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Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York
Received January 31, 2008; accepted April 15, 2008
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Abstract |
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; Moore et al., 2007). Receptor binding by arrestins is enhanced severalfold when the receptor is both agonist-bound (i.e., "active") and phosphorylated (Gurevich and Gurevich, 2006). Vertebrates express four arrestins: two are confined to rods or cones, and two, known as arrestins 2 and 3 (or β-arrestin 1 and 2, respectively), are ubiquitously expressed. How only two arrestins can regulate the very diverse family of GPCRs is an ongoing question.
Arrestins were originally identified as proteins that stop signaling by binding receptors. Numerous proteins that bind arrestin, in addition to GPCRs, have since been identified, revealing arrestin as a signaling scaffold and not only a steric inhibitor of receptor-G protein binding (DeWire et al., 2007). Several GPCRs activate extracellular signal-regulated kinase 1/2 via arrestin, and arrestin 2 has recently been shown to translocate to the nucleus, where it regulates gene expression (for review, see DeWire et al., 2007). In addition, direct interaction between arrestin and clathrin and AP-2 is required for internalization of many GPCRs (Ferguson, 2001; Moore et al., 2007), and arrestin-dependent ubiquitination is necessary for normal postendocytic degradation of receptors (Shenoy, 2007).
Although arrestin is often required for GPCR desensitization, internalization, and extracellular signal-regulated kinase activation, it is now clear that some receptors require arrestin for only a subset of these behaviors. For example, arrestin is required for protease-activated receptor 1 to desensitize but not to internalize (Paing et al., 2002), whereas arrestin is dispensable for uncoupling N-formyl peptide receptor from G protein but not for receptor recycling to the plasma membrane (Bennett et al., 2001; Vines et al., 2003). Indeed, as reviewed by Gurevich and Gurevich (2006), numerous combinations of arrestin-dependence and -independence have been described for GPCRs, making it clear that the consequences of arrestin binding are not "all or nothing."
The type 1 thyrotropin-releasing hormone (TRH) receptor is expressed in the anterior pituitary, where it controls synthesis and secretion of thyrotropin. When bound to TRH, the TRH receptor activates G
q/11, leading to the production of inositol 1,4,5-trisphosphate and diacylglycerol by phospholipase Cβ. Downstream signaling includes the release of calcium from internal stores and the activation of protein kinase C. Through the use of phosphosite-specific antibodies and site-directed mutagenesis, we previously defined a region in the TRH receptor C-terminal tail that is phosphorylated in response to agonist binding and is essential for receptor internalization and desensitization (Jones et al., 2007). Because arrestin is important for TRH receptor desensitization and internalization (Jones and Hinkle, 2005), we hypothesized that receptors lacking these key phosphosites would be defective in other arrestin-dependent behaviors because of an overall inability to bind arrestin. We coexpressed TRH receptors with or without arrestins in fibroblasts from mice lacking both arrestins 2 and 3 [Arr2/3KO mouse embryo fibroblasts (MEFs)] to distinguish between arrestin-dependent and -independent effects. Contrary to our expectation, we report that a mutant receptor lacking key phosphosites remains coupled to G protein even though it recruits and stably interacts with arrestin. We also provide evidence that desensitization and internalization require a conformational change in the arrestin molecule that is induced by specific receptor-bound phosphates.
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Materials and Methods |
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LIELD/F391A was from Dr. Jeffrey Benovic (Thomas Jefferson University, Philadelphia, PA); arrestin 3-GFP was from Dr. Marc Caron (Duke University, Durham, NC). All other arrestin-encoding plasmids were based on arrestin 3 and obtained from Dr. Vsevolod Gurevich (Vanderbilt University, Nashville, TN). All arrestins were bovine except intact Flag-tagged arrestins, which were rat. N-terminal hemagglutinin (HA)-tagged TRH receptors were described previously (Jones et al., 2007). When tested, expression levels of wild-type and 4Ala receptors were found to be similar and not affected by coexpression with different arrestins, as determined by labeling cell-surface receptors with anti-HA antibody or total receptor on Western blot, whereas surface expression of 4AlaStop receptor was typically 50 to 70% that of wild type (data not shown). TRH Receptor Internalization. MEFs expressing TRH receptors with two N-terminal HA tags were treated with or without 100 nM TRH for up to 30 min and fixed with 3% paraformaldehyde. Fixed cells were incubated with 1:1000 monoclonal anti-HA antibody (Covance Research Products, Princeton, NH) in 5% goat serum in phosphate-buffered saline without detergent to label only the receptors remaining at the surface, then incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (Bio-Rad Laboratories, Hercules, CA) and BM Blue POD Substrate (Roche Diagnostics). The colorimetric reaction was terminated with 5% sulfuric acid, and the absorbance at 450 nm was measured. Internalization was defined as the percentage of receptor lost from the surface after addition of TRH. Background obtained without antibody or in untransfected cells was normally less than 10% of total signal and was subtracted.
Arrestin Translocation. Arr2/3KO MEFs grown on glass coverslips were transiently transfected with TRH receptor and arrestin-3-GFP. Cells were placed in Hanks' balanced salt solution and 15 mM HEPES, pH 7.4, at room temperature and imaged before and after addition of 1 µM TRH. GFP was detected by excitation with a 488-nm argon laser, 543-nm bandpass emission filter, on a Nikon C1 visible light laser scanning confocal microscope with a 60x (1.4 numerical aperture) oil immersion objective. All images were processed identically using Metamorph Imaging Software (Molecular Devices, Sunnyvale, CA).
Coimmunoprecipitation and Immunoblotting. HEK293 cells in 6-cm culture dishes were transiently transfected with HA-tagged TRH receptor and FLAG-tagged arrestin, incubated in Hanks' balanced salt solution and 15 mM HEPES, pH 7.4, at room temperature, and stimulated with or without 1 µM TRH for 2 min. Proteins were cross-linked with 2 mM dithiobis(succinimidyl)propionate (Pierce, Rockford, IL), 30% dimethyl sulfoxide for 30 min at room temperature. HEK293 cells were used because the transfection efficiency of Arr2/3KO MEFs was low, and the MEFs did not produce enough protein for Western blot. The reaction was quenched by washing with 20 mM Tris and 500 mM NaCl, pH 7.4, and cells were lysed on ice in 1 ml of radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA, 10 mM NaF, 100 nM sodium orthovanadate, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate, pH 8.0) plus 1:1000 protease inhibitor cocktail III (Calbiochem, La Jolla, CA). Lysates were vortexed twice and centrifuged at 10,000g for 10 min at 4°C. Receptor complexes were immunoprecipitated from the supernatant with 1:5000 anti-HA antibody and 20 µl of protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA). Complexes were denatured, and cross-linker was cleaved by boiling in lithium dodecyl sulfate sample buffer (Invitrogen) plus 100 mM dithiothreitol. Proteins were resolved by SDS-PAGE on PAGEr Gold precast gels Lonza Rockland, Inc. (Rockland, ME). FLAG-arrestin was immunoblotted with 1:5000 M2 anti-FLAG antibody (Sigma, St. Louis, MO) and 1:1000 TrueBlot anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (eBioscience, San Diego, CA), which does not recognize denatured IgG heavy chain that runs near FLAG-arrestin (
50 kDa), followed by chemiluminescence detection with Western Lightning (PerkinElmer Life and Analytical Sciences, Waltham, MA). Gels were subsequently incubated in 0.1% sodium azide and reprobed for HA-TRH receptor using 1:5000 anti-HA primary antibody and 1:5000 goat anti-mouse secondary antibody (Bio-Rad Laboratories) to control for differences in receptor expression and gel loading. Lysates from untreated cells were also resolved by SDS-PAGE to normalize differences in arrestin expression. Densitometry was performed using Scion Image (Scion, Frederick, MD). For immunoblotting, cell lysates were resolved on 10% gels and blotted with polyclonal antibodies against cyclophilin B (1:10,000) or arrestin (1:500), both from Abcam (Cambridge, MA). The amino acid sequence recognized by the arrestin antibody is identical in mouse, rat and bovine arrestins but differs between arrestins 2 and arrestin 3.
Other. Measurement of inositol phosphate production, specifically bound [3H]MeTRH in acid/salt-resistant form, and affinity for [3H]MeTRH by Scatchard analysis were performed as described previously (Jones and Hinkle, 2005). [3H]MeTRH at 0.625 to 25 nM was used for Scatchard analysis. All experiments were performed at least three times, unless noted, and error bars show mean ± S.E. of triplicate determinations. Some error bars fell within symbol size. Differences were considered significant at p < 0.05 or 0.01, determined by one- or two-way analysis of variance and post hoc Tukey's test or Student's unpaired t test, as appropriate.
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Results |
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; Nobles et al., 2007) whereby its N and C domains close around the receptor like a clamp. A 12-residue "hinge" region links the N and C domains of arrestin; deletion of seven amino acids in the hinge (Hinge) inhibits its flexibility, greatly diminishing binding to receptors (Vishnivetskiy et al., 2002) but not to other proteins, such as microtubules (Hanson et al., 2006), JNK3, and Mdm2 (Song et al., 2006). We tested whether Hinge arrestin could inhibit receptor signaling through G proteins by coexpressing receptor and arrestin in Arr2/3KO MEFs and measuring total inositol phosphate production in cells metabolically labeled with [3H]inositol. Hinge arrestin was unable to desensitize TRH receptor, whereas wild-type arrestin caused a dramatic decrease in inositol phosphate production (Fig. 1A). Arrestin was not required for TRH stimulation of extracellular signal-regulated kinase at 5 or 35 min in Arr2/3KO MEFs (data not shown).
We previously identified phosphosites in the TRH receptor C-terminal tail that are required for internalization and desensitization (Jones et al., 2007). Mutation of these sites to Ala (4Ala receptor) reduces overall phosphorylation of the receptor by half. Truncation of 4Ala receptor just distal to this region (4AlaStop receptor) removes an additional 14 potential phosphosites, including several phosphorylated in response to TRH (Jones et al., 2007). 4Ala and 4AlaStop receptors were not desensitized by wild-type or Hinge arrestins (Fig. 1A and data not shown) (Jones et al., 2007). 4Ala receptors were desensitized, however, by an arrestin mutant, R169E, that binds activated receptors even if they are not phosphorylated (Fig. 1A). This indicates that the desensitization defect of the 4Ala receptor is due primarily to the absence of key phospho-Ser/Thr residues. R169E arrestin did not desensitize the 4AlaStop receptor, which is expected to have no arrestin binding sites in the cytoplasmic tail (Fig. 1A). Transfected arrestins were expressed at concentrations similar to those found for endogenous arrestins in wild-type MEFs (Fig. 1B).
TRH Receptor Internalization. The C-terminal tail of receptor-bound arrestin interacts with clathrin and AP2, thus recruiting the receptor to clathrin-coated pits in the plasma membrane. We previously reported that an arrestin that lacks clathrin and AP2 binding sites, LIELD/F391A arrestin, does not promote receptor endocytosis even though it effectively desensitizes the receptor (Jones and Hinkle, 2005). We asked whether a conformational change in the hinge region of arrestin is required for receptor endocytosis by expressing receptor and Hinge arrestin in Arr2/3KO MEFs. Cells were stimulated for various times with TRH and receptor remaining on the surface was quantified by enzyme-linked immunosorbent assay using an antibody against an N-terminal (extracellular) epitope on the receptor. A substantial fraction of TRH receptor was internalized in cells cotransfected with receptor and wild-type arrestin but not in cells cotransfected with LIELD/F391A arrestin, Hinge arrestin, or vector control (Fig. 2).
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; Hunyady et al., 1994; Böhm et al., 1997; Innamorati et al., 1998; Prossnitz et al., 1999; Drmota and Milligan, 2000; Kalatskaya et al., 2004). Because LIELD/F391A and Hinge arrestins do not support receptor internalization, we expected them to be deficient in promoting acid/salt resistance. We were surprised to find that both arrestin mutants increased acid/salt resistance in Arr2/3KO MEFs cotransfected with TRH receptor (Fig. 3A). Our results clearly indicate that acid/salt-resistant ligand is not necessarily internalized.
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). The two domains of Hinge arrestin cannot "clamp" around the receptor, suggesting that perhaps only one of the arrestin domains needs to interact with the receptor to promote high-affinity binding (described below) and acid/salt resistance. We expressed the N and C domains of arrestin either separately or together in Arr2/3KO MEFs to determine whether either domain alone could bind to the TRH receptor. When expressed together, the domains behaved essentially like wild-type arrestin. Neither domain on its own, however, had altered ligand binding or acid/salt resistance (Figs. 1A and 3, B and C). The data suggest that, despite steric restraints, both domains of Hinge arrestin interact with the receptor. The Flag-tagged N and C domains were expressed at high levels, particularly when coexpressed, relative to Flag-tagged full-length arrestins 2 and 3 (Fig. 3F).
Neither 4Ala receptor nor 4AlaStop receptor is desensitized by arrestin (Fig. 1A) or internalized in response to TRH (Jones et al., 2007), although the internalization defect of 4Ala receptor is partially overcome when arrestin is overexpressed (data not shown). We therefore expected that these mutant receptors would not form acid/salt-resistant complexes. Whereas 4AlaStop receptor behaved as predicted, 4Ala receptor showed very strong acid/salt resistance, albeit slightly reduced compared to wild-type receptor (Fig. 3D).
Some GPCRs bind preferentially to arrestin 3; others, including the TRH receptor, bind equally to arrestin 2 and 3 (Oakley et al., 2000). Because there is precedent for the arrestin binding preferences of a receptor changing after mutation of potential phosphosites (Qiu et al., 2007), we asked whether the phosphosite substitutions in the 4Ala and 4AlaStop receptors caused either to prefer one of the arrestins. We coexpressed wild type or mutant receptor with wild-type arrestin 2 or 3 and measured acid/salt resistance and found that none of the receptors showed a strong preference for either arrestin. The slightly reduced acid/salt resistance with the 4Ala receptor was seen whether it was coexpressed with arrestin 2 or 3 (Fig. 3E). 4AlaStop receptor did not exhibit acid-salt resistance regardless of coexpression of arrestin 2 or 3 (Fig. 3D and data not shown).
Receptor-Ligand Affinity. Arrestin preferentially binds to phosphorylated, agonist-bound receptors, thereby stabilizing the interaction between receptor and ligand (Gurevich et al., 1997). We examined the possibility that acid/salt resistance results simply from the formation a high-affinity agonist-receptor-arrestin complex. We measured the affinity of TRH receptors for radioligand by Scatchard analysis in Arr2/3KO MEFs expressing receptor with or without arrestin. Wild-type, LIELD/F391A, and Hinge arrestins all substantially increased the affinity of the wild-type receptor for TRH (Table 1). The affinities of wild-type and 4Ala receptors were identical in the absence of arrestin, and arrestin caused the same 13-fold increase in affinity when coexpressed with either receptor. This result shows that acid/salt resistance is not merely a reflection of enhanced affinity for ligand.
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Arrestin Translocation and Coimmunoprecipitation. To visualize arrestin recruitment to the plasma membrane, we coexpressed arrestin-GFP with wild-type TRH receptor in Arr2/3KO MEFs. Arrestin rapidly moved to the plasma membrane in response to TRH (Fig. 4B). 4Ala receptor also recruited arrestin (Fig. 4D), but 4AlaStop receptor did not (Fig. 4F).
To measure TRH-induced receptor clustering, we exposed cells to 100 nM TRH for 1 min, then added antibody to an N-terminal receptor epitope to label cell surface receptors exclusively and examined cells using immunofluorescence microscopy. Receptor clustering was not detected in the absence of arrestin. In the presence of arrestin, strong clustering of wild-type receptors, less intense clustering of 4Ala receptors, and negligible clustering of 4AlaStop receptors was observed (data not shown). Receptor aggregation therefore follows the same pattern as recruitment of arrestin-GFP (Fig. 4).
To monitor arrestin-receptor interaction biochemically, we transfected cells with HA-tagged TRH receptors and FLAG-tagged arrestins and coimmunoprecipitated after chemically cross-linking proteins. Wild-type arrestin coimmunoprecipitated with wild-type receptor in cells incubated with TRH, but less arrestin was recovered with 4Ala receptor (Fig. 5A and B). We saw no coimmunoprecipitation of arrestin with 4AlaStop receptor, consistent with this receptor's failure to recruit arrestin to the plasma membrane (Fig. 4F).
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Hinge arrestin increased receptor affinity for ligand and acid/salt resistance, we sought to determine whether this arrestin could also be coimmunoprecipitated with wild-type receptor. Although Hinge arrestin coimmunoprecipitated with wild-type receptor, the amount was reduced compared with wild-type arrestin (Fig. 5, C and D). In summary, 4AlaStop receptor is severely deficient in recruiting and binding to arrestin, whereas the 4Ala receptor forms intermediately stable complexes. Likewise, Hinge arrestin-receptor binding is reduced, but not abolished, in comparison to wild-type arrestin. |
Discussion |
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G protein uncoupling is believed to precede internalization of most GPCRs (Ferguson, 2001). We previously showed that TRH receptor desensitization is dependent on arrestin binding but not endocytosis, because receptor expressed with LIELD/F391A arrestin does not internalize but desensitizes normally (Jones and Hinkle, 2005). Nevertheless, desensitization and internalization were strongly correlated for every TRH receptor that we expressed with wild-type arrestin (Table 2), suggesting that desensitization and internalization of the TRH receptor require a similar—if not identical—interaction with arrestin. Conversely, overexpression of dominant-negative GRK2 results in a normal rate of β2-adrenergic receptor internalization but decreased desensitization, indicating that different phosphosites regulate the two events (Kong et al., 1994). Furthermore, mutation or deletion of key phosphosites in the m2 muscarinic, CB1 cannabinoid, n-formyl peptide, B2 bradykinin, complement 5a, and µ-opioid receptors leads to receptors that internalize but have reduced desensitization (Pals-Rylaarsdam et al., 1995; Jin et al., 1999; Maestes et al., 1999; Christophe et al., 2000; Blaukat et al., 2001; Celver et al., 2004).
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Although there is no crystal structure of an arrestin-receptor or G protein-receptor complex, the fact that G proteins and arrestins share some of the same receptor interaction sites suggests that arrestin inhibits G protein activation at least in part by direct competition. Two regions on receptors are crucial for both G protein and arrestin binding: first, the conserved Asp/Glu-Arg-Tyr motif, which is required for normal G protein activation, is also necessary for arrestin binding (Marion et al., 2006); second, a cavity between helices on the receptor's cytoplasmic side opens upon agonist binding, whereupon part of arrestin or G is inserted (Gurevich and Gurevich, 2006). Receptors are stabilized in an active conformation when elements from either G or arrestin bind in the transmembrane helix cavity.
By stabilizing a receptor's active conformation, arrestin increases its affinity for agonist (Gurevich et al., 1997; Key et al., 2001; Martini et al., 2002; Key et al., 2003; Jorgensen et al., 2005). As shown in Table 1, arrestin caused a profound increase in agonist affinity for all of the TRH receptors studied. This was predictable for the wild-type and 4Ala receptors, because both receptors recruit arrestin-GFP to the plasma membrane and coimmunoprecipitate chemically cross-linked arrestin. The ability of arrestin to increase agonist affinity of the 4AlaStop receptor was unexpected, because no interaction between this receptor and arrestin could be detected.
Arrestin is thought to sequentially probe, bind, and stabilize relevant elements of the receptor's active conformation and to undergo structural changes in its final receptor-bound state (Gurevich and Gurevich, 2004). In the case of the n-formyl peptide receptor, stepwise interactions with arrestin result in the formation of a ternary complex of ligand, receptor, and arrestin with high agonist affinity (Key et al., 2003). Key et al. (2003) proposed that the receptor first binds to arrestin through an activation-dependent binding site and through proximal phosphosites on the receptor tail, causing the release of the arrestin C tail that otherwise constrains arrestin in an "inactive" conformation. With its C tail released, arrestin is able to bind distal phosphosites on the receptor C tail, inducing additional conformational changes in arrestin that stabilize receptor-ligand binding. Thus, high-affinity ligand binding comes last in a series of interactions between n-formyl peptide receptor and arrestin. Our results with mutant receptors reveal a very different affinity spectrum, because the initial, weak interaction between arrestin and 4AlaStop receptor was sufficient to promote high receptor affinity for agonist but none of the other effects of arrestin (Table 2). Alternatively, arrestin may act indirectly to regulate receptor affinity.
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). Phosphosites in both proximal (355-365) and distal (370-412) regions of the TRH receptor tail are sufficient for strong arrestin binding, as shown by the ability of both 4Ala receptor (no proximal sites) and receptor truncated at residue 370 (no distal sites) to translocate arrestin-GFP and undergo chemical cross-linking to arrestin (Figs. 4 and 5 and data not shown). A more complete understanding of the role of arrestin will require future studies capable of quantifying the avidity of different arrestin-receptor interactions.
Even though distal sites in the TRH receptor are phosphorylated (Jones et al., 2007), only the proximal sites enable arrestin-dependent desensitization. Mutating the phosphosites in the proximal 355-to-365 region of intact or truncated receptor produced receptors that failed to undergo arrestin-mediated uncoupling or receptor endocytosis. Likewise, an arrestin mutant (Hinge) strongly increased agonist affinity but was completely ineffective at desensitizing or internalizing receptor (Table 2). Hinge arrestin is intact except for its inability to undergo phosphoreceptor-induced conformational changes. Our results suggest that the structural rearrangements that require flexibility in the hinge region of arrestin are not required to stabilize receptor-ligand binding but are essential to prevent interaction with G protein and expose AP2 and clathrin binding sites in arrestin. Incompletely phosphorylated receptors either lack the necessary phosphosites or bind arrestin in an alternate conformation that is unable to evoke all of the normal changes in arrestin. In effect, binding of wild-type arrestin to 4Ala receptor or Hinge arrestin to wild-type receptor "stalls" in its early stages, resulting in increased ligand affinity and acid/salt resistance but not desensitization or internalization (Fig. 6).
Development of acid/salt resistance and receptor endocytosis are distinct, because two arrestin mutants, LIELD/F391A and Hinge, which were completely ineffective at sequestering receptor, produced a substantial increase in acid/salt resistance. Acid/salt resistance does not simply reflect enhanced agonist affinity, because overexpression of wild-type arrestin did not increase acid/salt resistance of TRH bound to 4AlaStop receptor but did increase ligand affinity 5-fold. We conclude that phosphosites in either the proximal (355-365) or distal (370-412) half of the receptor's C-terminal tail are essential for acid/salt resistance but not for high agonist affinity (Table 2).
In addition to extending our understanding of TRH receptor-arrestin interaction, our report yields insight into how arrestins interact with GPCRs in general. Arrestin-receptor binding proceeds in a stepwise manner (Gurevich and Benovic, 1993; Gurevich and Gurevich, 2004) involving dramatic conformational changes in arrestin (Vishnivetskiy et al., 2002; Nobles et al., 2007) and possibly in the receptor as well (Kisselev et al., 2004). Our data demonstrate that each stage in this process has distinct functional consequences for the TRH receptor. The processive nature of arrestin-receptor interaction helps to explain why certain GPCRs bind arrestin but fail to display all of the canonical arrestin-dependent behaviors.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: GPCR, G protein-coupled receptor; TRH, thyrotropin-releasing hormone; Arr2/3KO MEF, mouse embryo fibroblast from arrestin 2 and 3 knock-out animals; HEK, human embryonic kidney; GFP, green fluorescent protein; HA, hemagglutinin; InsP, inositol phosphate; PAGE, polyacrylamide gel electrophoresis; ANOVA, analysis of variance.
Address correspondence to: Patricia M. Hinkle, Dept. of Pharmacology and Physiology, University of Rochester Medical Center, Box 711, Rochester, NY 14642. E-mail: patricia_hinkle{at}urmc.rochester.edu.
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