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Molecular Signature That Determines the Acute Tolerance of G Protein-Coupled Receptors

Desensitization and acute tolerance are terms used to describe the attenuation of receptor responsiveness by prolonged or intermittent exposure to an agonist. Unlike desensitization of G protein-coupled receptors (GPCRs), which is commonly explained by steric hindrance caused by the β-arrestins that are translocated to the activated receptors, molecular mechanisms involved in the acute tolerance of GPCRs remain unclear. Our studies with several GPCRs and related mutants showed that the acute tolerance of GPCRs could occur independently of agonist-induced β-arrestin translocation. A series of co-immunoprecipitation experiments revealed a correlation between receptor tolerance and interactions among receptors, β-arrestin2, and Gβγ. Gβγ displayed a stable interaction with receptors and β-arrestin2 in cells expressing GPCRs that were prone to undergo tolerance compared to the GPCRs that were resistant to acute tolerance. Strengthening the interaction between Gβγ and β-arrestin rendered the GPCRs to acquire the tendency of acute tolerance. Overall, stable interaction between the receptor and Gβγ complex is required for the formation of a complex with β-arrestin, and determines the potential of a particular GPCR to undergo acute tolerance. Rather than turning off the signal, β-arrestins seem to contribute on continuous signaling when they are in the context of complex with receptor and Gβγ.


INTRODUCTION
Upon agonist stimulation, most GPCRs undergo conformational changes leading to the dissociation of Ga from Gbg (Hepler and Gilman, 1992). Dissociated Gbg potentiates receptor phosphorylation by mediating the association of GPCR kinase 2 (GRK2) with the plasma membrane, and activation of GRK2 by affecting the Km and stoichiometry of phosphorylation (Benovic et al., 1986;Pitcher et al., 1992;Vickery and von Zastrow, 1999;Zheng et al., 2016). GRK2-mediated receptor phosphorylation increases the affinity of b-arrestins for receptors, and thereby uncouples the receptors from G proteins (Benovic et al., 1987;Lohse et al., 1990) or enhances receptor endocytosis (Ferguson et al., 1996).
Desensitization and tolerance of GPCRs are terms used to describe the attenuation of receptor responsiveness by prolonged or intermittent exposure to an agonist (Lohse et al., 1989;Hausdorff et al., 1990;Williams et al., 2013). Desensitization and acute tolerance of GPCRs have different temporal resolutions; i.e., desensitization is induced between seconds and minutes after agonist stimulation, whereas acute tolerance is induced between minutes and an hour (Williams et al., 2013). According to the current paradigm based on studies of the b2 adrenergic receptor (b2AR), receptor phosphorylation and b-arrestin translocation are suggested to be two key cellular events responsible for GPCR desensitization. b-Arrestins which are recruited to the activated receptors sterically interfere with the interaction between receptors and G proteins (uncoupling), resulting in a decrement of signaling efficiency. However, molecular mechanism of acute tolerance is yet not clear. Acute tolerance occurs over a longer period of time than desensitization, and its molecular mechanism should include desensitization and subsequent cellular events, such as endocytosis and recycling.
In preliminary studies with five GPCRs and related mutants with different tolerance properties, agonist-induced b-arrestin translocation that mediated desensitization did not explain the acute tolerance. The aim of the current study was to under-

Reporter gene assay
Cellular cAMP was measured by an indirect reporter gene method (Cho et al., 2010a;Zheng et al., 2011). Briefly, a reporter system with a plasmid containing the firefly luciferase gene under the control of multiple cAMP responsive elements, and with a pRL-TK control vector, was used. Transfected cells were seeded in 24-well plates, and each transfection set was organized into three identical groups. For D2R, D3R, and D4R, cells were treated with 2 mM forskolin and Quin (10 -12 -10 -8 M) for 4 h and harvested. Relative luciferase expression was measured using a dual luciferase assay kit (Promega, Madison, WI, USA). For b1AR and b2AR, cells were treated with increasing concentrations of ISO.

Induction of tolerance
Tolerance was induced as described previously (Min et al., 2013). Cells expressing the corresponding GPCR were treated with agonist for 1-5 min (first treatment) to trigger tolerance, and washed five times with serum-free medium (5 min per wash) at 37°C to remove all of the receptor-bound agonist. Cells were then re-challenged with agonist to induce the second response. The occurrence of tolerance was determined by comparing the amplitudes of the second response from the cells pre-treated with vehicle or agonist in the first treatment step.

Immunoprecipitation
Immunoprecipitation was conducted as described previously (Zheng et al., 2016). Cells were lysed with RIPA buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) for 1 h at 4°C with gentle mixing. A slurry of agarose beads coated with anti-FLAG antibodies (25 mL) was mixed with the supernatant for 2-3 hr on a rotation wheel at 4°C. Beads were washed with washing buffer (50 mM Tris, pH 7.4, 137 mM NaCl, 10% glycerol, 1% NP-40) three times for 5 min each at 4°C. The resulting immunoprecipitates were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and the membrane was blotted with antibodies to corresponding target proteins.

Confocal microscopy
One day after the transfection, cells were seeded onto the 35-mm confocal dishes, which contain a 1-cm well in the center sealed by glass coverslip on the bottom, and they were allowed to recover for 1 day. Then, the cells were examined with a TCS SP5/AOBS/Tandem laser scanning confocal microscope (Leica, Jena, Germany).

Statistical analysis
All results are expressed as means ± SEM. Dose-response curves with more than three experimental groups were compared via two-way analysis of variance (ANOVA) with Bonferroni post-hoc tests. Student's t-test was used for some results. p-values of less than 0.05 were considered significant.

Rapid desensitization and acute tolerance are caused by different molecular mechanisms
Dopamine D2 and D3 receptors (D2R, D3R) show high homology in amino acid composition (46% overall amino acid homology and 78% identity in the transmembrane domain) (Giros et al., 1990) and share most of their signaling pathways (Cho et al., 2010b). Nevertheless, D2R and D3R display different intracellular trafficking properties and employ different regulatory mechanisms for given signaling pathways (Cho et al., 2007). It was reported that D2R but not D3R is phosphorylated in response to agonist stimulation (Kim et al., 2001). Similarly, b-arrestin translocation was observed in cells expressing D2R but not D3R (Fig. 1A), suggesting that D2R but not D3R would undergo desensitization. If the same molecular mechanism is involved in the development of desensitization and acute tolerance, D2R but not D3R is expected to undergo tolerance. As shown in Fig. 1B, D3R but not D2R showed tolerance.
Results in Fig. 1A and 1B suggest that molecular mechanisms other than b-arrestin translocation could be involved in the development of acute tolerance of GPCRs. To determine whether this observation is generalizable, five different GP-CRs (D2R, D3R, D4R, b1AR, b2AR) were selected. In addition, five mutant receptors of D2R and D3R, in which the tolerance properties or b-arrestin translocation propensity were altered, were utilized. Using these receptors, the correlation between b-arrestin translocation and their acute tolerance was evaluated.
D3R activation of D 2 R or D 3 R in HEK-293 cells. Cells stably expressing D 2 R or D 3 R (2.5 and 3.3 pmol/mg protein, respectively) were transfected with 2 mg b-arrestin2-GFP per 100 mm culture dish. Cells were stimulated with 10 mM DA for 5 min. (B) Determination of tolerance development in cells expressing D 2 R and D 3 R. HEK-293 cells expressing D 2 R or D 3 R were pre-treated with either serum-free medium containing 100 mM ascorbic acid (Veh) or 10 mM DA dissolved in Veh for 5 min. Cells were washed five times with warm (37°C) serum-free medium and treated with increasing concentrations of Quin. The cellular levels of cAMP were then measured using the CRE-luciferase reporter gene assay. "Forsk" represents forskolin. **p<0.01, ***p<0.001 when the D 3 R/Veh group was compared with other experimental groups. (C) Cells expressing D 3 R, D 3 R-[IL2-D 2 R], or D 3 R-[IL3-D 2 R] were treated with 10 mM DA for 5 min to induce tolerance. The results for the Veh-treated group significantly differed from those for the DA-treated groups of D 3 R or D 3 R-[IL2-D 2 R] expressing cells at treatment concentrations between 10 -10 -10 -8 M (p<0.001). (D) Cells expressing D 4 R were treated with 10 mM DA for 5 min to induce tolerance. (E, F) Cells expressing either b 1 AR (E) or b 2 AR (F) were treated with vehicle (Veh) or 10 mM epinephrine (Epi) for 5 min, washed three times with serum-free media at 37°C, then treated with increasing concentrations of isoproterenol to obtain dose-response curves. The Veh group of b 1 AR was significantly different from the Epi group at treatment concentrations between 3×10 -7 -3×10 -5 M (p<0.001). The results for the Veh group of b 2 AR significantly differed from those for the corresponding Epi group at treatment concentrations between 10 -8 -3×10 -5 M (p<0.001). For (C-F) receptor expression levels were maintained between 1.5 and 2.5 pmol/mg protein.

Stable interaction between receptor and Gbg is observed in the cells that express the GPCRs which are prone to undergo acute tolerance
When the interaction between receptors and Gbg was tested, all receptors that underwent acute tolerance (D3R, K149C-D2R, b2AR, and b1AR) showed a stronger basal interaction with Gbg than did receptors that did not undergo acute tolerance (D2R, D4R, D3R-[IL3-D2R], and C147K-D3R) (Fig. 3).
These results suggest that the interaction between receptors and Gbg could be closely related to the development of receptor tolerance.

Stable interaction between Gbg and b-arrestin is observed in the cells that express the GPCRs which are prone to undergo acute tolerance
Next, interaction between Gbg and b-arrestin2 was tested. There was a stronger interaction between Gbg and b-arrestin in cells expressing the GPCRs that showed acute tolerance (D3R, b1AR, K149C-D2R, D3R-[IL2-D2R], b2AR) than in cells expressing GPCRs that did not undergo acute tolerance (D2R, C147K-D3R, D4R) (Fig. 4). The results shown in Fig. 3 and 4 suggest that the presence of b-arrestin2 in the receptor-Gbg complex determines the ability of a particular GPCR to undergo tolerance. These results are summarized in Table 2.

The N domain of b-arrestin2 is involved in its interaction with D 3 R in collaboration with Gbg
Because the simultaneous interaction of each GPCR with Gbg and b-arrestin was required for acute tolerance, we investigated whether each pair of proteins from the receptor-Gbg-barrestin complex could interact independently, or whether all three binding partners were required. To test this possibility, interactions between D3R and Gbg or b-arrestin2 were determined in the absence of either b-arrestin or Gbg, respectively. When cellular Gbg was sequestered by co-expression of GRK2-CT, the interaction between D3R and b-arrestin was abolished (Fig. 5A). In the reverse situation (Fig. 5B), the interaction between D3R and Gbg was abolished when cellular barrestins were knocked down (Min et al., 2013). These results b-arrestin2 and/or GRK2-CT. Cell lysates were immunoprecipitated with FLAG beads. Cell lysates were immunoprecipitated with FLAG beads. Samples were analyzed on SDS-PAGE. Immunoprecipitates were immunoblotted with antibodies against FLAG; co-immunoprecipitates and lysates were immunoblotted with antibodies against b-arrestin2. The ratios of immunoprecipitation are: 5.49 ± 1.32, Mock; 1.02 ± 0.29, GRK2-CT (p<0.01, n=3). (B) Con-KD and b-arrestin1/2-KD cells expressing FLAG-D 3 R were transfected with YFP-Gb1. Cell lysates were immunoprecipitated with FLAG beads. Immunoprecipitates were immunoblotted with antibodies against FLAG; co-immunoprecipitates and lysates were immunoblotted with antibodies against GFP. The ratios of immunoprecipitation are 5.71 ± 1.28, Con-KD; 0.51 ± 0.03, b-Arr-KD (p<0.01, n=3). (C) A GST pull-down assay was conducted to determine the requirement of b-arrestin for the interaction between the third cytoplasmic loop of D 3 R (IL3-D 3 R) and Gbg. For production of GST fusion proteins, 0.5 mM of IPTG was added to the bacterial cultures containing each plasmid for 2 hrs. Bacterial cell lysates containing GST or GST-IL3-D 3 R (Zheng et al., 2011) were incubated with cell lysates from control knockdown (Con-KD) and b-arrestin1/2-KD cells that expressed YFP-Gb1. Lower panel shows an SDS-PAGE analysis of the afterwash of bacterial cell lysates. The ratios of pull-down are 3.39, Con-KD; 1.0, b-Arr-KD. The data represent results of two independent experiments with similar outcomes. (D) Interaction between b-arrestin2 and Gb1 was determined by a GST pull-down assay. Bacterial lysates containing the GST fusion proteins of the full-length (GST-b-arr2-FL), N-domain (GST-b-arr2-N), C-domain (GST-b-arr2-C), or Cdomain plus carboxyl tail of rat b-arrestin2 (GST-b-arr2-CCT) (Zheng et al., 2011) were mixed with the cell lysates of HEK-293 cells transfected with YFP-Gb1. After three washes, GST beads were incubated with SDS sample buffer. The eluents were analyzed with SDS-PAGE and blotted with antibodies to GFP (GST pull-down part).   were also confirmed by GST pull-down assays. Interaction between the third intracellular loop of D3R and Gbg was inhibited when the expression of cellular b-arrestins was lowered (Fig.  5C). Overall, these results suggested that Gbg and b-arrestin are mutually required for their interaction with D3R. In agreement with these results, receptors are able to associate with b-arrestins and Gbg through distinct residues (Bornancin and Parker, 1997;Mahon et al., 2006). In addition, b-arrestins can interact with Gb subunits (Yang et al., 2009) through residues that do not necessarily contribute to the association between Gb and GPCRs (Shenoy and Lefkowitz, 2005;Mahon et al., 2006), thus allowing b-arrestins to associate simultaneously with GPCRs and the Gbg dimer.
To determine the regions of b-arrestin2 that interact with Gbg, b-arrestin2 was divided into three fragments, comprising its N-, C-, and CT-domains (Zheng et al., 2011), and GST pulldown assay was conducted for the interaction with Gb1. As shown in Fig. 5D, the N-domain of b-arrestin2 and C-domain to a less extend interacted with Gb1. These results suggested that the N domain of b-arrestin2 mainly interacts with D3R in a Gbg-dependent manner (Fig. 5E).

Strengthening the interaction between Gbg and b-arrestin enhances the acute tolerance of receptors
Our results suggested that basal interactions of each GPCR tested with Gbg, as well as the interaction between Gbg and b-arrestin, were correlated with acute tolerance. It was noticeable that D4R and D3R-[IL3-D2R], which did not undergo acute tolerance (Fig. 1C, 1D), had an abundant interaction with b-arrestin2 (Fig. 2D, 2H) but a weak interaction with Gbg (Fig. 3A , 3B). In addition, a weak interaction was observed between Gbg and b-arrestin2 in cells expressing these receptors (Fig. 4D). Therefore, we wanted to determine whether enforcement of the receptor/Gbg/b-arrestin complex by strengthening the interaction between Gbg and b-arrestin endowed GPCRs with the propensity to undergo acute tolerance. For this determination, we prepared a DNA sequence encoding the fusion protein Gb1-Arr3 where b-arrestin2 was attached to the carboxyl tail of Gb1.
Expression of Gb1-Arr3, but not simultaneous expression of Gb1 and b-arrestin2 in b-arrestin1/2-knockdown cells, rendered D4R prone to acute tolerance (Fig. 6A). Similar results were obtained with D3R-[IL3-D2R] (Fig. 6B). When this strategy was applied to D2R, which neither underwent tolerance nor was heavily bound to b-arrestin2, receptor tolerance was not induced (Fig. 6C). Thus, it could be postulated that the stable complex with Gbg and b-arrestin is required for the development of receptor tolerance.
Overall, these results showed that the simultaneous interaction between the receptors, and both Gbg and b-arrestin may be related to their acute tolerance properties.

DISCUSSION
Rapid desensitization and acute tolerance are usually distinguished based on the different time points when functional changes appeared after agonist treatment. Thus, proper establishment of time windows is needed to correctly determine the molecular mechanisms and functional consequences of rapid desensitization and acute tolerance.
While GRK2-mediated receptor phosphorylation and sub- sequent b-arrestin translocation toward activated receptors are believed to be responsible for GPCR desensitization, recent studies of b2AR suggest that this paradigm might need reinterpretation or correction. For example, knockdown of GRK2 or GRK6 resulted in a partial decrement of b2AR tolerance, but did not affect the agonist-induced association between b2AR and b-arrestin2 (Nobles et al., 2011). In addition, another study showed that GRK2 was not involved in the rapid desensitization of b2AR (Violin et al., 2008). Thus, it is clear that b-arrestins are involved in both rapid desensitization and acute tolerance. On the other hand, more data are required to generalize the roles of GRK2 in the rapid attenuation of GPCR signaling.
Results in the current study show that b-arrestins are required for the interaction between Gbg and D3R, and that formation of a stable b-arrestin-receptor complex does not prevent, but rather favors, continuous signaling of D3R. b-arrestins might have different roles in the regulation of GPCR signaling if they are in different context; for example, associated with Gbg. For example, recent studies examining the parathyroid hormone receptor (PTHR) and vasopressin type 2 receptor Wehbi et al., 2013) have shown that these receptors forms a ternary complex with b-arrestin2 and Gbg, which enables more persistent signaling of the receptors through the Ga subunit.
Tolerance represents a gradual decrement of response to a drug treatment following its repeated use, thus increasingly larger doses are required to obtain similar therapeutic effects obtained by administration of initial dose. Compared to rapid desensitization, detailed molecular mechanism of acute tolerance is not clear. Thus, the present study has revealed that potentialities to undergo tolerance can be predicted. GPCRs have the tendency to undergo tolerance if they are predisposed to mediate the formation of a stable protein complex with b-arrestins and Gbg.