A Functional Enhanced Green Fluorescent Protein (EGFP)-Tagged Angiotensin II AT1A Receptor Recruits the Endogenous G{alpha}q/11 Protein to the Membrane and Induces Its Specific Internalization Independently of Receptor- G 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}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{alpha}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{alpha}q/11 protein intracellular translocation, which presents a similar kinetic pattern but occurs predominantly in a different compartment; and 3) This G{alpha}q/11 protein cellular translocation is dependent on receptor internalization process, but 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 {Delta}329) for which the coupling and internalization functions are modified.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha} helices, 2) a transducer, corresponding to a heterotrimeric GTP-binding protein and associating a ß{gamma}-complex, which is anchored in membranes via a prenylated cystein and an {alpha}-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 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{alpha}q/11protein, phospholipase C (ß-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, 5, 6, 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{alpha}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{alpha}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1Go). 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 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. 1Go).


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Table 1. Pharmacological Characterization of the AT1A-EGFP Receptor in HEK-293 Cells

 


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Figure 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.

 
In addition, the AT1A-EGFP receptor is specifically immunodetected using an anti-EGFP antibody both by immunoprecipitation and Western blot (Fig. 2Go). The glycosylated form of AT1A receptor corresponds to a diffuse band from 70 to 90 kDa (Fig. 2Go, 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. 2Go, 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. 2Go, 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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Figure 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 AT1A-EGFP receptor is located at the plasma membrane as shown on a large confocal view of the cells (Fig. 3AGo) or for a typical HEK-AT1A-EGFP cell (Fig. 3BGo). 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 incubating 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. 3CGo) or a typical AT1A-EGFP-internalized cell (Fig. 3DGo). Optical sectioning of the cells by 1 µm thick serial confocal slices confirmed the presence of fluorescent internalization vesicles since their average size was less than 1 µm 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.



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Figure 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 µm; C and D, scale bar = 5 µm. 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.

 
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. 3CGo).

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. 3AGo), 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. 3AGo) with similar levels of receptor expression corresponding to 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 2Go).


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Table 2. Effect of Ang II on Cellular Distribution of the AT1A-EGFP, the D74N-AT1A-EGFP, {Delta}329-EGFP Receptors, and the G{alpha}q/11 Subunit

 
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. 3DGo and Table 2Go), 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. 3EGo). 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. 3EGo), 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 3Go), and nonpeptide ligands do not induce this process, again independently of their agonist or an- 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, 13, 14), is the key event that triggers internalization.


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Table 3. Pharmacology of the AT1A-EGFP Receptor Internalization

 
The Endogenous G{alpha}q/11 Protein Is Colocalized with the AT1A-EGFP Receptor at the Cell Surface
Since the AT1A receptor is coupled to the G{alpha}q/11 protein, it was interesting to analyze the distribution of the endogenous G protein parallel to the recombinant AT1A-EGFP receptor in HEK-AT1A-EGFP cells. The G{alpha}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 HEK-AT1A-EGFP cells, intense labeling indicates the presence of endogenous G{alpha}q/11 protein both in the cytoplasm and at the cell surface (Fig. 4BGo, 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).



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Figure 4. Colocalization of the G{alpha}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{alpha}q/11 protein (S'/C' ratio of G{alpha}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{alpha}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{alpha}q/11 protein (b3, d4, and h). Scale bar = 10 µm for panels B–E and 5 µm for panels F–I. (C) S'/C' ratio for G{alpha}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{alpha}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{alpha}q/11 protein. This hypothesis was investigated further using the heterogeneous expression of the AT1A-EGFP receptor in HEK cells and by measuring the S'/C' ratios for both AT1A-EGFP and G{alpha}q/11 fluorescence 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{alpha}q/11 protein (Fig. 4AGo) (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. 4BGo, panels a–d) or single cell images (Fig. 4BGo, panels e-h) showing a majority of cells (but not all) with parallel levels of AT1A-EGFP receptor and G{alpha}q/11 protein at the cell surface. Some cells present high surface levels of both AT1A-EGFP (Fig. 4BGo, panels a1, c2, and e) and membrane-bound G{alpha}q/11 protein (Fig. 4BGo, panels b1, d2, and f) or a low level of the receptor (Fig. 4BGo, panels a3, c4, and g) and membrane-bound G{alpha}q/11 protein (Fig. 4BGo, panels b3, d4, and h). In addition, the S'/C' ratio for G{alpha}q/11-associated fluorescence was also higher on a whole population of cells stably expressing either the wild-type AT1A receptor or another G{alpha}q/11-coupled receptor, the V1b vasopressin receptor (Fig. 4CGo).

All these data suggest that the membrane recruitment of the G{alpha}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{alpha}q/11 and the total expression of AT1A-EGFP or G{alpha}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{alpha}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{alpha}q/11 Protein and the AT1A-EGFP Receptor Is Synchronized but Occurs in Different Compartments
The AT1A receptor (Fig. 5AGo, left upper panel) and the G{alpha}q/11 protein (Fig. 5AGo, 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. 5AGo, right upper panel). Therefore, it was interesting to investigate the cellular distribution of G{alpha}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{alpha}q/11-associated fluorescence, n = 13 for each) (Table 2Go and Fig. 5AGo, lower panels). The loss of plasma membrane fluorescence was not associated with a loss of total cellular fluorescence intensity (Table 2Go).



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Figure 5. Kinetics of Ang II-Induced Cytoplasmic Translocation of the AT1A-EGFP Receptor and the G{alpha}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{alpha}q/11 protein fluorescence; lane III, fluorescence overlay. C, Changes in the S'/C' ratio of EGFP-associated ({diamondsuit}) and G{alpha}q/11-associated (•) fluorescences over time. The results are expressed as means ± SEM of three independent experiments. Scale bar = 5 µm for panel A and 2.5 µm for panel B.

 
The kinetics of intracellular translocations for both the AT1A-EGFP receptor and the G{alpha}q/11 proteins were analyzed next by sequential observation of the cells for 20 min. Figure 5BGo 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{alpha}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{alpha}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{alpha}q/11-associated fluorescence is more diffuse in the cytoplasm and corresponds to fine granules, without any superimposition with the receptor, as indicated by the absence of yellow labeling in the overlay (Fig. 5BGo, lane III, lower panels). Confocal images were quantified and the kinetics of intracellular translocation for both the AT1A-EGFP receptor and the G{alpha}q/11 protein were found to be identical (Fig. 5CGo). Internalization of 50% of the proteins was obtained after similar periods of time for the two proteins (t1/2 = 2.7 min for AT1A-EGFP; t1/2 = 3.2 min for G{alpha}q/11 protein; n = 4 for each point). Therefore, the AT1A-EGFP receptor and the G{alpha}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{alpha}q/11 protein is translocated inside the cell in a specific manner after the AT1A-EGFP activation for two reasons: 1) no G{alpha}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{alpha}s/olf protein, an unrelated G protein (data not shown).

Ang II-Induced G{alpha}q/11 Protein Translocation Is Dependent on Receptor Internalization but Not on Receptor Coupling and Signal Transduction
The association of this G{alpha}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, 16) but is still internalized after ligand binding (same references) and the truncated {Delta}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{alpha}q/11 protein without activating the receptor and the signaling pathway (Fig. 6AGo). The nonpeptide ligands Losartan (Fig. 6BGo) and L162,313 (Fig. 6CGo) are unable to induce translocation either of the receptor or of the G{alpha}q/11-protein independently of their agonist/antagonist status.



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Figure 6. Dependency of G{alpha}q/11 Translocation on Internalization but not Signal Transduction of the AT1A-EGFP Receptor

A, Pharmacology of the AT1A-EGFP receptor and the G{alpha}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{alpha}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 {Delta}329 mutant and its G{alpha}q/11 protein : HEK-{Delta}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 µm.

 
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 colocalized at the cell surface with the G{alpha}q/11 protein (Fig. 6DGo and Table 2Go) 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. 6EGo and Table 2Go). This normal internalization of the mutant is quantified by reductions of 90% and 67% of the S'/C' ratios of the D74N-AT1A-EGFP- and G{alpha}q/11-associated fluorescences, respectively, as compared with 90% and 74% reductions for the wild-type receptor.

The {Delta}329-EGFP mutant stably expressed in HEK-293 cells presents the same properties as the {Delta}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 {Delta}329-EGFP mutant). The {Delta}329-EGFP mutant is colocalized at the cell surface with the G{alpha}q/11 protein in basal conditions (Fig. 6FGo and Table 2Go). After a 100 nM Ang II treatment, the {Delta}329-EGFP mutant is partly internalized as measured by a reduction of only 54% of the S'/C' ratio (Fig. 6GGo, left, and Table 2Go) and despite Ang II-induced receptor activation, the G{alpha}q/11 is still localized at the cell surface, as measured by a reduction of only 8% of the S'/C' ratio (Fig. 6GGo, middle, and Table 2Go).

The pharmacological data obtained with agonist and antagonist ligands and those for the signaling- and the internalized-defective mutants show that subcellular translocation of the G{alpha}q/11 protein is dependent on receptor internalization but not on the activation state of the AT1A-EGFP receptor.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}1B-adrenergic (22), the ß2-adrenergic (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. 3Go of this paper the AT1A-EGFP 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.

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{alpha}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{alpha}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{alpha}q/11-coupled receptors, such as the vasopressin V1b receptor (Fig. 4CGo). In the literature, G{alpha} 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, 27, 28, 29, 30). Thus, G{alpha} 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 Gß{gamma} and G{alpha}z cellular distributions, indicating that the Gß{gamma} recruits the G{alpha}z to the proper cellular compartment (31). Altogether, these data suggest strongly that the expression of both the ß{gamma}-subunits and of the cognate receptors induces the recruitment of an adapted fraction of the G{alpha} 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{alpha} protein. Two possibilities exist: either the GDP form of the G{alpha} protein interacts with the inactive forms of the receptor with a moderate affinity or this G{alpha} 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{alpha}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{alpha}q/11 favor the hypothesis of an association of the inactive forms of GPCR and G{alpha}q/11 rather than association due to constitutive activity of the receptor.

The intracellular translocation of the G{alpha}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 {Delta}329-EGFP mutants. This suggests that intracellular translocation of the G{alpha}q/11 protein is not the consequence of dissociation from the receptor and ß{gamma}-subunits but follows other cellular processes linked to internalization. G{alpha}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{alpha}s in response to activation of the ß2-adrenergic receptor (26) and for G{alpha}q/11 in response to TRH binding to its receptor (32, 33, 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 ß2-adrenergic receptor and G{alpha}s protein, or similar for the TRH receptor and G{alpha}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{alpha}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{alpha}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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {Delta}329-EGFP mutant was constructed by PCR. The sequence of the {Delta}329 was amplified using the {Delta}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 {Delta}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{Delta}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 µg/ml streptomycin (all from Life Technologies, Inc., Gaithersburg, MD). For stable and transient expression, HEK-293 cells were tranfected with 1 µg/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-AT1A-EGFP, {Delta}329-EGFP, and V1b-EGFP receptors were selected for resistance to 750 µg/ml G418 (Life Technologies, Inc.) and cloned by limiting dilution.

Pharmacological and Signaling Properties of the AT1A-EGFP, D74N-AT1A-EGFP, {Delta}329-EGFP, and V1b- EGFP 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. 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 µg 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 µg/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 µl of 1% SDS and heated at 100 C for 3 min. H2O (225 µl) 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 ß-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.

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 µM 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 MgCl2·6 H2O, 25 mM HEPES, pH 7.6) supplemented with 0.1% BSA, 0.01% glucose, 70 µM 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 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 ice- cold 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{alpha}q/11 or G{alpha}s/olf (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a concentration of 2 µg/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 x 1,024 pixels) were obtained using a 63x oil-immersion 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 cross-talk 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%.

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{alpha}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/nih-image/). 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.

Statistics
Results are expressed as means ± SEM. Statistical significance was assessed by Student’s t-test.


    ACKNOWLEDGMENTS
 
We are grateful to Collette Auzan, Sabine Bardin, and Marie-Ange Ventura for methodological assistance and Sophie Conchon and Catherine Monnot for helpful discussions.


    FOOTNOTES
 
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: eric.clauser{at}college-de-france.fr

This study was supported by a grant from the Fond de Recherche Hoescht Marion Roussel (FR98CVS008) and also by the Institut national pour la santé and la recherche médicale.

Received for publication November 29, 1999. Revision received October 4, 2000. Accepted for publication November 1, 2000.


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