Here, we used MS-based quantitative proteomic approaches to map phosphorylation sites on the β2AR, determined the GRKs responsible for phosphorylation of the sites, and delineated conformation-specific β-arrestin capabilities imparted by these specific phosphorylation events. We found that in HEK293 cells, GRK2 sites were primarily responsible for β2AR internalization, GRK6 sites contributed to β-arrestin–mediated ERK activation, and both GRKs contributed to desensitization. We also demonstrated that a β-arrestin–biased ligand, carvedilol, induced a phosphorylation pattern distinct from that of an unbiased, full agonist, isoproterenol. Finally, our data are consistent with the model that different phosphorylation patterns on the β2AR elicited by either GRK2 or GRK6 can induce distinct β-arrestin conformations. These data are consistent with the receptor phosphorylation barcode hypothesis, whereby the distinct pattern of phosphorylation triggers specific downstream signaling. Our data further support the idea that distinct patterns of multisite phosphorylation on a receptor by different GRKs differentially enable β-arrestin functions by inducing distinct β-arrestin conformations.
The present study found roles for both GRK2 and GRK6, whereas Violin et al.
had previously reported that in HEK293 cells, GRK6 was the primary GRK responsible for mediating β2
AR desensitization as assessed by a quantitative analysis of real-time cAMP dynamics (10
). However, the experimental approach used in the present work differs in a number of respects. Violin et al.
monitored changes in cAMP abundance over a 12-min time period after stimulation with 1 µM isoproterenol to study the kinetics of cAMP accumulation and degradation during this constant exposure to isoproterenol. In contrast, here we prestimulated the
cells for 5 min with 100 nM isoproterenol, then washed the cells and rechallenged them with varying concentrations of isoproterenol (1 nM to 10 µM) 15 min after the initial stimulation. We measured cAMP within 5 min, representing a single ~20-min time point after the initial stimulation. Thus, in Violin et al.
), the experiment ended with the desensitization that had occurred within 12 min, whereas here we examined desensitization at a single later time point, 20 min. It is possible that there are temporal differences in the mechanisms of desensitization with GRK6 important at early times and both GRK2 and GRK6 important later. In addition, there may be differences in the contributions of the GRKs in conditions of continuous versus transient exposure to ligand. Moreover, Violin et al.
used the PKA antagonist H89 to block phosphodiesterase (PDE)–mediated cAMP degradation, a process activated by PKA. PKA promotes the translocation of GRK2, but not GRK6, to the membrane and thereby enhances its activity toward the receptor (19
), and the use of H89 may have obscured a contribution of GRK2 in the previous study.
Distinct signaling in response to specific GRK phosphorylation is supported by studies of several other GPCRs, including the vasopressin 2 receptor (V2R), the chemokine receptor CXCR4, and the angiotensin 1A receptor (AT1A
R), for which GRK6 or GRK5 promotes β-arrestin–mediated ERK activation, whereas GRK2 or GRK3 opposes it (7
). In the case of the CCR7 chemokine receptor overexpressed in HEK293 cells, although both endogenous ligands CCL19 and CCL21 induce G protein activation, calcium mobilization, and G protein–dependent and β-arrestin2–dependent ERK activation with equal potency, only activation by CCL19 promotes robust desensitization (21
). CCL19 leads to robust CCR7 phosphorylation and β-arrestin2 recruitment catalyzed by both GRK3 and GRK6, whereas CCL21 activates GRK6 alone and leads to weaker β-arrestin2 recruitment (22
). Although these data suggested a correlation between specific CCR7 phosphorylation and β-arrestin2–dependent activities, the relevant sites phosphorylated by different GRKs were not determined.
A study of the chemokine receptor CXCR4, with MS in conjunction with antibodies that recognized site-specific phosphorylation, mapped phosphorylation sites upon stromal cell–derived factor 1 stimulation in HEK293 cells stably transfected with the CXCR4 (20
).Of the 18 potential serine and threonine phosphorylation sites on the C terminus of CXCR4, 3 sites were identified as phosphorylated by MS, and an additional 4 were identified with phosphorylation site–specific antibodies. GRK6 accounted for most of the phosphorylation sites identified. Although no GRK2 or GRK3 sites were found, multiple GRKs regulate CXCR4 signaling, including GRK2. Silencing of either GRK2 or GRK6 by siRNA increased calcium mobilization, whereas knockdown of GRK3 or GRK6 decreased ERK1/2 activation. GRK2 knockdown enhanced ERK1/2 activation, suggesting coordination among the GRKs in terms of signaling, although no mechanistic explanation could be deduced in the absence of identified GRK2 phosphorylation sites.
Synthetic phosphopeptides corresponding to the C-terminal sequences of two GPCRs bind to and induce conformational changes in β-arrestins (23
). Moreover, phosphopeptides derived from the sequence of the V2
R tail (a member of the “class B” receptors that bind β-arrestins tightly) induce conformational changes in β-arrestin distinct from those observed with phosphopeptides from β2
AR (a member of the “class A” receptors that bind β-arrestin much less tightly) (12
). β-Arrestins in these distinct conformations also interact differently with E3 ubiquitin ligases and deubiquitinases, and this may explain the differences in trafficking of class A receptors (rapidly recycle after dissociation from β-arrestins) versus class B receptors (recycle slowly and remain tightly bound to β-arrestins). Furthermore, the trafficking behavior of β2
AR could be converted from that of a class A receptor to that of a class B receptor by transfection of GRK5 or GRK6, but not by GRK2, which suggests that the sites phosphorylated by GRK6 promote a more stable interaction between β-arrestin and the β2
Biased ligands of GPCRs that activate β-arrestin signaling in the absence of G protein activation induce conformations of β-arrestin2 that are distinct from those induced by unbiased ligands, as assessed by an intramolecular β-arrestin2 BRET biosensor (15
). That the biased ligand carvedilol triggered a pattern of receptor site phosphorylation distinct from that obtained with the unbiased agonist isoproterenol is consistent with these findings. Carvedilol is a β blocker that is effective in the treatment of heart failure and selectively stimulates β-arrestin–mediated signaling (5
). This signaling may contribute to the unique clinical efficacy of carvedilol in the treatment of heart failure. Therefore, carvedilol may serve as a prototype for a new generation of therapeutic β2
AR ligands. One hypothesis to explain the selectivity of carvedilol for β-arrestin–mediated β2
AR signaling is that it induces a specific conformation of the receptor, leading to receptor phosphorylation by specific GRK subtypes. We determined that stimulation of the β2
AR with carvedilol induced phosphorylation only of Ser355
by GRK6, which contrasts with the change in phosphorylation at all 13 identified sites, including those for GRK2 and GRK6, in response to the full agonist isoproterenol. This result suggests that, whereas isoproterenol stimulation recruits both GRK2 and GRK6 to the receptor, carvedilol stimulation recruits only GRK6. This fits the notion that membrane association and activation of GRK2 occur through its interaction with Gβγ subunits (25
). Without activation of G proteins during carvedilol stimulation, it is likely that GRK2 is not targeted to the membrane. These data further suggest that biased ligands, by inducing distinct receptor conformations and G protein coupling, are able to recruit distinct GRKs.
We tested the hypothesis that distinct receptor phosphorylation patterns established by the different GRKs induce structurally and functionally distinct conformations of the bound β-arrestins. Using an intramolecular β-arrestin2 BRET biosensor, we found that GRK2 siRNA treatment (resulting in GRK-mediated phosphorylation by GRK6 on Ser355
, the phosphorylation pattern of carvedilol) produced the same directionally negative change in the BRET ratio as we previously demonstrated with several β-arrestin–biased ligands in multiple GPCR systems (15
). In addition, GRK6-siRNA treatment led to no change in the BRET signal despite robust recruitment of β-arrestin, which suggests yet a third distinct β-arrestin conformation. These data indicate that distinct phosphorylation patterns on a 7TMR result in unique β-arrestin conformations.
The barcode mechanism of specific signaling is not supported by all studies. Mutants of the AT1A
R in which all potential C-terminal phosphorylation sites are removed by truncation or substitution with alanine still recruit β-arrestins, albeit in a weaker class A pattern than its wild type counterpart that induces a strong class B pattern (27
). The mutant receptors activate ERK to the same extent as do wild-type AT1A
R. However, in the absence of receptor phosphorylation, β-arrestin–mediated desensitization and endocytosis of AT1A
Rs are largely abrogated. In contrast, when all of the phosphorylation sites on the β2
AR are mutated to alanine, the receptor neither binds β-arrestin nor stimulates ERK in a β-arrestin–dependent manner (27
). Thus, for the β2
AR, phosphorylation appears to be a prerequisite for β-arrestin recruitment and β-arrestin–mediated signaling, whereas this does not appear to be the case for the AT1A
A limited number of studies reporting the mapping of specific phosphorylation sites on 7TMRs are available. Trester-Zedlitz et al.
reported that a peptide (residues 339 to 369) of the human β2
AR purified and subsequently trypsin-digested from HEK293 cells contained multiple sites and showed that the net phosphorylation of this peptide increased with agonist stimulation (28
). However, they were unable to assign specific phosphorylation sites or to detect any of the distal C-terminal sites. Fredericks et al.
used purified, recombinant human β2
AR in conjunction with GRK2 or GRK5 to delineate overlapping patterns of phosphorylation sites with these two GRKs in vitro (29
). However, the physiological relevance of these studies is somewhat uncertain because of the high concentrations of receptors and GRKs used in these in vitro experiments. Mutation of all serine and threonine residues to alanines or glycines in the C terminus of the β2
AR prevents agonist-stimulated phosphorylation (30
), the interaction between the receptor and β-arrestin (31
), and β-arrestin–mediated processes of desensitization (32
), internalization (31
), and ERK activation (27
A number of studies targeting various combinations of four phosphorylation sites (Ser355
, and Ser364
) on the β2
AR have shown that this region is important for β-arrestin binding and that loss of these sites impairs, to varying extents, receptor desensitization and internalization (31
). Krasel et al.
showed that although phosphorylation of these four sites (two of which we assigned as GRK6 sites here) can promote β-arrestin2 interaction with the β2
AR, it is phosphorylation distal to residue 381 (assigned as GRK2 sites here) that is required for a high-affinity interaction between the receptor and β-arrestin2 (31
). However, at least for β2
AR trafficking, those four sites are not the only contributors. A Leu381
AR truncation mutant demonstrated strong interaction with β-arrestin2 but failed to internalize, suggesting that β-arrestin binding in and of itself is not sufficient for receptor internalization (31
). Deletion of only the last eight residues of the β2
AR C terminus (ΔASN405) also results in the failure of receptor internalization (31
). These data suggest that, whereas both the distal and the proximal phosphorylation residues of the β2
AR are important for β-arrestin binding, it is the distal residues (assigned as GRK2 sites here) that confer high-affinity binding and also coordinate protein-protein interactions that facilitate internalization. These data are also consistent with our finding that silencing of GRK2 leads to more marked impairment of β2
AR internalization than does silencing of GRK6.
In summary, we have quantitatively mapped sites on the β2AR phosphorylated in response to stimulation with an unbiased agonist, isoproterenol, and a β-arrestin–biased ligand, carvedilol. We demonstrate that of the 13 sites phosphorylated in response to isoproterenol, only 2 (S355 and S356) are phosphorylated in response to carvedilol. Moreover, these correspond to the only sites for which phosphorylation is mediated by GRK6. Phosphorylation of the different sets of sites by the two GRKs engenders the distinct functionality of β-arrestin by inducing different conformations of the receptor-bound β-arrestin. These findings are consistent with a model where the patterning of receptor phosphorylation sites by different GRKs establishes a barcode that determines the conformation of the bound β-arrestins and, subsequently, its functional capabilities. Understanding such barcodes for various receptors may be useful in screening for therapeutic agents.