of cells expressing both
high amounts of the AT1A-EGFP (a1, c2, and e) receptor and of G␣q/11 protein (b1, d2, and f) at the plasma membrane or
both low amounts of the AT1A-EGFP receptor (a3, c4, and g) and of G␣q/11 protein (b3, d4, and h). Scale bar ⫽ 10 ␮m for
panels B–E and 5 ␮m for panels F–I. (C) S⬘/C⬘ ratio for G␣q/11-associated fluorescence on a whole-cell population of
untransfected HEK-293 and on cells expressing the AT1A (n ⫽ 46), the AT1A-EGFP (n ⫽ 26), and the V1b-EGFP receptor (n ⫽
22). *, P ⬍ 0.01 vs. untransfected HEK-293 cells. The results are expressed as means ⫾ SEM of three independent
experiments.
Surprisingly, a whole population of untransfected
HEK-293 cells presents very little membrane expression of the G␣q/11 protein (S⬘/C⬘ 0.63 ⫾ 0.05)
whereas this ratio was almost twice higher (S⬘/C⬘
0.89 ⫾ 0.07) in a comparable population of cells
transfected with the AT1A-EGFP receptor. This ratio
increases again (S⬘/C⬘ 1.31 ⫾ 0.16) when the population of HEK-AT1A-EGFP cells is selected for a
total EGFP fluorescence ranking between 6 and 12.
Altogether, these results suggest that the membrane
expression of the AT1A receptor may be a determinant for the cell surface localization of the G␣q/11
protein. This hypothesis was investigated further
using the heterogeneous expression of the AT1AEGFP receptor in HEK cells and by measuring the
S⬘/C⬘ ratios for both AT1A-EGFP and G␣q/11 fluo-
rescence in about 200 individual cells expressing
different levels of the AT1A-EGFP receptor (S⬘/C⬘
ratio from 0.39 to 20.75; see above). The positive
correlation between the level of cell surface expression of the AT1A receptor and the fraction of membrane-bound G␣q/11 protein (Fig. 4A) (r ⫽ 0.394,
P ⬍ 0.0001) is in favor of our hypothesis. This correlation is also illustrated by two representative examples of overall confocal views (Fig. 4B, panels
a–d) or single cell images (Fig. 4B, panels e-h) showing a majority of cells (but not all) with parallel levels
of AT1A-EGFP receptor and G␣q/11 protein at the
cell surface. Some cells present high surface levels
of both AT1A-EGFP (Fig. 4B, panels a1, c2, and e)
and membrane-bound G␣q/11 protein (Fig. 4B, panels b1, d2, and f) or a low level of the receptor (Fig.
MOL ENDO · 2001
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4B, panels a3, c4, and g) and membrane-bound
G␣q/11 protein (Fig. 4B, panels b3, d4, and h). In
addition, the S⬘/C⬘ ratio for G␣q/11-associated fluorescence was also higher on a whole population of
cells stably expressing either the wild-type AT1A
receptor or another G␣q/11-coupled receptor, the
V1b vasopressin receptor (Fig. 4C).
All these data suggest that the membrane recruitment of the G␣q/11 is at least partly dependent on the
level of membrane expression of its cognate receptor
and not on artifacts such as intercellular variations of
protein synthesis. Indeed, there was no correlation
between the cell surface fluorescence of G␣q/11 and
the total expression of AT1A-EGFP or G␣q/11 in either
HEK-293 or HEK-AT1A-EGFP cells (data not shown). In
addition, in HEK-AT1A-EGFP cells, a constant total
cellular expression of the G␣q/11 protein was measured despite a heterogeneous expression of the
AT1A-EGFP receptor, indicating that the two levels of
expression are independent.
Cytoplasmic Translocation of the G␣q/11 Protein
and the AT1A-EGFP Receptor Is Synchronized but
Occurs in Different Compartments
The AT1A receptor (Fig. 5A, left upper panel) and the
G␣q/11 protein (Fig. 5A, middle upper panel) are colocalized at the cell surface in basal conditions as
indicated by the yellow color of the plasma membrane
in the overlay of the two fluorescences (Fig. 5A, right
upper panel). Therefore, it was interesting to investigate the cellular distribution of G␣q/11 protein after
Ang II addition and receptor internalization. After 20
min at 37 C in the presence of 100 nM Ang II, both
proteins are translocated from the plasma membrane
to intracellular locations with major changes in the
S’/C’ ratios of both proteins (from 6.87 to 0.70 for
EGFP-associated fluorescence and from 1.31 to 0.34
for G␣q/11-associated fluorescence, n ⫽ 13 for each)
(Table 2 and Fig. 5A, lower panels). The loss of plasma
membrane fluorescence was not associated with a
loss of total cellular fluorescence intensity (Table 2).
The kinetics of intracellular translocations for both
the AT1A-EGFP receptor and the G␣q/11 proteins
were analyzed next by sequential observation of the
cells for 20 min. Figure 5B shows a representative
example of this observation on a plasma membrane
segment and its adjacent cytoplasm using a high resolution imaging on confocal microscope. During the
first 2 or 3 min after Ang II application, the AT1A-EGFP
receptor (green fluorescence on lane I) and the G␣q/11
protein (red fluorescence on lane II) remain colocalized
at the plasma membrane, as indicated by the yellow
labeling on the overlay (lane III). Starting at 3 min after
Ang II application and maximum at 5 min, both the
AT1A-EGFP receptor and the G␣q/11 are intracellular.
However, these localizations appear predominantly
different since the AT1A-EGFP receptor is essentially
located in large internalization vesicles, and the G␣q/
11-associated fluorescence is more diffuse in the cy-
Vol. 15 No. 2
toplasm and corresponds to fine granules, without any
superimposition with the receptor, as indicated by the
absence of yellow labeling in the overlay (Fig. 5B, lane
III, lower panels). Confocal images were quantified and
the kinetics of intracellular translocation for both the
AT1A-EGFP receptor and the G␣q/11 protein were
found to be identical (Fig. 5C). Internalization of 50%
of the proteins was obtained after similar periods of
time for the two proteins (t1/2 ⫽ 2.7 min for AT1AEGFP; t1/2 ⫽ 3.2 min for G␣q/11 protein; n ⫽ 4 for
each point). Therefore, the AT1A-EGFP receptor and
the G␣q/11 protein are both translocated after agonist
application, and, even more interestingly, their intracellular localizations seem to be predominantly
different.
In addition, the receptor-associated G␣q/11 protein
is translocated inside the cell in a specific manner after
the AT1A-EGFP activation for two reasons: 1) no
G␣q/11 translocation was observed after Ang II application in untransfected HEK-293 cells, which do not
express the receptor (data not shown), and 2) Ang
II-activated AT1A-EGFP receptor is unable to translocate a G␣s/olf protein, an unrelated G protein (data not
shown).
Ang II-Induced G␣q/11 Protein Translocation Is
Dependent on Receptor Internalization but Not
on Receptor Coupling and Signal Transduction
The association of this G␣q/11 translocation, which
depends on Ang II binding and on the expression of
the AT1A-EGFP receptor at the cell surface, with either
receptor activation, coupling and intracellular signaling, or receptor internalization was investigated next,
using several tools: 1) the previously described pharmacological tools for dissociating activation (agonists
vs. antagonists) and internalization (peptides vs. nonpeptides) and 2) two mutants of the AT1A receptor
presenting major dissociations between signaling and
internalization functions: the D74N mutant, which presents a major defect in inositol phosphate-calcium
signaling (90–100% reduction according to Refs. 15
and 16) but is still internalized after ligand binding
(same references) and the truncated ⌬329 mutant (9),
which presents a default of internalization after Ang II
treatment but signals even better than the wild-type
receptor.
The peptide antagonist [Sar1,Ile8]Ang II induces the
translocation of both the AT1A-EGFP receptor and
the G␣q/11 protein without activating the receptor
and the signaling pathway (Fig. 6A). The nonpeptide
ligands Losartan (Fig. 6B) and L162,313 (Fig. 6C) are
unable to induce translocation either of the receptor or
of the G␣q/11-protein independently of their agonist/
antagonist status.
The D74N-AT1A-EGFP mutant stably expressed in
HEK-293 cells presents the same properties as the
D74N-AT1A receptor: it binds Ang II and it is unable to
activate IP production significantly (data not shown). In
this cell line, the D74N-AT1A-EGFP mutant is colocal-
AT1A Receptor and G␣q/11 Protein Cellular Localizations in Response to Ang II
301
Fig. 5. Kinetics of Ang II-Induced Cytoplasmic Translocation of the AT1A-EGFP Receptor and the G␣q/11 Protein
A, Confocal microscopy images of individual cells incubated for 20 min at 37 C without (upper lane) or with (lower lane) 100
nM Ang II. B, Detail of confocal images (part of plasma membrane and cytoplasm) of cells incubated for various periods of time
with 100 nM Ang II; lane I, AT1A-EGFP receptor fluorescence; lane II, G␣q/11 protein fluorescence; lane III, fluorescence overlay.
C, Changes in the S⬘/C⬘ ratio of EGFP-associated (⽧) and G␣q/11-associated (F) fluorescences over time. The results are
expressed as means ⫾ SEM of three independent experiments. Scale bar ⫽ 5 ␮m for panel A and 2.5 ␮m for panel B.
ized at the cell surface with the G␣q/11 protein (Fig. 6D
and Table 2) in basal conditions. The translocation of
both proteins is similar to what is observed for the
wild-type receptor in the presence of 100 nM Ang II,
despite the defect in receptor activation and signal
transduction (Fig. 6E and Table 2). This normal internalization of the mutant is quantified by reductions of
90% and 67% of the S⬘/C⬘ ratios of the D74N-AT1AEGFP- and G␣q/11-associated fluorescences, respectively, as compared with 90% and 74% reductions for the wild-type receptor.
The ⌬329-EGFP mutant stably expressed in HEK293 cells presents the same properties as the ⌬329
mutant: it binds Ang II, induces an increase in IP production, and has a default of internalization as measured by the biochemical acid wash procedure (80%
of Ang II-induced internalization for the wild-type receptor and 40% for the ⌬329-EGFP mutant). The
⌬329-EGFP mutant is colocalized at the cell surface
with the G␣q/11 protein in basal conditions (Fig. 6F
and Table 2). After a 100 nM Ang II treatment, the
⌬329-EGFP mutant is partly internalized as measured
MOL ENDO · 2001
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Vol. 15 No. 2
Fig. 6. Dependency of G␣q/11 Translocation on Internalization but not Signal Transduction of the AT1A-EGFP Receptor
A, Pharmacology of the AT1A-EGFP receptor and the G␣q/11 protein cytoplasmic translocation: HEK-AT1A-EGFP cells were
incubated for 20 min at 37 C with [Sar1,Ile8] Ang II (10⫺7 M) (panel A), Losartan (10⫺6 M) (panel B), and L162,313 (10⫺5 M) (panel C). D
and E, Cellular distribution of the D74N-AT1A mutant and its G␣q/11 protein: HEK-D74N-AT1A-EGFP cells were incubated without (panel
D) or with (panel E) 100 nM Ang II and observed by confocal microscopy. F and G, Cellular distribution of the ⌬329 mutant and its
G␣q/11 protein : HEK-⌬329-EGFP cells were incubated without (panel F) or with (panel G) 100 nM Ang II and observed by confocal
microscopy. These images are typical of the images obtained in three independent experiments. Scale bar ⫽ 5 ␮m.
AT1A Receptor and G␣q/11 Protein Cellular Localizations in Response to Ang II
by a reduction of only 54% of the S⬘/C⬘ ratio (Fig. 6G,
left, and Table 2) and despite Ang II-induced receptor
activation, the G␣q/11 is still localized at the cell surface, as measured by a reduction of only 8% of the
S⬘/C⬘ ratio (Fig. 6G, middle, and Table 2).
The pharmacological data obtained with agonist
and antagonist ligands and those for the signalingand the internalized-defective mutants show that
subcellular translocation of the G␣q/11 protein is dependent on receptor internalization but not on the
activation state of the AT1A-EGFP receptor.
DISCUSSION
Since the cloning of the Ang II AT1 receptors, much
information on its structure/function relationships has
been obtained using site-directed mutagenesis. This
includes the molecular determinants of the binding site
for natural ligands and nonpeptide antagonists (14,
17), of the receptor activation and G protein coupling
(7, 15, 16), and of its internalization (9, 18, 19), phosphorylation (10, 11), and desensitization (20, 21). In
contrast, very little is known about the biosynthesis,
the biochemical structure, and the intracellular trafficking of this seven-transmembrane domain receptor,
probably as a consequence of the very hydrophobic
structure of the protein and the difficulty to produce
antibodies.
To achieve the morphological analysis of AT1A trafficking, we created and expressed a chimeric AT1A
receptor with a C-terminal extension corresponding to
a variant of the green fluorescent protein of the jelly
fish Aequoria victoria. To be utilizable, this tool had to
fulfil two main conditions: functionality and both direct
and indirect specific detectability.
Despite the 27-kDa extension, the AT1A-EGFP receptor is expressed at the plasma membrane, and its
binding, signaling, and internalization are comparable
to that of the wild-type receptor. Similar results have
been obtained for other GPCR C-terminally tagged
with EGFP, such as the ␣1B-adrenergic (22), the ␤2adrenergic (23), and the TRH receptors (24).
The immunoreactivity of this fusion protein was also
verified. We found that it could be specifically detected
in transfected cells by Western blot and immunoprecipitation, with an anti-EGFP antibody. The heterogeneity of the bands observed (ranging from 70 to 90
kDa) is reminiscent of what has been previously described for this receptor, using either a polyclonal antibody raised against the C-terminal segment of AT1A
(10) or a short epitope (hemagglutin)-tagged receptor
(11). Finally, as shown in Fig. 3 of this paper the AT1AEGFP receptor is directly and again specifically visualized by confocal microscopy.
This functional and fluorescent AT1A-EGFP receptor
is therefore the ideal tool for analyzing the interactions
of this receptor with signaling and trafficking proteins
and for the direct characterization of the internalization
process.
303
The parallel development of a software for image
analysis and quantification of the fluorescence in
various cellular compartments was also of key importance for this study and was validated by a perfect correlation with the measurement of internalization by classical biochemical acid wash techniques.
This direct morphological analysis of AT1A receptor
internalization allows testing of a large variety of
unlabeled ligands; extending the concept of previous studies (4, 25). Thus this analysis demonstrated
that this process is dependent on binding to the
peptide ligand binding site, but not to the nonpeptide ligand binding site, and is independent of coupling between G protein and the receptor since both
peptide antagonist and G protein coupling-deficient
mutants of the receptor undergo internalization. It
also shows definitively and directly that the internalization of GPCR involves not only the disappearance
of binding sites from the surface of the cell, but also
the real intracellular translocation of the receptors
contained in small vesicles with no contact with the
cell surface. This model was also the perfect tool for
evaluating the parallel traffic of the receptor and its
cognate G proteins during receptor activation and
signal transduction.
The fluorescent AT1A-EGFP receptor was observed in cells and is mostly localized at the cell
surface. The opportunity to directly observe the receptor and the natural variation of the receptor expression from one cell to another allowed one interesting observation: the more the receptor is
expressed at the cell surface, the more the cell
surface fraction of its cognate G␣q/11 protein increases. Such a recruitment could not be demonstrated previously on cellular models where both the
receptor and the G proteins were overexpressed.
Therefore, this observation is one of the first experimental data that sustains the hypothesis that a
GPCR may recruit its G protein to the cell surface.
This G␣q/11 recruitment is not an artifact due to
overexpression of the AT1A-EGFP receptor, since it
was made also for the nontagged wild-type receptor
and can be generalized to other G␣q/11-coupled
receptors, such as the vasopressin V1b receptor
(Fig. 4C). In the literature, G␣ proteins are shown to
be distributed in both cytosolic and membrane compartments, with most G protein immunoreactivity
associated with the plasma membrane, microsomal,
ER, or Golgi membranes and the cytoskeleton (26–
30). Thus, G␣ proteins can be addressed either to
intracellular compartments or to the cell surface,
depending on the presence or absence of posttranslational lipid modifications but also on the expression and targeting of its associated proteins. A recent and interesting observation was made for the
G␤␥ and G␣z cellular distributions, indicating that
the G␤␥ recruits the G␣z to the proper cellular compartment (31). Altogether, these data suggest
strongly that the expression of both the ␤␥-subunits
and of the cognate receptors induces the recruit-
MOL ENDO · 2001
304
ment of an adapted fraction of the G␣ protein to the
cell surface.
In the basal state (i.e. without any ligand) it is not
known how the receptor physically interacts with the
G␣ protein. Two possibilities exist: either the GDP
form of the G␣ protein interacts with the inactive
forms of the receptor with a moderate affinity or this
G␣ protein interacts only with the small fraction of
the receptor that is in its active state even in the
basal conditions, but this time with a strong affinity.
The similar recruitment of G␣q/11 by the wild-type
and the D74N mutant, which presents a major defect
of activation, and inefficacy of Losartan to modify
the membrane recruitment of G␣q/11 favor the hypothesis of an association of the inactive forms of
GPCR and G␣q/11 rather than association due to
constitutive activity of the receptor.
The intracellular translocation of the G␣q/11 protein after interaction with the cognate receptor is
another original and very interesting observation of
this study. This translocation is dependent on receptor internalization but not on its functional
coupling as demonstrated by experiments with the
coupling-defective D74N-AT1A-EGFP and the internalization-defective ⌬329-EGFP mutants. This suggests that intracellular translocation of the G␣q/11
protein is not the consequence of dissociation from
the receptor and ␤␥-subunits but follows other cellular processes linked to internalization. G␣q/11
translocation also occurs in a different cellular location compared with that of the internalized receptor.
Such intracellular translocations of G proteins after
receptor binding have been demonstrated for G␣s in
response to activation of the ␤2-adrenergic receptor
(26) and for G␣q/11 in response to TRH binding to its
receptor (32–34). These studies were carried out in
HEK-293 cells transfected with both the receptor
and its cognate G protein cDNAs. The intracellular
localizations were found either different, for the ␤2adrenergic receptor and G␣s protein, or similar for
the TRH receptor and G␣q/11. For the AT1A receptor, further studies using accurate markers of the
different intracellular compartments and their components should provide new information on the fate
of G proteins after their activation and intracellular
translocation.
In conclusion, this study demonstrates that endogenous G␣q/11 proteins are recruited by the AT1A
receptors from an intracellular pool, depending on
the level of expression of these cognate receptors at
the surface of the cell. Peptide binding to the receptor induces a translocation of the G protein from the
membrane to the intracellular pools, simultaneously
to the receptor internalization. The translocation of
G␣q/11 is dependent on the internalization rather
than on the activation of the receptor. In the near
future, the in vivo labeling of proteins should make it
possible to follow in real time the kinetics of interactions and the cellular translocation of these proteins during signal transduction.
Vol. 15 No. 2
MATERIALS AND METHODS
Construction of the Wild-Type and Mutated cDNAs
A chimeric cDNA encoding the AT1A receptor with EGFP at its
C terminus was constructed by PCR. The entire coding sequence of the AT1A receptor cDNA was amplified using
peAT1A (35) as a template and the primers 5⬘-(C T A T T C C
A G A A G T A G T G A G G A) and 3⬘-(T G T G G A T C C A
C C T C A A A A C A A G A C G C). The 5⬘-primer binds
upstream from the HindIII site of the peAT1A and the 3⬘-primer
replaces the stop codon with an alanine codon introducing a
BamHI site. The PCR fragment was digested with HindIII and
BamHI and inserted between the HindIII and BamHI sites of
pEGFP-N1 (CLONTECH Laboratories, Inc., Palo Alto, CA),
this construct was called pAT1A-EGFP. The D74N point mutation in the AT1A receptor was obtained by exchanging the
0.8-kb HindIII-EcoRI wild-type fragment with the equivalent
fragment from peAT1A-D74N, described previously (15); this
construct was called pD74N-AT1A-EGFP. The ⌬329-EGFP
mutant was constructed by PCR. The sequence of the ⌬329
was amplified using the ⌬329 mutant (9) as a template and
the primers 5⬘-(A A G C T T A C C A T G G C C C T T A C C
T) and 3⬘-(C C C G G G C C G A G T G G G A C T T G G C
C T T T). The 5⬘-primer binds upstream from the HindIII site
of the ⌬329 and the 3⬘- primer introduces a XmaI site. The
PCR fragment was digested with HindIII and XmaI and inserted between the HindIII and XmaI sites of pEGFP-N1
(CLONTECH Laboratories, Inc.); this construct was called
p⌬329-EGFP. The sequences of the constructs were verified
using fluorescent dideoxynucleotide sequencing on an ABI
Prism 377 sequencer (Perkin-Elmer Corp., Norwalk, CT). The
V1b-EGFP construct was a gift from M. A. Ventura (M. A.
Ventura and E. Clauser, in preparation).
Cell Culture and Transfection
HEK-293 cells were obtained from ATCC (Manassas, VA;
F-14742, 1573-CRL) and were grown in DMEM supplemented with 7.5% FCS, 0.5 mM glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin (all from Life Technologies,
Inc., Gaithersburg, MD). For stable and transient expression,
HEK-293 cells were tranfected with 1 ␮g/500,000 cells of the
plasmid of interest, using a liposomal transfection reagent
(Dosper from Roche Molecular Biochemicals, Indianapolis,
IN). Cell lines stably expressing the AT1A-EGFP, D74N-AT1AEGFP, ⌬329-EGFP, and V1b-EGFP receptors were selected
for resistance to 750 ␮g/ml G418 (Life Technologies, Inc.) and
cloned by limiting dilution.
Pharmacological and Signaling Properties of the
AT1A-EGFP, D74N-AT1A-EGFP, ⌬329-EGFP, and V1bEGFP Receptors
Binding experiments, including saturation and displacement
experiments with [125I]-labeled Ang II, were performed on
intact cells, essentially as previously described (7). Binding
data were analyzed by linear regression using the Excel 5
program (Microsoft Corp., Bellvue, WA).
The production of second messengers, inositol phosphates (IP), was determined by the extraction of IP and
separation on a Dowex AG1-X8 (Bio-Rad Laboratories, Inc.,
Hercules, CA) column, after incubation of the cells with myo[3H]inositol as previously described (7).
The signaling properties of the wild-type tagged receptor
were also tested in an integrated biological assay, the stimulation of a reporter gene (luciferase) placed under the control
of a minimal promoter and a multimer of a 12-O-tetradecanoylphorbol 13-acetate (TPA) (phorbol ester)-responsive element (CGTCA) (see Ref. 36 for details of the construction).
Cells were transiently transfected with the various constructs.
AT1A Receptor and G␣q/11 Protein Cellular Localizations in Response to Ang II
Two days after transfection, cells were seeded in 96-well
white opaque plates (Packard Instruments, Meriden, CT) and
stimulated for 24 h with various concentrations of Ang II, and
then rinsed twice with PBS and assayed for luciferase activity
by adding LucLite (Packard Instruments) substrate as described by the manufacturer. Luminescence was measured
using a Top Count (Packard Instruments) in single photon
counting mode.
Immunoprecipitation, Western-Blot Analyses of the
AT1A and the AT1A-EGFP Receptors, and PNGase
F Treatment
Transfected cells were scraped into solubilization buffer
(50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 1%
Triton X100, complete protease inhibitor from Roche Molecular Biochemicals). Cells (106) were incubated in 1 ml
solubilization buffer for 2 h at 4 C with slight agitation.
Insoluble material was removed by centrifugation, and the
supernatant was incubated overnight at 4 C with protein A
sepharose coupled to 25 ␮g anti-GFP antibody (anti-GFP
antibody from Roche Molecular Biochemicals) per condition (corresponding to 106 cells). Immune complexes were
collected by centrifugation and washed four times in washing buffer (37) and then eluted into Laemmli sample buffer
containing 8% SDS and heated for 5 min at 95 C. Proteins
were resolved by 10% SDS-PAGE and transferred to a
nitrocellulose membrane, which was processed as previously described (37). The membrane was incubated overnight with 0.4 ␮g/ml of GFP antibody, and then incubated
with horseradish peroxidase-conjugated goat antimouse
(1:20,000; Amersham Pharmacia Biotech, Arlington
Heights, IL). Immune complexes were detected using enhanced chemiluminescence (ECL) reagents (Amersham
Pharmacia Biotech), and the membrane was placed
against X-Omat film (Eastman Kodak Co., Rochester, NY).
Deglycosylation of the AT1A-EGFP receptor was performed on immunoprecipitated material. This material was
suspended in 25 ␮l of 1% SDS and heated at 100 C for 3 min.
H2O (225 ␮l) was added, and the samples were heated again
at 100 C for 1 min. After centrifugation, the supernatant
containing the proteins was adjusted to 40 mM sodium phosphate, pH 7.5, 1% Triton, 20 mM EDTA, and 0.144 M
␤-mercaptoethanol. After incubation with PNGase F (Roche
Molecular Biochemicals) (2 U per sample) for 17 h at 37 C,
samples were resuspended in Laemmli buffer containing 8%
SDS and analyzed on a 10% SDS-PAGE.
305
of time in the case of Ang II and for 20 min at 37 C in the case
of other peptide and nonpeptide ligands in Earle’s complete
buffer. At the end of the incubation, cells were rinsed in icecold Earle’s buffer and fixed by incubation for 10 min in 100%
methanol at 4 C.
Immunofluorescent Labeling of the G Proteins
Cells fixed and permeabilized by incubation in 100% methanol for 10 min at 4 C were incubated for 30 min at room
temperature with 5% normal goat serum (Sigma) in PBS ⫹
0.1% BSA to saturate nonspecific binding sites. Cells were
then incubated overnight at 4 C with an antibody directed
against G␣q/11 or G␣s/olf (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) at a concentration of 2 ␮g/ml in PBS ⫹ 0.1%
BSA. Cells were washed (PBS ⫹ 0.1% BSA) four times for 10
min each at room temperature and incubated with antirabbit
IgG coupled to cyanine-3 (Cy3) (Sigma) at a dilution of 1:800
for 60 min at room temperature in the dark. Cells were
washed a further four times and analyzed.
Confocal Microscopy
Cells were examined with a TCS NT confocal laser scanning
microscope (Leica Corp., Deerfield, IL) configured with a
Leica Corp. DM IRBE inverted microscope equipped with an
argon/krypton laser. EGFP fluorescence was detected after
100% excitation at 488 nm using a spectrophotometer set
with a window between 530 and 560 nm. For double detection of EGFP and Cy3 fluorescences, after excitation at 488
nm (excitation set to 100%) and 568 nm (excitation set to
50%), respectively, the fluorescences were detected in windows of 500–550 nm and 580–630 nm, respectively. Dual
excitation at 488 nm and 568 nm and the acquisition of the
emission were done simultaneously. Images of individual
cells (1,024 ⫻ 1,024 pixels) were obtained using a 63⫻ oilimmersion objective. Each image was done on a crosssection through the cells. Laser output power and photonmultipicator settings were kept at similar levels throughout
all experiments, so that the intensity values for different
experiments could be compared (see below). Lack of crosstalk between the green and the red channels was verified
as follows: 1) an absence of red fluorescence was observed
in HEK-AT1A-EGFP cells unlabeled with Cy3; 2) in HEKAT1A-EGFP cells labeled with Cy3, no loss of red fluorescence intensity was observed when excitation at 488 nm was
set to 0%.
Internalization Assays
Internalization of the AT1A-EGFP receptor was measured by
two different procedures using the stable HEK-AT1A-EGFP
cell line.
1. A biochemical acid wash procedure performed essentially as previously described (7) except that the acid wash
was done in 0.2 M acetic acid, 0.5 M NaCl in binding buffer.
2. A confocal microscopy procedure: chambered coverglass with eight wells (Nunc, Roskilde, Denmark) was treated
for 1 h with 0.1 mg/ml polyallylamine (Aldrich , Milwaukee, WI)
and rinsed twice with water. Cells were seeded (50,000 cells
per well), treated for 1 h at 37 C with 70 ␮M cycloheximide
(Sigma, St. Louis, MO), and preincubated for 15 min at 4 C in
Earle’s buffer (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.9 mM
MgCl2䡠6 H2O, 25 mM HEPES, pH 7.6) supplemented with
0.1% BSA, 0.01% glucose, 70 ␮M cycloheximide, and 0.8 mM
1–10 phenantrolin. Cells were then incubated for 30 min at 4
C with various concentrations of ligand. At this point, as a
control for the ligand specificity of internalization, some cells
were subjected to an acid wash procedure (0.2 M acetic acid,
0.5 M NaCl in Earle’s buffer, 2 min at 4 C). Internalization was
promoted by incubating the cells at 37 C for various periods
Quantification of the Subcellular Distribution of
the Fluorescence
Digital image analysis allowed us to measure the subcellular
distribution of the AT1A-EGFP receptor and the G␣q/11 protein, using a slightly modified version of a specific micro
software (40) derived from the public domain NIH Image
program (developed by the U.S. National Institute of Health
and available on the Internet at http://rsb.info.nih.gov/nihimage/). First, grayscale conversion and median filtering of
the confocal image were performed. Then the mean density
of total cellular fluorescence was determined for each cell,
and mean pixel grayscale density was automatically sampled
at 36 different locations on the cell plasma membrane, yielding the mean surface fluorescence (S) value. The mean grayscale densities of the cytoplasm (C) and the nucleus (N) were
measured after manually selecting the corresponding areas.
The background fluorescence (N) was subtracted from the C
and S values to give the S⬘ and C⬘ values. The S⬘/C⬘ ratio
provides reliable information about the level of cell surface
expression and the internalization state of the autofluorescent receptor or membrane-bound proteins.
MOL ENDO · 2001
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Vol. 15 No. 2
Statistics
Results are expressed as means ⫾ SEM. Statistical significance was assessed by Student’s t-test.
12.
Acknowledgments
We are grateful to Collette Auzan, Sabine Bardin, and MarieAnge Ventura for methodological assistance and Sophie
Conchon and Catherine Monnot for helpful discussions.
13.
Received November 29, 1999. Re-revision received October 4, 2000. Accepted November 1, 2000.
Address requests for reprints to: Dr. Eric Clauser, Institut
national de la santé et de la recherche médicale U36, Collège
de France, 3, rue d’Ulm 75005 Paris France. E-mail:
[email protected].
This study was supported by a grant from the Fond de
Recherche Hœscht Marion Roussel (FR98CVS008) and also
by the Institut national pour la santé and la recherche
médicale.
14.
15.
16.
REFERENCES
1. Clauser E, Curnow KM, Davies E, Conchon S, Teutsch B,
Vianello B, Monnot C, Corvol P 1996 Angiotensin II
receptors: protein and gene structures, expression and
potential pathological involvements. Eur J Endocrinol
134:403–411
2. Gilman AG 1987 G proteins: transducers of receptorgenerated signals. Annu Rev Biochem 56:615–649
3. Marrero MB, Schieffer B, Paxton WG, Heerdt L, Berk BC,
Delafontaine P, Bernstein KE 1995 Direct stimulation of
Jak/STAT pathway by the angiotensin II AT1 receptor.
Nature 375:247–250
4. Conchon S, Monnot C, Teutsch B, Corvol P, Clauser E
1994 Internalization of the rat AT1a and AT1b receptors:
pharmacological and functional requirements. FEBS Lett
349:365–370
5. Hunyady L, Merelli F, Baukal AJ, Balla T, Catt KJ 1991
Agonist-induced endocytosis and signal generation in
adrenal glomerulosa cells. A potential mechanism for
receptor-operated calcium entry. J Biol Chem 266:
2783–2788
6. Crozat A, Penhoat A, Saez JM 1986 Processing of angiotensin II (A-II) and (Sar1,Ala8)A-II by cultured bovine
adrenocortical cells. Endocrinology 118:2312–2318
7. Conchon S, Barrault MB, Miserey S, Corvol P, Clauser E
1997 The C-terminal third intracellular loop of the rat
AT1A angiotensin receptor plays a key role in G protein
coupling specificity and transduction of the mitogenic
signal. J Biol Chem 272:25566–25572
8. Claing A, Perry SJ, Achiriloaie M, Walker JK, Albanesi JP,
Lefkowitz RJ, Premont RT 2000 Multiple endocytic pathways of G protein-coupled receptors delineated by GIT1
sensitivity. Proc Natl Acad Sci USA 97:1119–1124
9. Conchon S, Peltier N, Corvol P, Clauser E 1998 A noninternalized nondesensitized truncated AT1A receptor
transduces an amplified ANG II signal. Am J Physiol
274:E336–345
10. Smith RD, Hunyady L, Olivares-Reyes JA, Mihalik B,
Jayadev S, Catt KJ 1998 Agonist-induced phosphorylation of the angiotensin AT1a receptor is localized to a
serine/threonine-rich region of its cytoplasmic tail. Mol
Pharmacol 54:935–941
11. Oppermann M, Freedman NJ, Alexander RW, Lefkowitz
RJ 1996 Phosphorylation of the type 1A angiotensin II
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
receptor by G protein- coupled receptor kinases and
protein kinase C. J Biol Chem 271:13266–13272
Groblewski T, Maigret B, Nouet S, Larguier R, Lombard
C, Bonnafous JC, Marie J 1995 Amino acids of the third
transmembrane domain of the AT1A angiotensin II receptor are involved in the differential recognition of peptide and nonpeptide ligands. Biochem Biophys Res
Commun 209:153–160
Ji H, Leung M, Zhang Y, Catt KJ, Sandberg K 1994
Differential structural requirements for specific binding of
nonpeptide and peptide antagonists to the AT1 angiotensin receptor. Identification of amino acid residues that
determine binding of the antihypertensive drug losartan.
J Biol Chem 269:16533–16536
Monnot C, Bihoreau C, Conchon S, Curnow KM, Corvol
P, Clauser E 1996 Polar residues in the transmembrane
domains of the type 1 angiotensin II receptor are required
for binding and coupling. Reconstitution of the binding
site by co-expression of two deficient mutants. J Biol
Chem 271:1507–1513
Bihoreau C, Monnot C, Davies E, Teutsch B, Bernstein
KE, Corvol P, Clauser E 1993 Mutation of Asp74 of the rat
angiotensin II receptor confers changes in antagonist
affinities and abolishes G-protein coupling. Proc Natl
Acad Sci USA 90:5133–5137
Hunyady L, Baukal AJ, Balla T, Catt KJ 1994 Independence of type I angiotensin II receptor endocytosis from
G protein coupling and signal transduction. J Biol Chem
269:24798–24804
Yamano Y, Ohyama K, Chaki S, Guo DF, Inagami T 1992
Identification of amino acid residues of rat angiotensin II
receptor for ligand binding by site directed mutagenesis.
Biochem Biophys Res Commun 187:1426–1431
Chaki S, Guo DF, Yamano Y, Ohyama K, Tani M, Mizukoshi M, Shirai H, Inagami T 1994 Role of carboxyl tail of
the rat angiotensin II type 1A receptor in agonist-induced
internalization of the receptor. Kidney Int 46:1492–1495
Thomas WG, Thekkumkara TJ, Motel TJ, Baker KM 1995
Stable expression of a truncated AT1A receptor in
CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling
or desensitization. J Biol Chem 270:207–213
Gunther S, Gimbrone Jr MA, Alexander RW 1980 Regulation by angiotensin II of its receptors in resistance
blood vessels. Nature 287:230–232
Abdellatif MM, Neubauer CF, Lederer WJ, Rogers TB
1991 Angiotensin-induced desensitization of the phosphoinositide pathway in cardiac cells occurs at the level
of the receptor. Circ Res 69:800–809
Awaji T, Hirasawa A, Kataoka M, Shinoura H, Nakayama
Y, Sugawara T, Izumi S, Tsujimoto G 1998 Real-time
optical monitoring of ligand-mediated internalization of
␣1b-adrenoceptor with green fluorescent protein. Mol
Endocrinol 12:1099–1111
Barak LS, Ferguson SS, Zhang J, Martenson C, Meyer T,
Caron MG 1997 Internal trafficking and surface mobility
of a functionally intact ␤2-adrenergic receptor-green fluorescent protein conjugate. Mol Pharmacol 51:177–184
Drmota T, Gould GW, Milligan G 1998 Real time visualization of agonist-mediated redistribution and internalization of a green fluorescent protein-tagged form of the
thyrotropin-releasing hormone receptor. J Biol Chem
273:24000–24008
Thomas WG, Thekkumkara TJ, Baker KM 1996 Molecular mechanisms of angiotensin II (AT1A) receptor endocytosis. Clin Exp Pharmacol Physiol Suppl 3:S74–80
Wedegaertner PB, Bourne HR, von Zastrow M 1996 Activation-induced subcellular redistribution of Gs ␣. Mol
Biol Cell 7:1225–1233
Astesano A, Regnauld K, Ferrand N, Gingras D, Bendayan M, Rosselin G, Emami S 1999 Cellular and subcellular expression of Golf/Gs and Gq/G11 ␣-subunits in
AT1A Receptor and G␣q/11 Protein Cellular Localizations in Response to Ang II
28.
29.
30.
31.
32.
33.
34.
rat pancreatic endocrine cells. J Histochem Cytochem
47:289–302
Dosanjh A 1998 Immunolocalization of G␣i-3 in foetal
lung fibroblasts. Eur Respir J 12:1137–1140
Murga C, Esteban N, Ruiz-Gomez A, Mayor Jr F 1997
The basal subcellular distribution of beta-adrenergic receptor kinase is independent of G-protein ␤ ␥ subunits.
FEBS Lett 409:24–28
Tran D, Stelly N, Tordjmann T, Durroux T, Dufour MN,
Forchioni A, Seyer R, Claret M, Guillon G 1999 Distribution of signaling molecules involved in vasopressininduced Ca2⫹ mobilization in rat hepatocyte multiplets.
J Histochem Cytochem 47:601–616
Fishburn CS, Pollitt SK, Bourne HR 2000 Localization of
a peripheral membrane protein: G␤␥ targets G␣(Z). Proc
Natl Acad Sci USA 97:1085–1090
Svoboda P, Kim GD, Grassie MA, Eidne KA, Milligan G
1996 Thyrotropin-releasing hormone-induced subcellular redistribution and down-regulation of G11␣: analysis
of agonist regulation of coexpressed G11␣ species variants. Mol Pharmacol 49:646–655
Drmota T, Novotny J, Kim GD, Eidne KA, Milligan G,
Svoboda P 1998 Agonist-induced internalization of the G
protein G11␣ and thyrotropin-releasing hormone receptors proceed on different time scales. J Biol Chem 273:
21699–21707
Drmota T, Novotny J, Gould GW, Svoboda P, Milligan G
1999 Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone recep-
35.
36.
37.
38.
39.
40.
307
tor-1 and Gq␣/G11␣ induced by agonist stimulation. Biochem J 340:529–538
Teutsch B, Bihoreau C, Monnot C, Bernstein KE, Murphy
TJ, Alexander RW, Corvol P, Clauser E 1992 A recombinant rat vascular AT1 receptor confers growth properties
to angiotensin II in Chinese hamster ovary cells. Biochem
Biophys Res Commun 187:1381–1388
Parnot C, Le Moullec JM, Cousin MA, Guedin D, Corvol
P, Pinet F 1997 A live-cell assay for studying extracellular
and intracellular endothelin-converting enzyme activity.
Hypertension 30:837–844
Towbin H, Staehelin T, Gordon J 1979 Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc
Natl Acad Sci USA 76:4350–4354
Anderson KM, Murahashi T, Dostal DE, Peach MJ 1993
Morphological and biochemical analysis of angiotensin II
internalization in cultured rat aortic smooth muscle cells.
Am J Physiol 264:C179–188
Griendling KK, Delafontaine P, Rittenhouse SE,
Gimbrone Jr MA, Alexander RW 1987 Correlation of
receptor sequestration with sustained diacylglycerol
accumulation in angiotensin II-stimulated cultured
vascular smooth muscle cells. J Biol Chem 262:
14555–14562
Lenkei Z, Beaudet A, Chartrel N, De Mota N, Irinopoulou
T, Braun B, Vaudry H, Llorens-Cortes C 2000 A highly
sensitive quantitative cytosensor technique for the identification of receptor ligands in tissus extracts. J Histochem Cytochem 48:1553–1564