Protein-independent Signaling Research Articles

Effects of carvedilol on human prostate tissue contractility and stromal cell growth pointing to potential clinical implications

Abstract Background Apart from antagonizing ß-adrenoceptors, carvedilol antagonizes vascular α1-adrenoceptors and activates G protein-independent signaling. Even though it is a commonly used antihypertensive and α1-adrenoceptors are essential for the treatment of voiding symptoms in benign prostatic hyperplasia, its actions in the human prostate are still unknown. Here, we examined carvedilol effects on contractions of human prostate tissues, and on stromal cell growth. Methods Contractions of prostate tissues from radical prostatectomy were induced by electric field stimulation (EFS) or α1-agonists. Growth-related functions were examined in cultured stromal cells. Results Concentration-response curves for phenylephrine, methoxamine and noradrenaline were right shifted by carvedilol (0.1–10 µM), around half a magnitude with 100 nM, half to one magnitude with 1 µM, and two magnitudes with 10 µM. Right shifts were reflected by increased EC50 values for agonists, with unchanged Emax values. EFS-induced contractions were reduced by 21–54% with 0.01–1 µM carvedilol, and by 94% by 10 µM. Colony numbers of stromal cells were increased by 500 nM, but reduced by 1–10 µM carvedilol, while all concentrations reduced colony size. Decreases in viability were time-dependent with 0.1–0.3 µM, but complete with 10 µM. Proliferation was slightly increased by 0.1–0.5 µM, but reduced with 1–10 µM. Conclusions Carvedilol antagonizes α1-adrenoceptors in the human prostate, starting with concentrations in ranges of known plasma levels. In vitro, effect sizes resemble those of α1-blockers used for the treatment of voiding symptoms, which requires concentrations beyond plasma levels. Bidirectional and dynamic effects on the growth of stromal cells may be attributed to "biased agonism".

Significant Functional Differences Between Dopamine D4 Receptor Polymorphic Variants Upon Heteromerization with α1A Adrenoreceptors

The functional role of the dopamine D4 receptor (D4R) and its main polymorphic variants has become more evident with the demonstration of heteromers of D4R that control the function of frontal cortico-striatal neurons. Those include heteromers with the α2A adrenoceptor (α2AR) and with the D2R, localized in their cortical somato-dendritic region and striatal nerve terminals, respectively. By using biophysical and cell-signaling methods and heteromer-disrupting peptides in mammalian transfected cells and rat brain slice preparations, here we provide evidence for a new functionally relevant D4R heteromer, the α1AR-D4R heteromer, which is also preferentially localized in cortico-striatal glutamatergic terminals. Significant differences in allosteric modulations between heteromers of α1AR with the D4.4R and D4.7R polymorphic variants could be evidenced with the analysis of G protein-dependent and independent signaling. Similar negative allosteric modulations between α1AR and D4R ligands could be demonstrated for both α1AR-D4.4R and α1AR-D4.7R heteromers on G protein-independent signaling, but only for α1AR-D4.4R on G protein-dependent signaling. From these functional differences, it is proposed that the D4.4R variant provides a gain of function of the α1AR-mediated noradrenergic stimulatory control of cortico-striatal glutamatergic neurotransmission, which could result in a decrease in the vulnerability for impulse control-related neuropsychiatric disorders and increase in the vulnerability for posttraumatic stress disorder.

Identification of potential antagonists of CRF1R for possible treatment of stress and anxiety neuro-disorders using structure-based virtual screening and molecular dynamics simulation

G protein-coupled-receptors (GPCRs) are the largest family of cell surface receptors with tremendous therapeutic potential. They mediate signal transduction activities via G protein-dependent signaling pathways, G protein-independent signaling pathways, and other complicated regulatory processes. The corticotropin-releasing factor receptor type 1 (CRF1R) is a member of class B GPCRs that is predominantly found in the central nervous system, where it plays a key role in stress-related neuro-disorders. To date, no drug targeting this receptor has been approved, partly due to inadequate understanding of the activation mechanism of class B GPCRs. Previously, using MD simulation, we demonstrated that the CRF1R complexed with a small-molecule antagonist CP-376395 maintains a conformation of its transmembrane domain (TMD). Here, using the most abundant structures derived from those simulations, we carried out a structure-based virtual screening of ZINC15 “Druglike” library containing approximately 17 million compounds. The docking complexes of the CRF1R with the top 30 hits were submitted to MD simulation to examine the stability of ligand binding mode. Furthermore, MM-GBSA binding energy calculations were performed on all the complexes to rank them with improving accuracy. Hit 1 (ZINC000046079839) and hit 20 (ZINC000032907937) span the allosteric site of the CRF1R, persistently forming interactions with transmembrane helices 3 and 6. These interactions are likely to keep the receptor in an inactive state since both transmembrane helices play a critical role in the activation of the receptor.

Beta‐agonist Profiling Reveals Novel Biased Signaling Phenotypes for the Beta 2‐adrenergic Receptor with Implications in the Treatment of Asthma

Asthma is characterized by chronic airway inflammation and airway smooth muscle (ASM) cell contraction. Current treatments for asthma include beta‐agonists which activate the beta 2‐adrenergic receptor, a G protein‐coupled receptor (GPCR) that promotes Gs‐dependent production of cAMP leading to ASM relaxation. Unfortunately, beta‐agonists can also promote severe side effects including an increased risk of having a severe asthmatic attack that can result in death. Such side effects have been correlated with the action of beta‐arrestins, scaffolding proteins that mediate GPCR desensitization and internalization as well as G protein independent signaling. In this scenario, biased ligands that promote selective receptor interaction with Gs should be beneficial in promoting airway relaxation while attenuating beta‐arrestin‐induced side effects. To test this, we have used high‐throughput screening to identify Gs‐biased beta‐agonists. The initial lead candidates were further analyzed for their ability to modulate receptor induced Gs and Gi interaction, cAMP production, and beta‐arrestin interaction using bioluminescence resonance energy transfer (BRET) analysis. We identified three compounds with Gs‐biased properties which showed minimal beta‐arrestin recruitment. These compounds were also able to decrease beta‐arrestin mediated outputs including receptor internalization and ERK activation. These compounds also showed reduced GRK‐mediated phosphorylation of the receptor as well as decreased agonist‐promoted desensitization in a physiological model of asthma (human ASM cells). In conclusion, these Gs‐biased beta‐agonists may pave the way for the development of more effective drugs for the treatment of asthma and may help to clarify the structural determinants of receptor mediated biased signaling.

Multiple roles of β‐Arrestin2 in initiating and sustaining D3R‐mediated ERK1/2 phosphorylation by G protein‐biased and unbiased D3R agonists

β‐arrestins play key roles in both terminating G protein signaling and activating G protein‐independent signaling across many G protein coupled receptors (GPCRs); however, the roles of β‐arrestins in dopamine D3 receptor (D3R)‐mediated ERK phosphorylation/signaling are poorly characterized. Our previous studies showed that novel compounds SK609 and its analog SK608 are selective D3R agonists and display Gi/o biased signaling without significant recruitment of β‐arrestin2 (βarr2) compared with dopamine which can potently activate G proteins and recruit β‐arrestins. We hypothesize that βarr2 plays multiple roles in initiating and sustaining D3R‐mediated ERK1/2 phosphorylation by G protein‐biased and unbiased D3R agonists. We tested this hypothesis using neuron‐like SH‐SY5Y cells stably expressing D3R (SH‐SY5Y‐D3R cells) with βarr2 knockdown (βarr2KD) in vitro. Our results show: SK609 and SK608 or pramipexole (PRX) time‐dependently elicited both the early and late phases of mono phosphorylation of ERK1/2; SK608 or SK609 only elicited the early phase of dual phosphorylation, whereas PRX induced both the early and late phases and sustained for 1‐4 h; βarr2KD partially reduced mono ERK1/2 phosphorylation by PRX but not SK608 or SK609; βarr2KD significantly enhanced both the early and late phases of dual ERK1/2 phosphorylation by SK608 or SK609 whereas it partially reduced the early phase by PRX and completely abolished the late phase; Gi/o inhibitor PTX abolished PRX‐ or SK608 and SK609‐induced mono ERK1/2 phosphorylation and SK608‐ and SK609‐induced the early phase of dual ERK1/2 phosphorylation, whereas the early phase of dual phosphorylation by PRX was abolished but the late phase was not affected; Gbg inhibitor Galllein had no effect on PRX‐ induced dual phosphorylation of ERK1/2 but inhibited SK609‐ or SK608‐indued dual phosphorylation; PKC inhibitor Go6983 greatly reduced or completely abolished PRX‐ or SK608‐ and SK609‐induced the mono phosphorylation, and blocked SK608‐ or SK609‐induced the dual phosphorylation in SH‐SY5Y‐D3R cells. In contrast, Go6983 abolished PRX‐induced late phase of dual phosphorylation, it only partially reduced PRX‐induced early phase; PTX plus Go6983 completely abolished PRX‐ or SK608‐ and SK609‐induced mono or dual ERK1/2 phosphorylation in SH‐SY5Y‐D3R/βarr2KD cells. In summary, βarr2 not only participates in inhibiting G protein‐biased D3R agonist‐induced early and late phases of dual ERK1/2 phosphorylation and activating unbiased D3R agonists‐induced the early and late phases of the dual phosphorylation, but also is required for Gi/o‐ or PKCs‐dependent the early phase of the mono and dual ERK1/2 phosphorylation. These findings support multiple roles in which βarr2 acts as both signal terminator for G protein biased agonist‐induced ERK1/2 signaling and signal transducer for unbiased D3R agonist‐induced signaling interactions among βarr2 and Gi/o and PKCs which collaboratively regulate ERK1/2 signaling.

Systems approach reveals distinct and shared signaling networks of the four PGE2 receptors in T cells.

Prostaglandin E2 (PGE2) promotes an immunosuppressive microenvironment in cancer, partly by signaling through four receptors (EP1, EP2, EP3, and EP4) on T cells. Here, we comprehensively characterized PGE2 signaling networks in helper, cytotoxic, and regulatory T cells using a phosphoproteomics and phosphoflow cytometry approach. We identified ~1500 PGE2-regulated phosphosites and several important EP1–4 signaling nodes, including PKC, CK2, PKA, PI3K, and Src. T cell subtypes exhibited distinct signaling pathways, with the strongest signaling in EP2-stimulated CD8+ cells. EP2 and EP4, both of which signal through Gαs, induced similar signaling outputs, but with distinct kinetics and intensity. Functional predictions from the observed phosphosite changes revealed PGE2 regulation of key cellular and immunological processes. Last, network modeling suggested signal integration between the receptors and a substantial contribution from G protein–independent signaling. This study offers a comprehensive view of the different PGE2-regulated phosphoproteomes in T cell subsets, providing a valuable resource for further research on this physiologically and pathophysiologically important signaling system.

Metabolic Functions of G Protein-Coupled Receptors and β-Arrestin-Mediated Signaling Pathways in the Pathophysiology of Type 2 Diabetes and Obesity.

Seven transmembrane receptors (7TMRs), often termed G protein-coupled receptors (GPCRs), are the most common target of therapeutic drugs used today. Many studies suggest that distinct members of the GPCR superfamily represent potential targets for the treatment of various metabolic disorders including obesity and type 2 diabetes (T2D). GPCRs typically activate different classes of heterotrimeric G proteins, which can be subgrouped into four major functional types: Gαs, Gαi, Gαq/11, and G12/13, in response to agonist binding. Accumulating evidence suggests that GPCRs can also initiate β-arrestin-dependent, G protein-independent signaling. Thus, the physiological outcome of activating a certain GPCR in a particular tissue may also be modulated by β-arrestin-dependent, but G protein-independent signaling pathways. In this review, we will focus on the role of G protein- and β-arrestin-dependent signaling pathways in the development of obesity and T2D-related metabolic disorders.

A new synthetic dual agonist of GPR120/GPR40 induces GLP-1 secretion and improves glucose homeostasis in mice

G-protein coupled receptors 40 and 120 (GPR40 and GPR120) are increasingly emerging as potential therapeutic targets for the treatment of altered glucose homeostasis, and their agonists are under evaluation for their glucagon-like peptide-1 (GLP-1)-mediated therapeutic effects on insulin production and sensitivity. Here, we characterized a new dual GPR40 and GPR120 agonist (DFL23916) and demonstrated that it can induce GLP-1 secretion and improve glucose homeostasis.Resulting from a rational drug design approach aimed at identifying new dual GPR120/40 agonists able to delay receptor internalization, DFL23916 had a good activity and a very high selectivity towards human GPR120 (long and short isoforms) and GPR40, as well as towards their mouse orthologous, by which it induced both Gαq/11-initiated signal transduction pathways with subsequent Ca2+ intracellular spikes and G protein-independent signaling via β-arrestin with the same activity. Compared to the endogenous ligand alpha-linolenic acid (ALA), a selective GPR120 agonist (TUG-891) and a well-known dual GPR40 and GPR120 agonist (GW9508), DFL23916 was the most effective in inducing GLP-1 secretion in human and murine enteroendocrine cells, and this could be due to the delayed internalization of the receptor (up to 3 h) that we observed after treatment with DFL23916. With a good pharmacokinetic/ADME profile, DFL23916 significantly increased GLP-1 portal vein levels in healthy mice, demonstrating that it can efficiently induce GLP-1 secretion in vivo. Contrary to the selective GPR120 agonist (TUG-891), DFL23916 significantly improved also glucose homeostasis in mice undergoing an oral glucose tolerance test (OGTT).

NanoLuc-Based Methods to Measure β-Arrestin2 Recruitment to G Protein-Coupled Receptors.

Cytosolic β-arrestins are key regulators of G protein-coupled receptors (GPCRs) by sterically uncoupling G protein activation, facilitating receptor internalization, and/or acting as G protein-independent signaling scaffolds. The current awareness that GPCR ligands may display bias toward G protein signaling or β-arrestin recruitment makes β-arrestin recruitment assays important additions to the drug discovery toolbox. This chapter describes two NanoLuc-based methods to monitor β-arrestin2 recruitment to the human histamine H1 receptor by measuring bioluminescence resonance energy transfer and enzyme-fragment complementation in real-time on living cells with reasonable high throughput. In addition to the detection of agonism, both assay formats can be used to qualitatively evaluate the binding kinetics of antihistamines on the human histamine H1 receptor.

OR24-03 GRK2 Mediates Beta-Adrenergic Receptor Crosstalk to Enhanced Adrenocortical AngII-Dependent Aldosterone Production

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to hyperkalemia or angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone synthesis and secretion via both G proteins and the GPCR adapter proteins βarrestins. AZG cells express all three subtypes of β-adrenergic receptor (AR) and respond to catecholamines by producing aldosterone. Being GPCRs, both activated βARs and AT1Rs are phosphorylated by GPCR-kinases (GRKs), followed by βarrestin binding to initiate G protein-independent signaling. Herein, we investigated whether the major adrenal GRKs, GRK2 and GRK5, are involved in catecholaminergic regulation of AngII-dependent aldosterone production. We used the human AZG cell line H295R, in which we measured aldosterone secretion via ELISA and synthesis via real-time PCR for steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA levels. Isoproterenol (Iso, a βAR full agonist) treatment significantly augmented AngII-dependent aldosterone synthesis (2.2+0.8-fold CYP11B2 & 1.6+0.5-fold StAR mRNA inductions over AngII alone; p<0.05, n=4), as well as secretion (2.3+0.8-fold of vehicle with Iso; 3.2+1.1-fold of vehicle with AngII; 7.4+1.1-fold of vehicle with Iso+AngII, p<0.05 vs. either agent alone; n=5) in H295R cells. Importantly, GRK2, but not the other major GRK isoform expressed in human adrenals GRK5, was indispensable for the catecholamine-mediated enhancement of aldosterone production in response to AngII in H295R cells. Specifically, GRK2 inhibition with the small molecule Cmpd101 abolished Iso effects on AngII-induced aldosterone synthesis and secretion (Iso+AngII-induced aldosterone secretion: 8.1+2.3-fold of vehicle without Cmpd101; 2.8+0.8-fold of vehicle with Cmpd101; p<0.05, n=5). In contrast, GRK5 knockout via CRISPR/Cas9 did not affect the synergism between isoproterenol and AngII in stimulating aldosterone production. Mechanistically, βAR-activated GRK2, but not GRK5, phosphorylated and activated the Ca2+-activated chloride channel anoctamine-1 (ANO1), also known as transmembrane member (TMEM)16A, ultimately increasing aldosterone production in H295R cells (Iso+10–6 M [Ca2+]-induced ANO1 activity of Cmpd101-pretreated cells: 55+15 % of non-Cmpd101-pretreated cells; p<0.05, n=5). AngII alone failed to stimulate GRK2 in H295R cells. In conclusion, GRK2 mediates a βAR-AT1R signaling crosstalk at the level of ANO1 activation, which results in enhanced aldosterone production in H295R cells. This finding suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems in the adrenal cortex and that GRK2 inhibition might be therapeutically advantageous for aldosterone suppression.

Not So Dry After All: DRY Mutants of the AT1A Receptor and H1 Receptor Can Induce G-Protein-Dependent Signaling.

G-protein-coupled receptors (GPCRs) are seven transmembrane spanning receptors that regulate a wide array of intracellular signaling cascades in response to various stimuli. To do so, they couple to different heterotrimeric G proteins and adaptor proteins, including arrestins. Importantly, arrestins were shown to regulate GPCR signaling through G proteins, as well as promote G protein-independent signaling events. Several research groups have reported successful isolation of exclusively G protein-dependent and arrestin-dependent signaling downstream of GPCR activation using biased agonists or receptor mutants incapable of coupling to either arrestins or G proteins. In the latter category, the DRY mutant of the angiotensin II type 1 receptor was extensively used to characterize the functional selectivity downstream of AT1AR. In an attempt to understand histamine 1 receptor signaling, we characterized the signaling capacity of the H1R DRY mutant in a panel of dynamic, live cell biosensor assays, including arrestin recruitment, heterotrimeric G protein activation, Ca2+ signaling, protein kinase C activity, GTP binding of RhoA, and activation of ERK1/2. Here, we show that both H1R DRY mutant and the AT1AR DRY mutant are capable of efficient activation of G protein-mediated signaling. Therefore, contrary to the common belief, they do not constitute suitable tools for the dissection of the arrestin-mediated, G protein-independent signaling downstream of these receptors.

GRK2-Mediated Crosstalk Between β-Adrenergic and Angiotensin II Receptors Enhances Adrenocortical Aldosterone Production In Vitro and In Vivo.

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone via both G proteins and the adapter protein βarrestin1, which binds the receptor following its phosphorylation by GPCR-kinases (GRKs) to initiate G protein-independent signaling. β-adrenergic receptors (ARs) also induce aldosterone production in AZG cells. Herein, we investigated whether GRK2 or GRK5, the two major adrenal GRKs, is involved in the catecholaminergic regulation of AngII-dependent aldosterone production. In human AZG (H295R) cells in vitro, the βAR agonist isoproterenol significantly augmented both AngII-dependent aldosterone secretion and synthesis, as measured by the steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA inductions. Importantly, GRK2, but not GRK5, was indispensable for the βAR-mediated enhancement of aldosterone in response to AngII. Specifically, GRK2 inhibition with Cmpd101 abolished isoproterenol’s effects on AngII-induced aldosterone synthesis/secretion, whereas the GRK5 knockout via CRISPR/Cas9 had no effect. It is worth noting that these findings were confirmed in vivo, since rats overexpressing GRK2, but not GRK5, in their adrenals had elevated circulating aldosterone levels compared to the control animals. However, treatment with the β-blocker propranolol prevented hyperaldosteronism in the adrenal GRK2-overexpressing rats. In conclusion, GRK2 mediates a βAR-AT1R signaling crosstalk in the adrenal cortex leading to elevated aldosterone production. This suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems at the level of the adrenal cortex and that its inhibition might be therapeutically advantageous in hyperaldosteronism-related conditions.

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Loss of β-arrestin2 in D2 cells alters neuronal excitability in the nucleus accumbens and behavioral responses to psychostimulants and opioids.

Psychostimulants and opioids increase dopamine (DA) neurotransmission, activating D1 and D2 G protein-coupled receptors. β-arrestin2 (βarr2) desensitizes and internalizes these receptors and initiates G protein-independent signaling. Previous work revealed that mice with a global or cell-specific knockout of βarr2 have altered responses to certain drugs; however, the effects of βarr2 on the excitability of medium spiny neurons (MSNs), and its role in mediating the rewarding effects of drugs of abuse are unknown. D1-Cre and D2-Cre transgenic mice were crossed with floxed βarr2 mice to eliminate βarr2 specifically in cells containing either D1 (D1βarr2-KO ) or D2 (D2βarr2-KO ) receptors. We used slice electrophysiology to characterize the role of βarr2 in modulating D1 and D2 nucleus accumbens MSN intrinsic excitability in response to DA and tested the locomotor-activating and rewarding effects of cocaine and morphine in these mice. Eliminating βarr2 attenuated the ability of DA to inhibit D2-MSNs and altered the DA-induced maximum firing rate in D1-MSNs. While D1βarr2-KO mice had mostly normal drug responses, D2βarr2-KO mice showed dose-dependent reductions in acute locomotor responses to cocaine and morphine, attenuated locomotor sensitization to cocaine, and blunted cocaine reward measured with conditioned place preference. Both D2βarr2-KO and D1βarr2-KO mice displayed an enhanced conditioned place preference for the highest dose of morphine. These results indicate that D1- and D2-derived βarr2 functionally contribute to DA-induced changes in MSN intrinsic excitability and behavioral responses to psychostimulants and opioids dose-dependently.

Calcium-Sensing Receptor Internalization Is β-Arrestin-Dependent and Modulated by Allosteric Ligands.

G protein-coupled receptor (GPCR) internalization is crucial for the termination of GPCR activity, and in some cases is associated with G protein-independent signaling and endosomal receptor signaling. To date, internalization has been studied in great detail for class A GPCRs; whereas it is not well established to what extent the observations can be generalized to class C GPCRs, including the extracellular calcium-sensing receptor (CaSR). The CaSR is a prototypical class C GPCR that maintains stable blood calcium (Ca2+) levels by sensing minute changes in extracellular free Ca2+ It is thus necessary that the activity of the CaSR is tightly regulated, even while continuously being exposed to its endogenous agonist. Previous studies have used overexpression of intracellular proteins involved in GPCR trafficking, pathway inhibitors, and cell-surface expression or functional desensitization as indirect measures to investigate CaSR internalization. However, there is no general consensus on the processes involved, and the mechanism of CaSR internalization remains poorly understood. The current study provides new insights into the internalization mechanism of the CaSR. We have used a state-of-the-art time-resolved fluorescence resonance energy transfer-based internalization assay to directly measure CaSR internalization in real-time. We demonstrate that the CaSR displays both constitutive and concentration-dependent Ca2+-mediated internalization. For the first time, we conclusively show that CaSR internalization is sensitive to immediate positive and negative modulation by the CaSR-specific allosteric modulators N-(3-[2-chlorophenyl]propyl)-(R)-α-methyl-3-methoxybenzylamine (NPS R-568) and 2-chloro-6-[(2R)-2-hydroxy-3-[(2-methyl-1-naphthalen-2-ylpropan-2-yl)amino]propoxy]benzonitrile (NPS 2143), respectively. In addition, we provide compelling evidence that CaSR internalization is β-arrestin-dependent while interestingly being largely independent of Gq/11 and Gi/o protein signaling. SIGNIFICANCE STATEMENT: A novel highly efficient cell-based real-time internalization assay to show that calcium-sensing receptor (CaSR) internalization is β-arrestin-dependent and sensitive to modulation by allosteric ligands.

The Role of β-Arrestin Proteins in Organization of Signaling and Regulation of the AT1 Angiotensin Receptor.

AT1 angiotensin receptor plays important physiological and pathophysiological roles in the cardiovascular system. Renin-angiotensin system represents a target system for drugs acting at different levels. The main effects of ATR1 stimulation involve activation of Gq proteins and subsequent IP3, DAG, and calcium signaling. It has become evident in recent years that besides the well-known G protein pathways, AT1R also activates a parallel signaling pathway through β-arrestins. β-arrestins were originally described as proteins that desensitize G protein-coupled receptors, but they can also mediate receptor internalization and G protein-independent signaling. AT1R is one of the most studied receptors, which was used to unravel the newly recognized β-arrestin-mediated pathways. β-arrestin-mediated signaling has become one of the most studied topics in recent years in molecular pharmacology and the modulation of these pathways of the AT1R might offer new therapeutic opportunities in the near future. In this paper, we review the recent advances in the field of β-arrestin signaling of the AT1R, emphasizing its role in cardiovascular regulation and heart failure.

Structural Mechanism of the Arrestin-3/JNK3 Interaction

Arrestins, in addition to desensitizing GPCR-induced G protein activation, also mediate G protein-independent signaling by interacting with various signaling proteins. Among these, arrestins regulate MAPK signal transduction by scaffolding mitogen-activated protein kinase (MAPK) signaling components such as MAPKKK, MAPKK, and MAPK. In this study, we investigated the binding mode and interfaces between arrestin-3 and JNK3 using hydrogen/deuterium exchange mass spectrometry, 19F-NMR, and tryptophan-induced Atto 655 fluorescence-quenching techniques. Results suggested that the β1 strand of arrestin-3 is the major and potentially only interaction site with JNK3. The results also suggested that C-lobe regions near the activation loop of JNK3 form the potential binding interface, which is variable depending on the ATP binding status. Because the β1 strand of arrestin-3 is buried by the C-terminal strand in its basal state, C-terminal truncation (i.e., pre-activation) of arrestin-3 facilitates the arrestin-3/JNK3 interaction.

Calcium‐sensing Receptor Internalization is β‐arrestin‐dependent and Modulated by Allosteric Ligands

G protein‐coupled receptor (GPCR) internalization is a complex process responsible for the transport of GPCRs from the cell‐surface into intracellular compartments. Receptor internalization is crucial for the termination of GPCR activity and in some cases linked to the initiation of G protein‐independent signalling pathways and endosomal receptor signalling. Internalization has been widely studied in class A GPCRs, where the most prevalent mechanism after receptor activation is initiated by kinase‐mediated phosphorylation of intracellular loops and the C‐terminus, followed by β‐arrestin recruitment and dynamin driven internalization via clathrin‐coated pits. However, the mechanism and clinical relevance in class B and C GPCRs remain largely unknown.The extracellular calcium‐sensing receptor (CaSR) is a prototypical class C GPCR that plays a pivotal role in calcium homeostasis due to continuous sensing of small changes in blood ionized calcium (Ca2+) levels. Impressively, CaSR is able to tightly control its activity despite being constantly exposed to its endogenous agonist. Previous studies investigating CaSR internalization were based on functional assays, and microscopy‐ or flow cytometry‐based analyses. However, there is no general agreement on the pathway(s) involved in CaSR internalization. Overall, the mechanism of CaSR remains to be poorly understood.In this study, we aimed to investigate CaSR internalization using the recently developed TR‐FRET‐based real‐time internalization assay for the first time. We demonstrate that Ca2+ induces CaSR internalization in a concentration‐dependent manner with an EC50 of 3.3 mM, which aligns well with the IP1 response with an EC50 of 3.2 mM. We show that internalization can be positively modulated by NPS R‐568 and negatively modulated by NPS 2143. Through the usage of CRISPR‐Cas9 edited HEK293 cells, we demonstrate that CaSR internalization is β‐arrestin dependent.Support or Funding InformationThis project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement NO 675228, the Lundbeck Foundation, the Carlsberg Foundation, the Augustinus Foundation and the Toyota Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biased G Protein-Independent Signaling of Dopamine D1-D3 Receptor Heteromers in the Nucleus Accumbens.

Several studies found in vitro evidence for heteromerization of dopamine D1 receptors (D1R) and D3 receptors (D3R), and it has been postulated that functional D1R-D3R heteromers that are normally present in the ventral striatum mediate synergistic locomotor-activating effects of D1R and D3R agonists in rodents. Based also on results obtained in vitro, with mammalian transfected cells, it has been hypothesized that those behavioral effects depend on a D1R-D3R heteromer-mediated G protein-independent signaling. Here, we demonstrate the presence on D1R-D3R heteromers in the mouse ventral striatum by using a synthetic peptide that selectively destabilizes D1R-D3R heteromers. Parallel locomotor activity and ex vivo experiments in reserpinized mice and in vitro experiments in D1R-D3R mammalian transfected cells were performed to dissect the signaling mechanisms of D1R-D3R heteromers. Co-administration of D1R and D3R agonists in reserpinized mice produced synergistic locomotor activation and a selective synergistic AKT phosphorylation in the most ventromedial region of the striatum in the shell of the nucleus accumbens. Application of the destabilizing peptide in transfected cells and in the shell of the nucleus accumbens allowed demonstrating that both in vitro and in vivo co-activation of D3R induces a switch from G protein-dependent to G protein-independent D1R-mediated signaling determined by D1R-D3R heteromerization. The results therefore demonstrate that a biased G protein-independent signaling of D1R-D3R heteromers localized in the shell of the nucleus accumbens mediate the locomotor synergistic effects of D1R and D3R agonists in reserpinized mice.

Role of Metabotropic Glutamate Receptors in Neurological Disorders.

Glutamate is a fundamental excitatory neurotransmitter in the mammalian central nervous system (CNS), playing key roles in memory, neuronal development, and synaptic plasticity. Moreover, excessive glutamate release has been implicated in neuronal cell death. There are both ionotropic and metabotropic glutamate receptors (mGluRs), the latter of which can be divided into eight subtypes and three subgroups based on homology sequence and their effects on cell signaling. Indeed, mGluRs exert fine control over glutamate activity by stimulating several cell-signaling pathways via the activation of G protein-coupled (GPC) or G protein-independent cell signaling. The involvement of specific mGluRs in different forms of synaptic plasticity suggests that modulation of mGluRs may aid in the treatment of cognitive impairments related to several neurodevelopmental/psychiatric disorders and neurodegenerative diseases, which are associated with a high economic and social burden. Preclinical and clinical data have shown that, in the CNS, mGluRs are able to modulate presynaptic neurotransmission by fine-tuning neuronal firing and neurotransmitter release in a dynamic, activity-dependent manner. Current studies on drugs that target mGluRs have identified promising, innovative pharmacological tools for the treatment of neurodegenerative and neuropsychiatric conditions, including chronic pain.