The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS)

In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395nm) using appropriate dual channel luminometry instruments (e.g., Fusion Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein-Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of beta-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2:beta-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2:beta-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R:RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand-GPCR interactions which can be applied to ligand identification for orphan receptors.     

Effect of enhanced Renilla luciferase and fluorescent protein variants on the Förster distance of Bioluminescence resonance energy transfer (BRET)

Bioluminescence resonance energy transfer (BRET) is an important tool for monitoring macromolecular interactions and is useful as a transduction technique for biosensor development. Förster distance (R₀), the intermolecular separation characterized by 50% of the maximum possible energy transfer, is a critical BRET parameter. R₀ provides a means of linking measured changes in BRET ratio to a physical dimension scale and allows estimation of the range of distances that can be measured by any donor–acceptor pair. The sensitivity of BRET assays has recently been improved by introduction of new BRET components, RLuc2, RLuc8 and Venus with improved quantum yields, stability and brightness. We determined R₀ for BRET¹ systems incorporating novel RLuc variants RLuc2 or RLuc8, in combination with Venus, as 5.68 or 5.55 nm respectively. These values were approximately 25% higher than the R₀ of the original BRET¹ system. R₀ for BRET² systems combining green fluorescent proteins (GFP²) with RLuc2 or RLuc8 variants was 7.67 or 8.15 nm, i.e. only 2–9% greater than the original BRET² system despite being ～30-fold brighter.     

Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage

Bioluminescence resonance energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. Two common implementations of BRET are BRET¹ with Renilla luciferase (RLuc) and coelenterazine h (CLZ, λ_em ∼ 475 nm) and BRET² with the substrate coelenterazine 400a (CLZ400A substrate, λ_em = 395 nm) as the respective donors. For BRET¹ the acceptor is yellow fluorescent protein (YFP) (λ_em ∼ 535 nm), a mutant of green fluorescent protein (GFP), and for BRET² it is GFP² (λ_em ∼ 515 nm). It is not clear from previous studies which of these systems has superior signal-to-background characteristics. Here we directly compared BRET¹ and BRET² by placing two different protease-specific cleavage sequences between the donor and acceptor domains. The intact proteins simulate protein-protein association. Proteolytic cleavage of the peptide linker simulates protein dissociation and can be detected as a change in the BRET ratios. Complete cleavage of its target sequence by thrombin changed the BRET² ratio by a factor of 28.9 ± 0.2 (relative standard deviation [RSD], n = 3) and changed the BRET¹ ratio by a factor of 3.05 ± 0.07. Complete cleavage of a caspase-3 target sequence resulted in the BRET ratio changes by factors of 15.45 ± 0.08 for BRET² and 2.00 ± 0.04 for BRET¹. The BRET² assay for thrombin was 2.9 times more sensitive compared with the BRET¹ version. Calculated detection limits (blank signal + 3σ_b, where σ_b = standard deviation [SD] of blank signal) were 53 pM (0.002 U) thrombin with BRET¹ and 15 pM (0.0005 U) thrombin with BRET². The results presented here suggest that BRET² is a more suitable system than BRET¹ for studying protein-protein interactions and as a potential sensor for monitoring protease activity.     

In-frame fusion of SUMO1 enhances βarrestin2's association with activated GPCRs as well as with nuclear pore complexes

Small ubiquitin like modifier (SUMO) conjugation or SUMOylation of βarrestin2 promotes its association with the clathrin adaptor protein AP2 and facilitates rapid β₂ adrenergic receptor (β₂AR) internalization. However, disruption of the consensus SUMOylation site in βarrestin2, did not prevent βarrestin2's association with activated β₂ARs, dopamine D₂ receptors (D₂Rs), angiotensin type 1a receptors (AT_1aRs) and V₂ vasopressin receptors (V₂Rs). To address the role of SUMOylation in the trafficking of βarrestin and GPCR complexes, we generated and characterized a yellow fluorescent protein (YFP) tagged βarrestin2-SUMO1 chimeric protein, which is resistant to de-SUMOylation. In HEK-293 cells, YFP-SUMO1 predominantly localized in the nucleus, whereas YFP-βarrestin2 is cytoplasmic. YFP-βarrestin2-SUMO1 in addition to being cytoplasmic, is localized at the nuclear membrane. Nonetheless, βarrestin2-SUMO1 associated robustly with agonist-activated β₂ARs as evaluated by co-immunoprecipitation, confocal microscopy and bioluminescence resonance energy transfer (BRET). βarrestin2-SUMO1 associated strongly with the D₂R, which forms transient complexes with βarrestin2. But, βarrestin2-SUMO1 and βarrestin2 showed equivalent binding with the V₂R, which forms stable complexes with βarrestin2. βarrestin2 expression level directly correlated with the steady state levels of the unmodified form of RanGAP1, which upon SUMOylation associates with nuclear membrane. On the other hand, βarrestin2-SUMO1 not only localized at the nuclear membrane, but also formed a macromolecular complex with RanGAP1. Taken together, our data suggest that SUMOylation of βarrestin2 promotes its protein interactions at both cell and nuclear membranes. Furthermore, βarrestin2-SUMO1 presents as a useful tool to characterize βarrestin2 recruitment to GPCRs, which form transient and unstable complex with βarrestin2.     

β-Arrestin2 directly or through GRK2 inhibits PKCβII activation in a ubiquitination-dependent manner

The GRK/β-arrestin and PKC/PKA mediate the homologous and heterologous regulation of G protein-coupled receptors (GPCRs), respectively. Interaction between the two pathways is one of the most important issues in understanding the regulation of GPCRs. The present study investigated the regulatory effect of GRK2 and β-arrestins on PKC activation. The roles of GRK2 and β-arrestins in the functional regulation of PKC were assessed by determining their influence on PKC autophosphorylation and intracellular translocation. Radioligand binding assay was utilized to characterize intracellular trafficking of dopamine D₂R, D₃R, and β₂ adrenergic receptor (β₂AR). The subdomains involved in the mutual interactions among GRK2, β-arrestin2, and PKCβII were determined by in vitro binding assay. Various point mutants of key regulatory players were combined with knockdown cells of GRK2, β-arrestins, and Mdm2 to functionally correlate the biochemical changes with functional outcomes. GRK2 and β-arrestin2 mutually inhibited the PKCβII autophosphorylation, a hallmark of PKCβII activation. β-Arrestin2 ubiquitination was required for the inhibitory activities of GRK2 as well as β-arrestin2. Furthermore, GRK2 facilitated β-arrestin2 ubiquitination, thus to enhance the inhibitory actions of β-arrestin2 on PKCβII activity. Aforementioned processes were also involved in the GRK2/β-arrestin2-mediated inhibition of the D₂R, D₃R, and β₂AR endocytosis. The present study provides new insights into the intricate interactions between the homologous and heterologous GPCR regulation pathways. In addition, a novel regulatory role of GRK2 was proposed for the ubiquitination of β-arrestin in the context of the PKC-mediated heterologous regulation of GPCRs.     

On the Existence of a Possible A2A-D2-β-Arrestin2 Complex: A2A Agonist Modulation of D2 Agonist-Induced β-Arrestin2 Recruitment

Given that coactivation of adenosine A_2A (A_2AR) and dopamine D₂ (D₂R) receptors results in the coaggregation, cointernalization, and codesensitization of the A_2AR and D₂R and the role of scaffolding protein β-arrestin2 in the desensitization, internalization, and signaling of G-protein-coupled receptors, in this study we explored the ability of the A_2AR agonist CGS21680 in A_2AR-D₂R-coexpressing cells to modulate the D₂R agonist-induced recruitment of β-arrestin2 to the D₂R by means of proximity-based bioluminescence resonance energy transfer (BRET²) and co-trafficking analysis. We found evidence that CGS21680 can increase the maximal BRET² signal between β-arrestin2^RLuc and D_2LR^GFP2 upon D₂R activation, by increasing the potency of the D₂R agonist to exert this action. In addition, this change was associated with an increased formation of cytoplasmic clusters containing β-arrestin2^GFP2 and D_2LR^YFP as seen from the co-trafficking analysis. Furthermore, the A_2AR agonist advanced the time for the increase in Akt phosphorylation obtained with the D₂R agonist. Finally, using a novel bioinformatics approach to predict the protein-protein interface, we have also found that amino acid pro-triplets TNY, LLS, RAF, and VSR may be crucial for the -induced β-arrestin2 recruitment by A_2AR-D₂R heteromers. Taken together, the results indicate that the antagonistic A_2AR-D₂R allosteric receptor-receptor interaction in A_2AR-D₂R heteromers favors β-arrestin2 recruitment to the D_2LR protomer with subsequent cointernalization associated with a reduced time onset of Akt phosphorylation followed by a rapid dephosphorylation. Thus, β-arrestin2 action becomes more rapid and short-lasting and, in this way, mimics G-protein-mediated signaling.     

Complementary roles of the DRY motif and C-terminus tail of GPCRS for G protein coupling and beta-arrestin interaction

β-arrestin mediates the desensitization of GPCRs and acts as an adaptor molecule to recruit the receptor complex to clathrin-rich regions. Class-A GPCRs subsequently dissociate from β-arrestin but class-B GPCRs internalize with β-arrestin in the endocytic vesicles. Here the dopamine D₂ and D₃ receptors, which have similar structural features but different intracellular trafficking properties, were used in an attempt to better understand the structural requirements for the classification of GPCRs. The C-terminus tail of the vasopressin type-2 receptor was added to the ends of D₂R and D₃R to increase their affinity to β-arrestin. A point mutation was introduced into the DRY motif to change their basal activation levels. Among a battery of constructs in which the C-terminus tail and/or DRY motif was altered, class-B behavior was observed with the constructs whose affinities for β-arrestin were increased complementarily and their signaling was either maintained or regained. In conclusion, the DRY motif and C-terminal tail of the GPCRs determine complementarily their intracellular trafficking behavior by regulating the affinity to β-arrestin and G protein coupling.     

Monitoring G protein-coupled receptor activation using an adenovirus-based β-arrestin bimolecular fluorescence complementation assay

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and are involved in a variety of pathological conditions including cancer and cardiovascular, metabolic, neurological, and autoimmune diseases. GPCRs are being intensively investigated as targets for therapeutic intervention, and the β-arrestin recruitment assay has become a popular tool for analyzing GPCR activation. Here, we report a high-throughput method for cloning GPCR cDNAs into adenoviral bimolecular fluorescence complementation (BiFC) vectors and performing the β-arrestin BiFC assay in cells transduced with recombinant adenoviruses. An analysis of the activation of somatostatin receptor 2 (SSTR2) with the adenovirus-based β-arrestin BiFC assay showed that the assay is suitable for quantifying SSTR2 activation in response to specific agonists or antagonists. Furthermore, the adenovirus-based β-arrestin BiFC assay was able to detect the activation of a broad range of GPCRs. Collectively, our data indicate that the adenovirus-based β-arrestin BiFC assay can serve as a simple and universal platform for studying GPCR activation and thus will be useful for high-throughput screening of drugs that target GPCRs.     

Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET2

Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET² pair consisting of the fusion proteins CK2α-Rluc8 and CK2α-GFP². This BRET² sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET² sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (G_αs) and the polycationic compound polylysine. G_αs, but not the CK2 substrate β-arrestin2, decreased the BRET² signal by up to 50 %. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50 %. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET² sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors.     

A rapid fluorogenic GPCR–β‐arrestin interaction assay

Detection of protein–protein interactions involved in signal transduction in live cells and organisms has a variety of important applications. We report a fluorogenic assay for G protein‐coupled receptor (GPCR)–β‐arrestin interaction that is genetically encoded, generalizes to multiple GPCRs, and features high signal‐to‐noise because fluorescence is absent until its components interact upon GPCR activation. Fluorescence after protease‐activated receptor‐1 activation developed in minutes and required specific serine–threonine residues in the receptor carboxyl tail, consistent with a classical G protein‐coupled receptor kinase dependent β‐arrestin recruitment mechanism. This assay provides a useful complement to other in vivo assays of GPCR activation.     

Dynamic modulation of FGFR1–5-HT1A heteroreceptor complexes. Agonist treatment enhances participation of FGFR1 and 5-HT1A homodimers and recruitment of β-arrestin2

New findings show that neurotrophic and antidepressant effects of 5-HT in brain can, in part, be mediated by activation of the 5-HT1A receptor protomer in the hippocampal and raphe FGFR1–5-HT1A heteroreceptor complexes enhancing the FGFR1 signaling. The dynamic agonist modulation of the FGFR1–5-HT1A heteroreceptor complexes and their recruitment of β-arrestin is now determined in cellular models with focus on its impact on 5-HT1AR and FGFR1 homodimerization in the heteroreceptor complexes based on BRET² assays. The findings show that coagonist treatment with 8-OH-DPAT and FGF2 but not treatment with the 5-HT1A agonist alone markedly increases the BRETmax values and significantly reduces the BRET50 values of 5HT1A homodimerization. The effects of FGF2 or FGF20 with or without the 5-HT1A agonist were also studied on the FGFR1 homodimerization of the heteroreceptor complexes. FGF2 produced a marked and rapid increase in FGFR1 homodimerization which partially declined over a 10 min period. Cotreatment with FGF2 and 5-HT1A agonist blocked this decline in FGFR1 homodimerization. Furthermore, FGF2 alone produced a small increase in the BRET² signal from the 5-HT1A-β-arrestin2 receptor–protein complex which was additive to the marked effect of 8-OH-DPAT alone. Taken together, the participation of 5-HT1A and FGFR1 homodimers and recruitment of β-arrestin2 was demonstrated in the FGFR1–5-HT1A heteroreceptor complexes upon agonist treatments.     

Src homology 2 (SH2) domain containing protein tyrosine phosphatase-1 (SHP-1) dephosphorylates VEGF Receptor-2 and attenuates endothelial DNA synthesis, but not migration*

Background

Sustained agonist-promoted ubiquitination of β-arrestin has been correlated with increased stability of the GPCR – β-arrestin complex. Moreover, abrogation of β-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation.

Results

Herein we report that agonist activation of M₁ mAChRs produces a sustained β-arrestin ubiquitination but no stable co-localization with β-arrestin. In contrast, sustained ubiquitination of β-arrestin by activation of M₂ mAChRs does result in stable co-localization between the M₂ mAChR and β-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of β-arrestin following agonist treatment, we sought to determine the effects of β-arrestin ubiquitination on M₁ and M₂ mAChR down-regulation. A constitutively ubiquitinated β-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of β-arrestin 2 (YFP-β-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M₁ and M₂ mAChRs, with the effect substantially higher on the M₂ mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the β-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M₂ mAChR was not affected by expression of β-arrestin lysine mutants lacking putative ubiquitination sites, β-arrestin 2^{K18R, K107R, K108R, K207R, K296R}, while down-regulation and stable co-localiztion of the receptor with this β-arrestin lysine mutant were significantly reduced. Interestingly, expression of β-arrestin 2^{K18R, K107R, K108R, K207R, K296R} increased the agonist-promoted down-regulation of the M₁ mAChR but did not result in a stable co-localiztion of the receptor with this β-arrestin lysine mutant.

Conclusion

These findings indicate that ubiquitination of β-arrestin has a distinct role in the differential trafficking and degradation of M₁ and M₂ mAChRs.     

Rapid dephosphorylation of G protein-coupled receptors by protein phosphatase 1β is required for termination of β-arrestin-dependent signaling

Termination of signaling of activated G protein-coupled receptors (GPCRs) is essential for maintenance of cellular homeostasis. It is well established that β-arrestin redistributes to phosphorylated GPCRs and thereby facilitates desensitization of classical G protein-dependent signaling. β-Arrestin in turn serves as a scaffold to initiate a second wave of signaling. Here, we report a molecular mechanism that regulates the termination of unconventional β-arrestin-dependent GPCR signaling. We identify protein phosphatase 1β (PP1β) as a phosphatase for the cluster of phosphorylated threonines ((353)TTETQRT(359)) within the sst(2A) somatostatin receptor carboxyl terminus that mediates β-arrestin binding using siRNA knock-down screening. We show that PP1β-mediated sst(2A) dephosphorylation is initiated directly after receptor activation at or near the plasma membrane. As a functional consequence of diminished PP1β activity, we find that somatostatin- and substance P-induced but not epidermal growth factor-induced ERK activation was aberrantly enhanced and prolonged. Thus, we demonstrate a novel mechanism for fine tuning unconventional β-arrestin-dependent GPCR signaling in that recruitment of PP1β to activated GPCRs facilitates GPCR dephosphorylation and, hence, leads to disruption of the β-arrestin-GPCR complex.     

Opsin Oligomerization in a Heterologous Cell System

Using bioluminescence resonance energy transfer (BRET) we studied opsin oligomerization in heterologous expression systems and quantitatively assessed its oligomerization state. BRET² saturation and competition experiments were performed with live COS-7 cells expressing Rluc-and GFP²-tagged receptor constructs. BRET² saturation curves obtained were hyperbolic, and the calculated oligomerization state (N = 1 for dimers) suggested that opsin (N = 1.34 ± 0.25) forms higher oligomers. Very high BRET² values obtained for the opsin homo-dimer pair indicated a large energy transfer efficiency (E) and for cases where E ? 0.1 a modified saturation curve was proposed. The existence of homo-dimer complexes was additionally supported by competition assay results and was also observed in HEK-293 cells. Furthermore, evidence was provided for homo-and hetero-dimerization of family A (β₂-adrenergic) and B (gastric inhibitory polypeptide, GIP) receptors. In summary, these experiments demonstrate homo-and hetero-dimerization for opsin, β ₂-adrenergic, and GIP receptors.     

Elucidating structural and molecular mechanisms of β-arrestin-biased agonism at GPCRs via MS-based proteomics

The discovery of β-arrestin-dependent GPCR signaling has led to an exciting new field in GPCR pharmacology: to develop “biased agonists” that can selectively target a specific downstream signaling pathway that elicits beneficial therapeutic effects without activating other pathways that elicit negative side effects. This new trend in GPCR drug discovery requires us to understand the structural and molecular mechanisms of β-arrestin-biased agonism, which largely remain unclear. We have used cutting-edge mass spectrometry (MS)-based proteomics, combined with systems, chemical and structural biology to study protein function, macromolecular interaction, protein expression and posttranslational modifications in the β-arrestin-dependent GPCR signaling. These high-throughput proteomic studies have provided a systems view of β-arrestin-biased agonism from several perspectives: distinct receptor phosphorylation barcode, multiple receptor conformations, distinct β-arrestin conformations, and ligand-specific signaling. The information obtained from these studies offers new insights into the molecular basis of GPCR regulation by β-arrestin and provides a potential platform for developing novel therapeutic interventions through GPCRs.     

β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent 5‐HT4 receptor signalling

G protein‐coupled receptors (GPCRs) have been found to trigger G protein‐independent signalling. However, the regulation of G protein‐independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein‐independent 5‐HT₄ receptor (5‐HT₄R)‐operated Src/ERK (extracellular signal‐regulated kinase) pathway, but not the G_s pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor’ C‐terminus in both human embryonic kidney (HEK)‐293 cells and colliculi neurons. This inhibition required two sequences of events: the association of β–arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C‐t domain and the phosphorylation, by GRK5, of β–arrestin1 (at Ser⁴¹²) bound to the receptor. Phosphorylated β‐arrestin1 in turn prevented activation of Src constitutively bound to 5‐HT₄Rs, a necessary step in receptor‐stimulated ERK signalling. This is the first demonstration that β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent signalling.     

Differential regulation of β2-adrenoceptor and adenosine A2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle

Generation of cAMP through G_s-coupled G protein-coupled receptor (GPCR) [e.g. β₂-adrenoceptor (β₂AR), adenosine A_2B receptor (A_2BR)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. G_s-coupled GPCR signalling is regulated by G protein-coupled receptor kinases (GRK) and arrestin proteins, and dysregulation of Gs/GPCR signalling is thought play a role in the development of hypertension, which may be a consequence of enhanced GRK2 and/or arrestin expression. However, despite numerous studies indicating that β₂AR and A_2BR can be substrates for GRK/arrestin proteins, currently little is known regarding GRK/arrestin regulation of these endogenous receptors in arterial smooth muscle. Here, endogenous GRK isoenzymes and arrestin proteins were selectively depleted using RNA-interference in rat arterial smooth muscle cells (RASM) and the consequences of this for β₂AR- and A_2BR-mediated adenylyl cyclase (AC) signalling were determined by assessing cAMP accumulation. GRK2 or GRK5 depletion enhanced and prolonged β₂AR/AC signalling, while combined deletion of GRK2/5 has an additive effect. Conversely, activation of AC by A_2BR was regulated by GRK5, but not GRK2. β₂AR desensitization was attenuated following combined GRK2/GRK5 knockdown, but not by depletion of individual GRKs, arrestins, or by inhibiting PKA. Arrestin3 (but not arrestin2) depletion enhanced A_2BR-AC signalling and attenuated A_2BR desensitization, while β₂AR-AC signalling was regulated by both arrestin isoforms. This study provides a first demonstration of how different complements of GRK and arrestin proteins contribute to the regulation of signalling and desensitization of these important receptors mediating vasodilator responses in arterial smooth muscle.     

Heterotrimeric G proteins demonstrate differential sensitivity to β-arrestin dependent desensitization

G15 is a heterotrimeric G protein of the Gq/11 family. In this study, we describe its exceptional poor sensitivity to the general regulatory mechanism of G protein-coupled receptor (GPCR) desensitization.Enhancing β2 adrenergic receptor desensitization by arrestin overexpression, did not affect signalling to G15. Similarly, increased levels of arrestin did not affect G15 signalling triggered by the activation of V2 vasopressin and δ opioid receptors.Furthermore, co-immunoprecipitation experiments showed that G15 α subunit (as opposed to Gαq and Gαs) is recruited to a V2 vasopressin receptor mutant that is constitutively desensitized by β-arrestin. Interestingly, co-expression of Gα15 partially rescued cell surface localization and signalling capabilities of the same mutant receptor and reduced β2 adrenergic receptor internalization.Taken together, these findings provide evidence for a novel mechanism whereby GPCR desensitization can be bypassed and G15 can support sustained signalling in cells chronically exposed to hormones or neurotransmitters.