Functional consequences of spatial, temporal and ligand bias of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) regulate every aspect of kidney function by mediating the effects of various endogenous and exogenous substances. A key concept in GPCR function is biased signalling, whereby certain ligands may selectively activate specific pathways within the receptor's signalling repertoire. For example, different agonists may induce biased signalling by stabilizing distinct active receptor conformations - a concept that is supported by advances in structural biology. However, the processes underlying functional selectivity in receptor signalling are extremely complex, involving differences in subcellular compartmentalization and signalling dynamics. Importantly, the molecular mechanisms of spatiotemporal bias, particularly its connection to ligand binding kinetics, have been detailed for GPCRs critical to kidney function, such as the AT1 angiotensin receptor (AT1R), V2 vasopressin receptor (V2R) and the parathyroid hormone 1 receptor (PTH1R). This expanding insight into the multifaceted nature of biased signalling paves the way for innovative strategies for targeting GPCR functions; the development of novel biased agonists may represent advanced pharmacotherapeutic approaches to the treatment of kidney diseases and related systemic conditions, such as hypertension, diabetes and heart failure.

G protein-coupled receptor endocytosis generates spatiotemporal bias in ß-arrestin signaling.

The stabilization of different active conformations of G protein-coupled receptors is thought to underlie the varying efficacies of biased and balanced agonists. Here, profiling the activation of signal transducers by angiotensin II type 1 receptor (AT1R) agonists revealed that the extent and kinetics of ß-arrestin binding exhibited substantial ligand-dependent differences, which were lost when receptor internalization was inhibited. When AT1R endocytosis was prevented, even weak partial agonists of the ß-arrestin pathway acted as full or near-full agonists, suggesting that receptor conformation did not exclusively determine ß-arrestin recruitment. The ligand-dependent variance in ß-arrestin translocation was much larger at endosomes than at the plasma membrane, showing that ligand efficacy in the ß-arrestin pathway was spatiotemporally determined. Experimental investigations and mathematical modeling demonstrated how multiple factors concurrently shaped the effects of agonists on endosomal receptor-ß-arrestin binding and thus determined the extent of functional selectivity. Ligand dissociation rate and G protein activity had particularly strong, internalization-dependent effects on the receptor-ß-arrestin interaction. We also showed that endocytosis regulated the agonist efficacies of two other receptors with sustained ß-arrestin binding: the V2 vasopressin receptor and a mutant ß2-adrenergic receptor. In the absence of endocytosis, the agonist-dependent variance in ß-arrestin2 binding was markedly diminished. Our results suggest that endocytosis determines the spatiotemporal bias in GPCR signaling and can aid in the development of more efficacious, functionally selective compounds.

Inflammation leads through PGE/EP3 signaling to HDAC5/MEF2-dependent transcription in cardiac myocytes.

The myocyte enhancer factor 2 (MEF2) regulates transcription in cardiac myocytes and adverse remodeling of adult hearts. Activators of G protein-coupled receptors (GPCRs) have been reported to activate MEF2, but a comprehensive analysis of GPCR activators that regulate MEF2 has to our knowledge not been performed. Here, we tested several GPCR agonists regarding their ability to activate a MEF2 reporter in neonatal rat ventricular myocytes. The inflammatory mediator prostaglandin E2 (PGE2) strongly activated MEF2. Using pharmacological and protein-based inhibitors, we demonstrated that PGE2 regulates MEF2 via the EP3 receptor, the ßγ subunit of Gi/o protein and two concomitantly activated downstream pathways. The first consists of Tiam1, Rac1, and its effector p21-activated kinase 2, the second of protein kinase D. Both pathways converge on and inactivate histone deacetylase 5 (HDAC5) and thereby de-repress MEF2. In vivo, endotoxemia in MEF2-reporter mice induced upregulation of PGE2 and MEF2 activation. Our findings provide an unexpected new link between inflammation and cardiac remodeling by de-repression of MEF2 through HDAC5 inactivation, which has potential implications for new strategies to treat inflammatory cardiomyopathies.

Novel mechanisms of G-protein-coupled receptors functions: AT1 angiotensin receptor acts as a signaling hub and focal point of receptor cross-talk.

AT1 angiotensin receptor (AT1R), a prototypical G protein-coupled receptor (GPCR), is the main receptor, which mediates the effects of the renin-angiotensin system (RAS). AT1R plays a crucial role in the regulation of blood pressure and salt-water homeostasis, and in the development of pathological conditions, such as hypertension, heart failure, cardiovascular remodeling, renal fibrosis, inflammation, and metabolic disorders. Stimulation of AT1R leads to pleiotropic signal transduction pathways generating arrays of complex cellular responses. Growing amount of evidence shows that AT1R is a versatile GPCR, which has multiple unique faces with distinct conformations and signaling properties providing new opportunities for functionally selective pharmacological targeting of the receptor. Biased ligands of AT1R have been developed to selectively activate the ß-arrestin pathway, which may have therapeutic benefits compared to the conventional angiotensin converting enzyme inhibitors and angiotensin receptor blockers. In this review, we provide a summary about the most recent findings and novel aspects of the AT1R function, signaling, regulation, dimerization or oligomerization and its cross-talk with other receptors, including epidermal growth factor (EGF) receptor, adrenergic receptors and CB1 cannabinoid receptor. Better understanding of the mechanisms and structural aspects of AT1R activation and cross-talk can lead to the development of novel type of drugs for the treatment of cardiovascular and other diseases.

Heterologous phosphorylation-induced formation of a stability lock permits regulation of inactive receptors by ß-arrestins.

ß-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and ß-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether ß-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of ß-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, Gq/11-coupled GPCR, or epidermal growth factor receptor stimulation promotes ß-arrestin2 recruitment to unliganded AT1 angiotensin receptor (AT1R). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and ß-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the ß-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters ß-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-ß-arrestin interaction, but also governs the structural rearrangements within ß-arrestins. Furthermore, we found that ß-arrestin2 binds to PKC-phosphorylated AT1R in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of ß-arrestins and reveal their novel role in receptor cross-talk.

Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors.

Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M2 muscarinic receptor, so that agonist activation of the M2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M2, whereas its interactions with other receptors, including the ß2-adrenergic receptor and the D1 and D2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the ß2-adrenergic and D2 dopamine receptors, while reducing its interaction with the D1 dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes.

Angiotensin type 1A receptor regulates ß-arrestin binding of the ß2-adrenergic receptor via heterodimerization.

Heterodimerization between angiotensin type 1A receptor (AT1R) and ß2-adrenergic receptor (ß2AR) has been shown to modulate G protein-mediated effects of these receptors. Activation of G protein-coupled receptors (GPCRs) leads to ß-arrestin binding, desensitization, internalization and G protein-independent signaling of GPCRs. Our aim was to study the effect of heterodimerization on ß-arrestin coupling. We found that ß-arrestin binding of ß2AR is affected by activation of AT1Rs. Costimulation with angiotensin II and isoproterenol markedly enhanced the interaction between ß2AR and ß-arrestins, by prolonging the lifespan of ß2AR-induced ß-arrestin2 clusters at the plasma membrane. While candesartan, a conventional AT1R antagonist, had no effect on the ß-arrestin2 binding to ß2AR, TRV120023, a ß-arrestin biased agonist, enhanced the interaction. These findings reveal a new crosstalk mechanism between AT1R and ß2AR, and suggest that enhanced ß-arrestin2 binding to ß2AR can contribute to the pharmacological effects of biased AT1R agonists.

Investigation of the fate of type I angiotensin receptor after biased activation.

Biased agonism on the type I angiotensin receptor (AT1-R) can achieve different outcomes via activation of G protein-dependent and -independent cellular responses. In this study, we investigated whether the biased activation of AT1-R can lead to different regulation and intracellular processing of the receptor. We analyzed ß-arrestin binding, endocytosis, and subsequent trafficking steps, such as early and late phases of recycling of AT1-R in human embryonic kidney 293 cells expressing wild-type or biased mutant receptors in response to different ligands. We used Renilla luciferase-tagged receptors and yellow fluorescent protein-tagged ß-arrestin2, Rab5, Rab7, and Rab11 proteins in bioluminescence resonance energy transfer measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. ß-arrestin binding and the internalization kinetics of the angiotensin II-stimulated AT1-R differed from those stimulated by the biased agonists. Similarly, angiotensin II-stimulated wild-type AT1-R showed differences compared with a biased mutant AT1-R (DRY/AAY AT1-R) with regards to ß-arrestin binding and endocytosis. We found that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane phosphatidylinositol 4,5-bisphosphate depletion. Moreover, the stability of the ß-arrestin binding is a major determinant of the later fate of the internalized AT1-R receptor.

Mutations in the 'DRY' motif of the CB1 cannabinoid receptor result in biased receptor variants.

The role of the highly conserved 'DRY' motif in the signaling of the CB1 cannabinoid receptor (CB1R) was investigated by inducing single-, double-, and triple-alanine mutations into this site of the receptor. We found that the CB1R-R3.50A mutant displays a partial decrease in its ability to activate heterotrimeric Go proteins (∼80% of WT CB1R (CB1R-WT)). Moreover, this mutant showed an enhanced basal ß-arrestin2 (ß-arr2) recruitment. More strikingly, the double-mutant CB1R-D3.49A/R3.50A was biased toward ß-arrs, as it gained a robustly increased ß-arr1 and ß-arr2 recruitment ability compared with the WT receptor, while its G-protein activation was decreased. In contrast, the double-mutant CB1R-R3.50A/Y3.51A proved to be G-protein-biased, as it was practically unable to recruit ß-arrs in response to agonist stimulus, while still activating G-proteins, although at a reduced level (∼70% of CB1R-WT). Agonist-induced ERK1/2 activation of the CB1R mutants showed a good correlation with their ß-arr recruitment ability but not with their G-protein activation or inhibition of cAMP accumulation. Our results suggest that G-protein activation and ß-arr binding of the CB1R are mediated by distinct receptor conformations, and the conserved 'DRY' motif plays different roles in the stabilization of these conformations, thus mediating both G-protein- and ß-arr-mediated functions of CB1R.