Association of ß-Arrestin 1 with the Type 1A Angiotensin II Receptor Involves Phosphorylation of the Receptor Carboxyl Terminus and Correlates with Receptor Internalization

Hongwei Qian, Luisa Pipolo and Walter G. Thomas

Molecular Endocrinology Laboratory, Baker Medical Research Institute, Melbourne 8008, Australia

Address all correspondence and requests for reprints to: Dr. Walter G. Thomas, Molecular Endocrinology Laboratory, Baker Medical Research Institute, PO Box 6492, St. Kilda Road Central, Melbourne Victoria 8008, Australia. E-mail: walter.thomas{at}baker.edu.au


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Arrestins bind to phosphorylated G protein-coupled receptors and participate in receptor desensitization and endocytosis. Although arrestins traffic with activated type 1 (AT1A) angiotensin II (AngII) receptors, the contribution of arrestins to AT1A receptor internalization is controversial, and the physical association of arrestins with the AT1A receptor has not been established. In this study, by coimmunoprecipitating AT1A receptors and ß-arrestin 1, we provide direct evidence for an association between arrestins and the AT1A receptor that was agonist- and time-dependent and contingent upon the level of ß-arrestin 1 expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of ß-arrestin 1 association, which correlated with decreases in receptor phosphorylation. Truncation of the AT1A receptor to lysine325 prevented AngII-induced phosphorylation and ß-arrestin 1 association as well as markedly inhibiting receptor internalization, indicating a close correlation between these receptor parameters. AngII-induced association was also dramatically reduced in a phosphorylation- and internalization-impaired receptor mutant in which four serine and threonine residues in the central portion of the AT1A receptor carboxyl terminus (Thr332, Ser335, Thr336, Ser338) were substituted with alanine. In contrast, substitutions in another serine/threonine-rich region (Ser346, Ser347, Ser348) and at three PKC phosphorylation sites (Ser331, Ser338, Ser348) had no effect on AngII-induced ß-arrestin 1 association or receptor internalization. While AT1A receptor internalization could be inhibited by a dominant-negative ß-arrestin 1 mutant (ßarr1319–418), treatment with hyperosmotic sucrose to inhibit internalization did not abrogate the differences in arrestin association observed between the wild-type and mutant receptors, indicating that arrestin binding precedes, and is not dependent upon, receptor internalization. Interestingly, a substituted analog of AngII, [Sar1Ile4Ile8]-AngII, which promotes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong ß-arrestin 1 association with the full-length AT1A receptor. These results identify the central portion of the AT1A receptor carboxyl terminus as the important determinant for ß-arrestin 1 binding and internalization and indicate that AT1A receptor phosphorylation is crucial for ß-arrestin docking.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ß-ARRESTINS WERE IDENTIFIED as proteins that associated with the phosphorylated form of the ß2-adrenergic receptor (1) and quenched signals by sterically hindering the coupling of receptor to heterotrimeric G protein. A family of arrestin proteins have been identified [arrestin1 = visual arrestin; arrestin 2 = ß-arrestin 1 (ßarr1); arrestin 3 = ß-arrestin 2 (ßarr2)], and they play an important role in regulating the activity of the large superfamily of G protein-coupled receptors (GPCRs) (2). High resolution x-ray structures of arrestin (3, 4), combined with extensive mutagenesis and in vitro assays, indicate that phosphorylation within the carboxyl-terminal region of GPCRs acts to disrupt the inactive state of arrestin (5, 6). Ensuing structural rearrangements in arrestin then permit interactions with additional determinants within the receptor that, in effect, shield the receptor from further association with G protein.

In addition to modulating G protein coupling, arrestins can also bind to components of the internalization machinery, such as clathrin (7) and adaptins (8), and may act as adaptors to promote receptor endocytosis. Accordingly, overexpression of arrestin dominant-negative mutants or arrestin fragments inhibits the endocytosis of some, but not all, GPCRs (9, 10, 11, 12). In contrast, overexpression of wild-type ßarr1 or ßarr2 can enhance the internalization of coexpressed GPCRs (9, 13). Moreover, the use of green fluorescent protein (GFP)-tagged arrestins and confocal microscopy has demonstrated that arrestins are recruited to the cell membrane after receptor activation and are then either trafficked with receptors to intracellular vesicles or rapidly separated from internalizing receptors and retained near the cell membrane (14, 15, 16). Serine- and threonine-rich regions within the carboxyl terminus of GPCRs appear to control this selective trafficking of arrestins (14) and whether a particular GPCR internalizes in an arrestin-, dynamin-, and clathrin-sensitive manner may be governed by additional regulators, such as the phosphoprotein, EBP50 (17), GIT1 [GPCR kinase(GRK)-interactor 1] (18), N-ethylmaleimide-sensitive protein (19), and ADP ribosylation factors (20), or by receptor heterodimerization (21). For some GPCRs, arrestins may also couple the activated receptors to tyrosine kinase and MAPK pathways during the process of receptor internalization (22, 23). Thus, arrestins play a central role in GPCR activation and deactivation.

Angiotensin II (AngII) is an important cardiovascular peptide hormone, the actions of which are mediated primarily by the type 1 angiotensin receptor (AT1). The AT1 receptor, which has two subtypes (AT1A and AT1B) in rodents, is a GPCR that couples primarily through G{alpha}q/11 to activate PLC, which stimulates PKC and releases calcium from intracellular stores. After activation, responses to AngII are rapidly terminated, presumably by receptor phosphorylation and internalization (24). The carboxyl terminus of the AT1A receptor is the site of receptor phosphorylation (25, 26) by GRKs and PKC (27, 28, 29) and is crucial for AngII-mediated receptor internalization (30, 31). The relationship between receptor phosphorylation, putative arrestin binding, internalization, and receptor function remains unclear, and recent evidence from constitutively active AT1A receptors and activation-selective analogs of AngII suggests that the processes of signaling, phosphorylation, and internalization can be differentiated, supporting the concept of multiple receptor transition states (32).

The contribution of arrestins to AT1A receptor desensitization and internalization is also unclear. While overexpressed arrestins can enhance AT1A receptor sequestration (10), dominant-negative mutants of arrestin have little effect on (10), or cause dramatic inhibition of (33), AT1A receptor endocytosis. Indirect evidence for an AngII-induced association of the AT1A receptor and arrestins comes from the impressive immunofluorescent colocalization of AT1A receptors and trafficking GFP-linked ßarr1 and ßarr2 (14, 34, 35). ßarr2-GFP was shown to rapidly translocate from the cytoplasm to the cell membrane in response to AT1A receptor activation and then colocalize with the receptor in endocytic vesicles after extended stimulation via a mechanism that depended upon the AT1A receptor carboxyl terminus. Whether arrestins contribute to AT1A receptor endocytosis or desensitization (35, 36) or if they merely traffic with the internalizing receptors remains to be established. Also unresolved is whether arrestins can physically associate with the AT1A receptor and the involvement of the AT1A carboxyl terminus, and its phosphorylation, to this process. Hence, in this study, we investigated the direct association of ßarr1 with AngII-activated AT1A receptors and examined the contribution of the receptor carboxyl terminus, activation status, and receptor phosphorylation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Assay for AT1A Receptor-ßarr1 Association
To determine whether ßarr1 associates with wild-type and mutated AT1A receptors (see Fig. 1Go), our strategy was to coexpress hemagglutin (HA)-tagged AT1A receptors and FLAG-tagged ßarr1 in CHO-K1 cells. After AngII stimulation, closely associated cellular proteins were covalently cross-linked with a membrane-permeable, cleavable agent dithiobis(succinimidyl)propionate (DSP) and AT1A receptors were immunoprecipitated from cell lysates with anti-HA monoclonal antibody. Immunoprecipitates were then Western blotted and probed with an anti-FLAG antibody to detect coimmunoprecipitation of ßarr1 with the stimulated AT1A receptor. In preliminary experiments, we made the unexpected observation (data not shown) that overexpressed ßarr1 coimmunoprecipitated equally well with the wild-type NHA-AT1A receptor and a truncated mutant, NHA-TK325, in which all carboxyl-terminal serine and threonine residues are removed and which is not phosphorylated in response to AngII (25). This unanticipated result was also peculiar because the association was not regulated by AngII stimulation for either receptor.



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Figure 1. The AT1A Carboxyl Terminus and Various Truncated and Site-Directed Mutants

Wild-type and mutant receptors were engineered to include an HA epitope tag at the N terminus (prefix, NHA-) to permit immunoprecipitation. The top sequence is the carboxyl terminus of the wild-type AT1A receptor (Leu305) through Glu359, indicating the boundary of the seventh transmembrane-spanning helix (TM7) and the start of the carboxyl terminus (C-T); asterisks label 13 carboxyl-terminal serine and threonine residues. The three truncated mutants (NHA-TD343, NHA-TK333, and NHA-TK325) are terminated after Asp343, Lys333, and Lys325, respectively. Triple or quadruple alanine mutations (bold/underlined) were introduced into the carboxyl-terminal region of the AT1A receptor at Thr332/Ser335/Thr336/Ser338/Ala, Ser346/Ser347/Ser348/Ala, and Ser331/Ser338/Ser348/Ala, which are abbreviated as NHA-TSTS/A, NHA-SSS/A, and NHA-PKC(SSS)/A, respectively.

 
The constitutive association of ßarr1 with both the wild-type and truncated AT1A receptors suggested an affinity of ßarr1 for the basal state of the AT1A receptor, which was exacerbated by ßarr1 overexpression and was independent of receptor activation, phosphorylation, or the presence of the carboxyl terminus. In an attempt to establish a level of ßarr1 expression at which constitutive (i.e. non-agonist-induced) association was minimized, we cotransfected variable amounts of FLAG-tagged ßarr1 (0, 0.003, 0.01, 0.03, 0.1, and 0.3 µg DNA/well) into CHO-K1 cells with a constant amount (0.3 µg DNA/well) of HA-tagged wild-type AT1A receptor (NHA-AT1A). Without stimulation by AngII, the transfected cells were directly treated with DSP for cross-linking, followed by detergent lysis and receptor immunoprecipitation. Increasing ßarr1 expression was confirmed by Western blot analysis of cell extracts that were removed before receptor immunoprecipitation (Fig. 2Go, top panel). Detection of ßarr1 after AT1A receptor immunoprecipitation (Fig. 2Go, middle panel) revealed that, at the highest ßarr1 DNA concentrations (0.1 and 0.3 µg DNA/well), significant amounts of ßarr1 were coimmunoprecipitated with the unstimulated receptor. Equal AT1A receptor expression was confirmed by reprobing with an HA antibody (Fig. 2Go, bottom panel).



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Figure 2. Variable Expression of ßarr1 Reveals a Nonspecific Binding of ßarr1 to the AT1A Receptor

HA-tagged wild-type AT1A receptor (0.3 µg; NHA-AT1A), a variable amount of ßarr1 (0, 0.003, 0.01, 0.03, 0.1, and 0.3 µg), and empty vector (PCDNA3.1) to a total of 0.6 µg plasmid DNA per well was simultaneously cotransfected into CHO-K1 cells. Without AngII stimulation, the transfected cells were directly treated with DSP for cross-linking, followed by detergent lysis and AT1A receptor immunoprecipitation. Ten microliters of cell extracts, removed before immunoprecipitation, were Western blotted (WB) and probed with antibodies to the FLAG-epitope (anti-FLAG) to confirm variable expression of ßarr1 (top panel). AT1A receptors were immunoprecipitated (IP) with anti-HA monoclonal antibodies (anti-HA), and immunoprecipitates were resolved on SDS-PAGE, Western blotted (WB), and probed with biotinylated anti-FLAG antibodies (anti-FLAG-bio) to determine whether ßarr1 was coimmunoprecipitated with the receptor (middle panel). Blots were then stripped and reprobed with antibody to the HA-tag (anti-HA, 3F10) to confirm receptor expression and immunoprecipitation (bottom panel). A representative blot from three separate experiments is shown.

 
Agonist-Dependent Association of ßarr1 with the AT1A Receptor
Having established a level of ßarr1 expression (<0.1 µg DNA/well) that minimized the amount of agonist-independent association, we next investigated whether AngII could induced ßarr1-AT1A receptor association and determined if the carboxyl terminus was involved in the association. For this, ßarr1 (0.06 µg DNA/well) was transiently cotransfected with vector, HA-tagged wild-type AT1A receptor (NHA-AT1A, 0.30 µg DNA/well) or NHA-TK325 (0.30 µg DNA/well). Forty eight hours later, the transfected cells were stimulated (or not stimulated) with AngII (100 nM, 10 min) followed by DSP cross-linking, immunoprecipitation, and Western blotting. Figure 3AGo (top panel) shows that when cells were cotransfected with both AT1A receptor and ßarr1, the amount of ßarr1 coimmunoprecipitated with the receptor was significantly increased after agonist stimulation. Truncation of the AT1A receptor (NHA-TK325) abolished AngII-induced arrestin association. Both the wild-type and truncated receptor, together with ßarr1, displayed a similar level of expression among the different groups tested (Fig. 3AGo, middle and bottom panels).



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Figure 3. Association of ßarr1 with the AT1A Receptor After Agonist Stimulation Depends on the Carboxyl Terminus and Is Time Dependent

A, CHO-K1 cells were cotransfected with HA-tagged wild-type AT1A receptor (NHA-AT1A) or truncated AT1A receptor (NHA-TK325) and ßarr1 (0.06 µg DNA/well). Cells were stimulated with AngII (100 nM) for 10 min, followed by cross-linking with DSP, detergent lysis, and immunoprecipitation (IP) of AT1A receptors. Immunoprecipitates were resolved on SDS-PAGE, Western blotted (WB), and probed with biotinylated anti-FLAG antibodies (anti-FLAG-bio) to visualize ßarr1 coimmunoprecipitation with the receptor (top panel). Blots were stripped and reprobed with anti-HA antibody (3F10) to confirmed receptor expression (middle panel), and samples of cell extracts before immunoprecipitation were probed with anti-FLAG antibodies to confirm equal expression of ßarr1 between groups (bottom panel). B, Cells coexpressing the wild-type NHA-AT1A receptor and ßarr1 were stimulated with 100 nM AngII for the indicated time and processed for receptor immunoprecipitation and probed with anti-FLAG antibodies. Results are representative blots from four separate experiments.

 
Next, we determined whether the agonist-regulated association of ßarr1 with the receptor was time-dependent. Cells cotransfected with ßarr1 and wild-type AT1A receptor were treated with AngII for 0, 0.5, 1, 5, 10, and 30 min, respectively, and ßarr1 coimmunoprecipitated with the receptor at each time point. Figure 3BGo shows that the level of the association was slightly increased above basal at 0.5 min, the earliest time point tested, followed by a greater increase at 1 min; maximal association was seen at 5 min, and this was maintained to 30 min. The kinetics of ßarr1 association correlate well with the phosphorylation and internalization (Ref. 25 and Fig. 4Go, C and D) kinetics previously determined for the AT1A receptor. Based on this experiment and previous information, we used a maximal 10-min stimulation with AngII for further ßarr1-AT1A receptor association experiments. Together, these results demonstrate that at moderate levels of expression, ßarr1 can associate with the AT1A receptor in an agonist-, time-, and carboxyl-terminal-dependent manner.



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Figure 4. The Central Region of the AT1A Receptor Carboxyl Terminus Is Important for AngII-Stimulated ßarr1 Association

A, CHO-K1 cells were cotransfected with vector, HA-tagged wild-type AT1A receptor (NHA-AT1A), or various truncated versions of the receptor (NHA-TD343, NHA-TK333 and NHA-TK325), and FLAG-tagged ßarr1. The transfected cells were stimulated (or not stimulated) with AngII (100 nM, 10 min) followed by DSP cross-linking, detergent lysis, and immunoprecipitation (IP) of AT1A receptors and probed for ßarr1 coimmunoprecipitation (top panel). Blots were stripped and reprobed to confirm receptor expression (middle panel), and cell extracts were tested for ßarr1 expression (bottom panel). B, AngII-induced ßarr1 association for the various receptor constructs was quantified from four experiments and presented as means ± SE. (**, P < 0.001 compared with wild-type receptor, NHA-AT1A). C, Receptor phosphorylation determined by incorporation of 32P into receptors after AngII stimulation (100 nM, 10 min) and immunoprecipitation, SDS-PAGE, and phosphoimaging. Data are representative of three separate experiments. D, Internalization kinetics for wild-type and truncated receptors determined by acid-sensitive binding of 125I-AngII. Results are the means ± SE from three experiments.

 
The ßarr1-AT1A Receptor Interaction Involves Phosphorylation of the Receptor Carboxyl Terminus
To further analyze the contribution of the AT1A receptor carboxyl terminus to ßarr1 association, we compared ßarr1 binding to full-length receptors and a series of mutant receptors truncated at the carboxyl terminus (listed in Fig. 1Go). HA-tagged wild-type AT1A receptor (NHA-AT1A), NHA-TD343 with a deletion of 16 amino acids, NHA-TK333 with a deletion of 26 amino acids, or NHA-TK325 with a deletion of 34 amino acids were transiently cotransfected with ßarr1 into CHO-K1 cells. The transfected cells were stimulated (or not stimulated) with AngII (100 nM, 10 min), and the receptors were immunoprecipitated and probed for ßarr1 association. As shown in Fig. 4AGo (top panel), serial truncation at the carboxyl terminus caused a commensurate decrease in ßarr1 association with the receptor. The binding of ßarr1 to the receptors was quantified and is shown in Fig. 4BGo. NHA-TD343, with a truncation to Asp343, showed a reduction in the association that was not statistically different from that of wild-type receptor. However, the association of ßarr1 with the NHA-TD333 and NHA-TK325 mutants was significantly reduced by greater than 70% and 85%, respectively. The results suggest that the carboxyl terminus of the AT1A receptor is an important determinant for ßarr1 binding. Interestingly, the level of association correlated with the degree of receptor phosphorylation (Refs. 25 and 26 and Fig. 4CGo) and internalization (Refs. 25 and 26 and Fig. 4DGo) in that the more truncated mutants (NHA-TK333 and NHA-TK325) showed dramatically reduced phosphorylation, arrestin binding, and internalization compared with the wild-type and NHA-TD343 receptors.

Serine/threonine-rich clusters at an appropriate distance (30–40 amino acids) from the cell membrane in the carboxyl terminus of GPCRs play an important role in ß-arrestin trafficking to the receptor, presumably through direct interaction between phosphorylated receptor and ß-arrestin (34). Oakley et al. (34) nominated two regions in the AT1A receptor (Thr332Ser335Thr336Ser338 and Ser346Ser347Ser348) as potential candidates for ß-arrestin binding. To test the importance of these separate sites on arrestin binding, we examined ßarr1 coimmunoprecipitation for wild-type receptor and mutated receptors (NHA-TSTS/A, NHA-SSS/A). In addition, we and others (27, 28, 29, 37, 38) have previously demonstrated that PKC is a major kinase that phosphorylates and regulates the AT1A receptor. A combined mutation of Ser331, Ser333, and Ser338 to alanine [NHA-PKC(SSS/A)] causes about 60% decrease in AngII-dependent phosphorylation (29). Hence, we also tested the binding of arrestin to NHA-PKC(SSS/A). As shown in Fig. 5Go, A and B, compared with the wild-type AT1A receptor, the level of AngII-stimulated binding of ßarr1 above basal was similar in the NHA-SSS/A mutant, was slightly, but not significantly, decreased in the NHA-PKC(SSS/A) mutant, and was dramatically reduced by about 84% in the NHA-TSTS/A mutant. The expression level of either receptor (wild-type and mutants) or ßarr1 were similar among the different groups (Fig. 5AGo, middle and bottom panel). The impaired capacity of the NHA-TSTS/A mutant suggests that Thr332, Ser335, Thr336, and Ser338 at the carboxyl terminus of the AT1A receptor is a critical site for ßarr1-receptor interaction. Previously, we have shown that the NHA-TSTS/A mutant displays a markedly decreased AngII-stimulated receptor phosphorylation (Ref. 25 and Fig. 5CGo) and significantly reduced receptor internalization (Ref. 25 and Fig. 5DGo). For the NHA-PKC(SSS/A) mutant, phosphorylation was reduced by about 60% but this receptor displays full internalization (Ref. 29 and Fig. 5DGo). In addition, the NHA-SSS/A mutant showed an equivalent phosphorylation and internalization to the wild-type AT1A receptor after AngII stimulation (Fig. 5Go, C and D). Taken together, and despite the caveat that the efficiency of DSP cross-linking may differ for the various AT1A receptor mutants due to slight differences in the relative conformation of intracellular regions, these results strongly indicate that the region from Thr332 to Ser338 is most important for ß-arrestin association and receptor internalization, presumably via phosphorylation in this region.



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Figure 5. Effect of Mutating Serine- and Threonine-Rich Regions of the AT1A Receptor Carboxyl Terminus on AngII-Induced ßarr1 Binding, Receptor Phosphorylation, and Internalization

A, The association of ßarr1 with wild-type (NHA-AT1A) and carboxyl-terminally mutated receptors was examined in response to AngII stimulation (100 nM, 10 min) (top panel), and receptor expression was confirmed by probing for the HA epitope (middle panel). ßarr1 expression was examined in cell extracts before immunoprecipitation (bottom panel). B, AngII-induced ßarr1 association for the various receptor constructs was quantified from seven experiments and presented as means ± SE (*, P < 0.05 compared with wild-type receptor, NHA-AT1A). C, Receptor phosphorylation determined by incorporation of 32P into receptors after AngII-stimulation (100 nM, 10 min) and immunoprecipitation, SDS-PAGE, and phosphoimaging. Data are representative of three separate experiments. D, Internalization kinetics for wild-type and truncated receptors determined by acid-sensitive binding of 125I-AngII. Results are the means ± SE from three experiments.

 
Correlation and Independence of AT1A Endocytosis and ßarr Association
The above data are consistent with the idea that the central carboxyl terminus is phosphorylated after AngII stimulation and serves to bind arrestin and mediate AT1A receptor internalization. However, because the various AT1A receptor mutants tested for arrestin binding possess differing capacities to internalize, one alternative interpretation could be that the internalization process itself stabilizes the receptor-ßarr1 complex. Hence, receptor mutants with affected internalization may have reduced ßarr association. To examine this, we sought to inhibit wild-type and mutant AT1A receptor internalization and examine ßarr1 association. If internalization is necessary to stabilize ßarr1 binding, then we would predict that abolishing internalization with hyperosmotic sucrose (39, 40) would inhibit AT1A-ßarr1 association and abrogate the differences observed between the wild-type and mutant receptors. As shown in Fig. 6AGo, pretreatment of cells expressing wild-type and mutated AT1A receptors with sucrose (0.45 M, 30 min, 37 C) led to a marked and equivalent inhibition of receptor internalization. In contrast, sucrose treatment, while having a slight inhibitory effect on overall arrestin binding (not shown), did not alter the pattern of differences in arrestin association between the wild-type and mutant receptors. Thus, these data suggest that internalization is not obligate for arrestin binding, although we cannot rule out some stabilization of arrestin binding during internalization.



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Figure 6. Differences in AngII-Induced Arrestin Association Between Wild-Type and Mutant AT1A Receptors Persists in the Presence of Hyperosmolar Sucrose That Blocks AT1A Receptor Internalization

A, CHO-K1 cells transfected with wild-type or mutant AT1A receptors were treated with 0.45 M sucrose at 37 C for 30 min before determination of receptor internalization by acid-sensitive binding of 125I-AngII (1 nM, 10 min). Results are the means ± SE (n = 3). B, CHO-K1 cells cotransfected with wild-type or mutant AT1A receptor and FLAG-tagged ßarr1 were treated with 0.45 M sucrose (37 C, 30 min) before AngII stimulation (100 nM, 10 min). After cross-linking with DSP and detergent lysis, AT1A receptors were immunoprecipitated (IP) with anti-HA antibody and resolved on SDS-PAGE, Western blotted (WB), and then probed with biotinylated anti-FLAG antibodies (anti-FLAG-bio) to visualize ßarr1 coimmunoprecipitation with the receptor. Data are representative of three experiments.

 
Dominant-negative mutants of arrestin have been used to inhibit endogenous arrestin action and identify an arrestin-mediating endocytic pathway. Zhang et al. (10) reported that ßarr1V53D inhibited ß2-adrenergic but not AT1 receptor internalization in HEK-293 cells. In contrast, very recently, Gáborik et al. (33) demonstrated that the overexpressed ßarr1V53D in CHO-K1 cells inhibited AT1A receptor internalization. Moreover, expression of ßarr11–349 markedly inhibited AT1 receptor internalization. To further examine whether AT1 receptor internalization is arrestin/clathrin mediated, we constructed ßarr1319–418, a mutant that lacks a capacity to bind to receptor but possesses motifs for interaction with AP2 (41) and clathrin (11), thereby inhibiting the capacity of endogenous arrestin to functionally couple to endocytosis. As shown in Fig. 7Go, when ßarr319–418 was cotransfected with HA-tagged wild-type AT1A receptor (NHA-AT1A) in CHO-K1 cells, the overexpressed ßarr319–418 markedly reduced AT1A receptor internalization. Thus, our immunoprecipitation (Figs. 3Go, 4Go, and 5Go) and ßarr1319–418 inhibitory (Fig. 7Go) data, combined with those of Gáborik et al. (33), strongly suggest that the AT1 receptor binds to arrestin to internalize via a clathrin-dependent pathway.



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Figure 7. ßarr1319–418 Inhibits Internalization of the AT1A Receptor

A, Internalization kinetics (acid-sensitive binding of 125I-AngII) were determined for CHO-K1 cells cotransfected with wild-type AT1A receptor (NHA-AT1A) and either vector (pcDNA6/Myc-His A) or the carboxyl-terminal arrestin fragment (ßarr1319–418); results are the means ± SE (n = 4). B, Expression of full-length ßarr1 and ßarr1319–418 confirmed by Western blotting. Cell extracts (5 µl) were Western blotted (WB) and probed with anti-FLAG antibodies for wild-type ßarr1 and with anti-MYC antibodies for ßarr1319–418.

 
[Sar1Ile4Ile8]-AngII Promotes AT1A-ßarr1 Association
The AT1A receptor is thought to transit through multiple conformational states in the process of receptor activation and regulation (i.e. signaling, phosphorylation, and internalization) (32). We have shown previously that an analog of AngII, [Sar1Ile4Ile8-AngII, that does not activate signaling through the inositol phosphate pathway, can nevertheless induce a robust (~85% of AngII) phosphorylation of the wild-type AT1A receptor (32). To examine whether receptor phosphorylation is sufficient for ß-arrestin association, we tested the capacity of [Sar1Ile4Ile8]-AngII to induce an association of ßarr1 with the wild-type AT1A receptor. Shown in Fig. 8Go is the association of ßarr1 with the wild-type AT1A receptor stimulated by either AngII or [Sar1Ile4Ile8]-AngII at concentrations 100 times the dissociation constant for the receptor (100 nM for AngII and 30 µM for [Sar1Ile4Ile8]-AngII). Both AngII and [Sar1Ile4Ile8]-AngII induced ßarr1 binding to the receptor. Compared with AngII, [Sar1Ile4Ile8]-AngII stimulated slightly less ßarr1 association (Fig. 8AGo), although this decrease was not statistically significant, which parallels the slightly reduced receptor phosphorylation in response to this analog (32). The physical association of ßarr1 with the AT1A receptor induced by [Sar1Ile4Ile8]-AngII suggests that receptor phosphorylation is sufficient for ßarr1 binding, although the active conformation of the receptor presumably enhances the affinity of the interaction.



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Figure 8. Receptor Phosphorylation Is Sufficient to Promote ßarr1 Binding to the AT1A Receptor

A, ßarr1 association with the wild-type receptor (NHA-AT1A) was determined in response to AngII (100 nM) or [Sar1Ile4Ile8]-AngII (30 µM) stimulation for 10 min. B, Quantification of ßarr1 association from 14 separate experiments is presented as means ± SE. The slightly reduced association after [Sar1Ile4Ile8]-AngII stimulation was not significant, compared with AngII.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The major finding of the present study is that ßarr1 associates with the AT1A receptor after AngII stimulation. To stabilize the receptor-arrestin complex during immunoprecipitation, we used a covalent, reversible cross-linking agent, DSP, as previously described (22, 42), which considerably improved the reproducibility of the procedure. Interestingly, vast overexpression of ßarr1 initially masked the agonist- and time-dependent association of receptor and ß-arrestin. This nonspecific association (in the absence of AngII stimulation) could be alleviated by careful titration of cellular ßarr1 levels to reveal a robust, carboxyl-terminal- and phosphorylation-dependent binding of arrestin to the activated AT1A receptor. Such nonspecific binding of arrestin to the basal (inactive) state of the receptor has been reported (5). Its lack of regulation, in our study, by receptor stimulation or the carboxyl terminus, indicates an inherent affinity of ß-arrestin for the cytoplasmic loops of the AT1A receptor. As cautioned by Mundell and Benovic (36), and exemplified by our observations, care should be taken in the interpretation of results from studies in which arrestin function is elucidated from untitrated arrestin overexpression.

Having established a reproducible coimmunoprecipitation approach for examining AngII-stimulated ßarr1 binding to the AT1A receptor, we next explored the contribution of the receptor carboxyl terminus and its phosphorylation to this association. Truncation of the AT1A receptor to remove only 16 amino acids from the receptor carboxyl-terminus did not significantly reduce AngII-induced arrestin binding compared with the full-length receptor, and this correlated with little effect of this truncation on receptor phosphorylation or internalization. In contrast, larger truncations of the receptor tail (to Lys333 and to Lys325) produced proportionally greater decreases in ßarr1 association with corresponding reductions in receptor phosphorylation and internalization. Together, these results indicate a high degree of correlation between the level of receptor phosphorylation and the degree of ßarr1 binding and identify the central region of the carboxyl terminus (Lys333 to Asp343) as most important for AngII-induced phosphorylation and ß-arrestin binding, as well as internalization. While we and others have previously identified this central region as important for phosphorylation and internalization (25, 26), the present study is the first to correlate these regulatory aspects with the degree of ß-arrestin binding. This correlation, as well as the rapid time-dependent ßarr1 association, the persistence of ßarr1-AT1A interactions in the presence of hyperosmotic sucrose that destabilizes clathrin cages and prevents internalization, and the inhibition of AT1A receptor endocytosis by the ßarr1319–418 mutant, strongly implicates AngII-induced ß-arrestin binding in the initiation and/or support of AT1A receptor endocytosis.

Oakley et al. (34) recently demonstrated that a ß-arrestin-GFP chimera trafficked to the plasma membrane and then into endocytic vesicles with activated V2 vasopressin receptors, while the same chimera was retained near the cell membrane and did not follow the activated ß2-adrenergic receptor into endosomes. The endocytic translocation of ß-arrestin was contingent upon clusters of phosphorylated serine/threonine residues within the V2 receptor carboxyl terminus. Swapping of carboxyl termini between the V2 and ß2 receptors revealed that both the presence and the contextual placement of these clusters (30–40 amino acids relative to the cytoplasmic face of the membrane) was critical for ß-arrestin endocytic trafficking as well as a resistance to receptor dephosphorylation, recycling, and resensitization. Moreover, such clusters were noted in appropriate positions in the carboxyl termini of the neurotensin 1 and the AT1A receptors (34), which, like the V2 vasopressin receptor, also traffic ß-arrestins into endocytic vesicles (14). Thus, a major aim of our study was to examine the relative contribution of these two potential candidate clusters (Thr332Ser335Thr336Ser338 and Ser346Ser347Ser348) to arrestin binding. We therefore tested alanine mutants of these regions (NHA-TSTS/A and NHA-SSS/A, respectively) for their capacity to inhibit AngII-induced ßarr1-receptor association as well as receptor phosphorylation and internalization. We observed dramatic decreases in ßarr1 binding to the NHA-TSTS/A mutant that correlated with a similar large inhibition of AngII-stimulated phosphorylation and internalization. In contrast, mutations of the more distal serine cluster (Ser346Ser347Ser348) did not significantly affect ßarr1 binding or receptor phosphorylation and internalization. Consistent with the idea that a GRK- (not PKC-) mediated phosphorylation in the central portion of the receptor carboxyl terminus recruits arrestins to activated AT1A receptors, a receptor mutated at three PKC phosphorylation sites [NHA-PKC(SSS/A)] retained full capacity for ßarr1 binding. As previously noted (29), substitution of the PKC phosphorylation sites significantly inhibits AngII-induced receptor phosphorylation (see also Fig. 6Go), suggesting that the reported role of PKC phosphorylation on receptor regulation and desensitization (27, 28, 29, 37, 38) may occur independently of ß-arrestin. Indeed, the NHA-PKC(SSS/A) mutant displays wild-type-like receptor internalization (Ref. 29 ; see also Fig. 6Go). Together, these results identify the central region of the AT1A carboxyl terminus (Thr332Ser335Thr336Ser338) as the crucial determinant for GRK-mediated phosphorylation, ß-arrestin binding, and internalization.

As introduced earlier, ambiguity has existed as to the role of arrestins and other mediators of clathrin-mediated endocytosis (e.g. the guanosine triphosphatase, dynamin) in AT1A receptor internalization. Dominant-negative mutants of ß-arrestin (ßarr1V53D) and dynamin (K44A), which inhibit the internalization of other GPCRs, were reported to have marginal effects on AT1A receptor internalization (10), suggesting that the AT1A receptor internalizes independently of ß-arrestin and dynamin. Recently, Claing et al. (18) suggested that GPCR internalization can be finger-printed based on sensitivity to overexpression of the GIT1 protein, ß-arrestin, and dynamin dominant-negative mutants and by inhibitors of clathrin-cage formation. Internalization of the AT1A receptor was insensitive to GIT1 overexpression, which correlated with an apparent divergence from the arrestin-, dynamin-, and clathrin-dependent pathway. Finally, an arrestin-antisense strategy was ineffective in blocking desensitization of an endogenous AT1A receptor-G{alpha}i signal in HEK293 cells (36). In direct contrast, a series of recent studies (14, 33, 35, 43, 44), including the present study, argue persuasively in favor of an arrestin-, dynamin-, and clathrin-dependent mechanism of AT1A receptor internalization. Indeed, there is irrefutable evidence from confocal microscopy (14, 35, 43) for an AngII- and AT1A-directed trafficking of ßarr2-GFP in HEK293 cells. ßarr2-GFP colocalized with an epitope-tagged AT1A receptor and both translocated to a common endocytic vesicle (14). That this trafficking was dependent on the AT1A receptor carboxyl terminus was convincingly demonstrated using a chimeric ß2-adrenergic receptor containing the AT1A receptor tail, which gained the capacity to translocate ßarr2-GFP to endocytic vesicles, in contrast to the parent ß2-adrenergic receptor. Moreover, AngII stimulation of carboxyl terminally truncated AT1A receptors led to ßarr2-GFP translocation to plasma membrane-localized coated pits but did not promote redistribution to endosomes (35). In the same study (35), agonist (isoproterenol)-induced coimmunoprecipitation from endocytic vesicles of ßarr2-GFP with a chimeric ß2-adrenergic receptor containing the AT1A receptor carboxyl terminus was reported. This chimeric receptor also displayed agonist-independent internalization that could be significantly inhibited by a dominant-negative arrestin mutant (ßarr1185–418). Moreover, dominant-negative mutants of arrestin (ßarr1V53D and ßarr11–349) as well as guanosine triphosphatase- and phosphatidylinositol-insensitive versions of dynamin have been recently reported to dramatically inhibit AngII-stimulated internalization of the wild-type AT1A receptor (33, 44). Finally, cellular context may also be important given that the dynamin I K44A mutant can indeed potently inhibit AT1A internalization when the AT1A receptor is coexpressed with the bradykinin B2 receptor, ostensibly as a consequence of AT1A/B2 receptor heterodimerization (21). In support of these studies, our data strongly suggest that ß-arrestin can directly interact with the AT1A receptor (via phosphorylation of the central carboxyl terminus) and thereby regulate receptor endocytosis, presumably via an arrestin-directed route. Our demonstration that AT1A receptor internalization is inhibited by coexpression of ßarr1319–418 is also consistent with an arrestin-, dynamin-, and clathrin-mediated pathway for AT1A receptor endocytosis.

Finally, ß-arrestin trafficking is thought to involve recruitment to the cell membrane and dephosphorylation (12), followed by binding to the phosphorylated carboxyl terminus of an activated GPCR and secondary interactions with other regions of the receptor. It is, however, still not clear what drives ß-arrestin recruitment to the membrane and whether this involves receptor-mediated signaling, phosphatase activation, or if agonist-mediated receptor phosphorylation itself is sufficient to serve this process. To test if receptor phosphorylation is sufficient to drive ßarr1 binding to the AT1A receptor, we stimulated cells expressing the NHA-AT1A receptor and FLAG-ßarr1 with an analog of AngII, [Sar1Ile4Ile8]-AngII. We have previously shown that this analog can induce a significant phosphorylation of the AT1A receptor despite an inability to activate phospholipase-mediated signaling (32), consistent with the idea that [Sar1Ile4Ile8]AngII is unable to promote the active, signaling conformation in the receptor. We observed a clear association of ßarr1 with the AT1A receptor after [Sar1Ile4Ile8]-AngII stimulation that was similar to that produced by AngII. Moreover, truncation of the receptor to Lys325, which retains signaling (i.e. can attain the active state) but not phosphorylation, showed no association with ßarr1 after AngII (Fig. 7Go) or [Sar1Ile4Ile8]-AngII stimulation (data not shown). These observations suggest the crucial requirement for ß-arrestin recruitment and binding is receptor phosphorylation and not the active, signaling state of the receptor or a signal emanating from the receptor. Nevertheless, the activated conformation of the receptor almost certainly contributes to high affinity binding of arrestin to the phosphorylated receptor, although our use of cross-linker to trap arrestin on the receptor precludes any significant analysis of relative affinities. Despite the capacity of [Sar1Ile4Ile8]-AngII to induce robust phosphorylation and significant ßarr1 binding to the AT1A receptor, it is intriguing that [Sar1Ile4Ile8]-AngII does not promote strong AT1A receptor internalization (32), as measured by an assay that involved ligand stimulation, surface receptor stripping, and rebinding with 125I-AngII. One interpretation could be that, in the absence of an active receptor conformation or an accompanying signal, ß-arrestin binding alone is not sufficient to instigate receptor endocytosis. Alternatively, more direct methods for determining receptor internalization (e.g. confocal microscopy of GFP-labeled receptors and arrestins), which do not rely on indirect stimulation/rebinding assays, may reveal slower, yet significant, AT1A receptor internalization in response to [Sar1Ile4Ile8]-AngII stimulation.

In conclusion, coimmunoprecipitation of phosphorylated AT1A receptors and ßarr1 involves key serine/threonine residues in the central portion of the receptor carboxyl-terminus and correlates with the capacity of receptors to rapidly internalize in response to AngII and for receptors to traffic with ß-arrestin inside the cell (14). Future studies will further examine the interplay between the active, signaling, and phosphorylated states of the AT1A receptor, ß-arrestin binding and trafficking, and receptor internalization, desensitization, and resensitization. The receptor mutants described in the present study, as well as constitutively active versions of the AT1A receptor that are poorly phosphorylated (32), and G protein-uncoupled AT1A receptor mutants [e.g. Asp74 (45) and Tyr302 (46)] that internalize with wild type-like kinetics will prove useful in these endeavors. In conjunction with confocal microscopy, the coimmunoprecipitation approach used here will provide important insights into the identity and hierarchy of proteins that interact with the AT1A receptor during the cycle of receptor activation and deactivation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
CHO-K1 cells were obtained from the American Type Culture Collection (Manassas, VA). Dr. R. J. Lefkowitz and Dr. M. G. Caron (Duke University Medical Center, Durham, NC) kindly supplied FLAG epitope-tagged versions of ß-arrestin. The ExSite Mutagenesis kit was purchased from Stratagene (La Jolla, CA) and DNA modifying enzymes were from Promega Corp. (Madison, WI). 5'-Phosphorylated oligonucleotides, {alpha}-MEM, OPTI-MEM, HBSS, okadaic acid, and lipofectAMINE were purchased from Life Technologies, Inc. (Gaithersburg, MD), while protein A-agarose and anti-HA high-affinity rat monoclonal antibody (3F10) were from Roche Molecular Biochemicals (Indianapolis, IN). The 12CA5 monoclonal antibody was purified from hybridoma culture media using an influenza HA antigen (HA)-peptide affinity column. Anti-FLAG M2 monoclonal antibody and anti-FLAG biotinylated M2 monoclonal antibody were from Sigma (St. Louis, MO), and anti-MYC antibody was from Calbiochem (La Jolla, CA). The avidin-horseradiash peroxidase conjugate was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Antirat IgG-horseradish peroxidase was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). SuperSignal West Pico Chemiluminescent substrate and the cross-linker, DSP, were purchased from Pierce Chemical Co. (Rockford, IL). 125I-Ang II (specific activity > 2,000 Ci/mmol) was provided by Austin Biomedical Services (Melbourne, Australia) and 32P-orthophosphate was from ICN Biochemicals, Inc. AngII and [Sar1Ile4Ile8]-AngII were from Auspep Pty. Ltd. (Melbourne, Australia). All other chemicals were from Sigma or BDH Laboratory Supplies (Kilsyth, Victoria, Australia).

Plasmid DNA Constructs
The construction of an HA epitope-tagged wild-type AT1A receptor, three carboxyl-terminal truncated versions (NHA-TD343, deletion of 16 amino acids; NHA-TK333, deletion of 26 amino acids; NHA-TK325, deletion of 34 amino acids), and two point-mutated versions (NHA-TSTS/A, T332/S335/T336/S338/A and NHA-PKC(SSS/A), S331/S338/S348/A) have been described previously (25, 29). Another mutant NHA-SSS/A, characterized by three consecutive substitutions of serine residues (Ser346, Ser347, Ser348) with alanine (see Fig. 1Go), was generated using PCR-based site-directed mutagenesis (ExSite). 5'-Phosphorylated oligonucleotides used for mutagenesis were (5'-3'):

S346/347AS: p-GCTGCGGCCAAAAAGCCTGCG (sense)

S346/347AA: p-TGCCATGTTATCCGAAGGCCG (antisense)

The primers were designed to generate a triple (S346/S347/S348A) mutant when the single mutant (S348A) (29) was used as a template. A silent PvuII (bold italic, formed after ligation of PCR product generated by primers S346/347AS and S346/347AA) restriction site was introduced to assist with the screening for mutated clones. Mutations from the original sequence are underlined. The mutant receptor was sequenced to confirm the entire coding region and the relevant nucleotide mutations.

The dominant-negative ßarr1 (ßarr1319–418) was constructed using PCR from pcDNA3-FLAG-ßarr1, a gift from Dr. R. J. Lefkowitz (Duke University Medical Center, Durham, NC). A sense primer (5'-ACAAGCTTACCATGGTTTCCTACAAAGTCAAAGTGAAGCTG-3') was designed to include a HindIII site (italics) and a minimal Kozak consensus sequence (underlined) around an initiator methionine, while the antisense primer (5'-AGTGGGCCCTCTGTTGTTGAGGTGTGGAGA-3') included an ApaI site (italics). The PCR fragment was digested with HindIII and ApaI and ligated to pcDNA6/Myc-His A vector (Invitrogen, San Diego, CA) cut with the same enzymes. Positive clones were selected and sequenced to confirm the integrity of the construct.

Transient Transfection of CHO-K1 Cells
CHO-K1 cells were maintained in {alpha}-MEM containing FBS (10%), penicillin G sodium (100 µg/ml), streptomycin sulfate (100 µg/ml), and amphotericin (0.25 µg/ml, Life Technologies, Inc., Gaithersburg, MD) (complete media), seeded in 12-well culture plates, and grown in complete media until 70–80% confluent. Cells were transiently transfected with a total 0.6 µg plasmid DNA/well, using lipofectAMINE, as previously described (25). For cotransfection, 0.3 µg DNA/well of either HA-tagged wild-type, truncated, or mutated AT1A receptor were used simultaneously with various amount of FLAG-tagged ßarr1 up to 0.3 µg DNA/well, and various amounts of pRc/CMV (empty vector) to a total of 0.6 µg DNA/well. After 5 h exposure to DNA-lipofectAMINE complexes, cells were washed and grown in complete media for a further 48 h.

Coimmunoprecipitation Assay for AT1A Receptor and ß-Arrestin1 Association
The procedure for immunoprecipitation of transiently transfected HA-tagged AT1A receptors in CHO-K1 cells (25) was modified to include a cross-linking step (22, 42) for coimmunoprecipitation of AT1A receptors and ßarr1, using the reversible cross-linker, DSP. Transfected cells in 12-well plates were serum-starved for 16 h and stimulated by the agonist (Ang II, 100 nM) or an analog ([Sar1Ile4Ile8]-AngII, 30 µM) for 10 min at 37 C. In some experiments, the cells were treated with 0.45 M sucrose (30 min, 37 C), before agonist stimulation, to determine the effect of inhibiting internalization on arrestin association. For the time course experiment, stimulation by AngII was for 0, 0.5, 1, 5, 10, and 30 min at 37 C. After stimulation, DSP was added to a final concentration of 2.5 mM, and the plates were incubated for an additional 20 min at room temperature. The plates were placed on ice, washed twice with 1 ml/well of HBSS (4 C), and solubilized by the addition of RIPA buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 50 mM sodium fluoride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) containing both phosphatase inhibitors (0.5 mM okadaic acid, 10 mM sodium pyrophosphate) and proteinase inhibitors (1 µg/ml of aprotinin, 5 µg/ml of leupeptin, and 1 µg/ml of pepstatin). Plates were rocked at 4 C for 1 h, and the cell lysates were harvested, clarified by centrifugation (14,000 x g for 15 min) and precleared by the addition of protein A-agarose and BSA. Precleared lysates were incubated with 2 µg of affinity-purified 12CA5 antibody and 20 µl of protein A-agarose and agitated overnight at 4 C to coimmunoprecipitate the epitope-tagged AT1A receptors and associated, cross-linked proteins. The immunoprecipitates were washed six times, resuspended in an urea-based SDS sample buffer containing 10% ß-mercaptoethanol, and incubated at 37 C for 90 min to cleave the DSP cross-linker and extract the receptor and any associated proteins. Samples were resolved by 10% SDS-PAGE, Western blotted, and probed with a biotinylated anti-FLAG antibody and an avidin-horseradish peroxidase conjugate to identify coimmunoprecipitated arrestin protein. Blots were then stripped and reprobed with a rat monoclonal anti-HA (3F10) antibody and an antirat IgG horseradish peroxidase conjugate to confirm receptor expression. Samples representing 0.03% of the total cell extract before immunoprecipitation were also Western blotted and probed with an anti-FLAG antibody and an antimouse IgG horseradish peroxidase conjugate to confirm arrestin expression. All blots were developed using enhanced chemiluminescence. Images were analyzed using Scion image software (Frederick, MD) and differences between group compared using t tests (P < 0.05 was considered significant).

AT1A Receptor Phosphorylation
In parallel to the ßarr1 association experiments, the AngII-induced phosphorylation of wild-type, truncated, and alanine-mutated AT1A receptors was determined. The procedure for the phosphorylation assay has been described previously (25). Briefly, CHO-K1 cells, transiently transfected with HA-tagged wild- type AT1A, truncated, or alanine-mutated receptor, were incubated for 1 h with 0.4 mCi/ml 32Pi and stimulated (or not stimulated) with AngII (100 nM, 10 min). Cells were lysed, and AT1A receptors were immunoprecipitated with the 12CA5 monoclonal antibody and resolved on SDS-PAGE. Gels were fixed, dried, and placed against type BAS-IIIs phosphoimaging plates (Fuji Photo Film Co., Ltd., Tokyo, Japan), which were read in a FUJIX Bio-imaging Analyzer BAS 1000 (Fuji Photo Film Co., Ltd.) and analyzed using MacBAS v1.0 software (Fuji Photo Film Co., Ltd.).

AT1A Receptor Internalization
Internalization kinetics for the wild-type and mutated AT1A receptors were determined from acid-insensitive 125I-AngII receptor binding, as previously described (25). In some experiments, the transfected CHO-K1 cells were treated with 0.45 M sucrose (30 min, 37 C) before agonist stimulation to examine the capacity of hyperosmotic sucrose to inhibit AT1A receptor internalization.


    ACKNOWLEDGMENTS
 
We are indebted to Drs. R. J. Lefkowitz and M.G. Caron for supplying FLAG-tagged ß-arrestin constructs. We thank the members of the Molecular Endocrinology Laboratory for comments and assistance.


    FOOTNOTES
 
This work was supported by A National Heart Foundation of Australia Grant-in-Aid to W.G.T. (G99M0301) and a National Health and Medical Research Council of Australia Block Grant to the Baker Medical Research Institute.

Abbreviations: AngII, Angiotensin II; AT1A, angiotensin II type 1A receptor; ßarr, ß-arrestin; DSP, dithiobis(succinimidyl)propionate; GFP, green fluorescent protein; GIT1, GRK-interactor 1; GPCR, G protein-coupled receptor; GRK, GPCR kinase; HA, hemagglutin; NHA-AT1A, N-terminal HA-tagged AT1A receptor.

Received for publication December 18, 2000. Accepted for publication June 27, 2001.


    REFERENCES
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 DISCUSSION
 MATERIALS AND METHODS
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