Kinetic Model for the Desensitization of G Protein-Coupled Receptor.

Desensitization of G-protein-coupled receptors (GPCR) is a general regulatory mechanism adopted by biological organisms against overstimulation of G protein signaling. Although the details of the mechanism are extensively studied, it is not easy to gain an overarching understanding of the process constituted by a multitude of molecular events with vastly differing time scales. To offer a semiquantitative yet predictive understanding of the mechanism, we formulate a kinetic model for the G protein signaling and desensitization by considering essential biochemical steps from ligand binding to receptor internalization. The internalization, followed by receptor depletion from the plasma membrane, attenuates the downstream signal. Together with the kinetic model and its full numerics of the expression derived for the dose-response relation, an approximated form of the expression clarifies the role played by the individual biochemical processes and allows us to identify four distinct regimes for the downregulation that emerge from the balance between phosphorylation, dephosphorylation, and the cellular level of ß-arrestin.

ArreSTick motif controls ß-arrestin-binding stability and extends phosphorylation-dependent ß-arrestin interactions to non-receptor proteins.

The binding and function of ß-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids responsible for establishing a stable interaction remains unclear. We employ a 1D sequence convolution model trained on GPCRs with established ß-arrestin-binding properties. With this approach, amino acid motifs characteristic of GPCRs that form stable interactions with ß-arrestins can be identified, a pattern that we name "arreSTick." Intriguingly, the arreSTick pattern is also present in numerous non-receptor proteins. Using proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between many non-receptor proteins and ß-arrestin2. The HIV-1 Tat-specific factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and its subcellular localization is influenced by ß-arrestin2. Our findings unveil a broader role for ß-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.

Generation of Comprehensive GPCR-Transducer-Deficient Cell Lines to Dissect the Complexity of GPCR Signaling.

G-protein-coupled receptors (GPCRs) compose the largest family of transmembrane receptors and are targets of approximately one-third of Food and Drug Administration-approved drugs owing to their involvement in almost all physiologic processes. GPCR signaling occurs through the activation of heterotrimeric G-protein complexes and ß-arrestins, both of which serve as transducers, resulting in distinct cellular responses. Despite seeming simple at first glance, accumulating evidence indicates that activation of either transducer is not a straightforward process as a stimulation of a single molecule has the potential to activate multiple signaling branches. The complexity of GPCR signaling arises from the aspects of G-protein-coupling selectivity, biased signaling, interpathway crosstalk, and variable molecular modifications generating these diverse signaling patterns. Numerous questions relative to these aspects of signaling remained unanswered until the recent development of CRISPR genome-editing technology. Such genome editing technology presents opportunities to chronically eliminate the expression of G-protein subunits, ß-arrestins, G-protein-coupled receptor kinases (GRKs), and many other signaling nodes in the GPCR pathways at one's convenience. Here, we review the practicality of using CRISPR-derived knockout (KO) cells in the experimental contexts of unraveling the molecular details of GPCR signaling mechanisms. To mention a few, KO cells have revealed the contribution of ß-arrestins in ERK activation, Gα protein selectivity, GRK-based regulation of GPCRs, and many more, hence validating its broad applicability in GPCR studies. SIGNIFICANCE STATEMENT: This review emphasizes the practical application of G-protein-coupled receptor (GPCR) transducer knockout (KO) cells in dissecting the intricate regulatory mechanisms of the GPCR signaling network. Currently available cell lines, along with accumulating KO cell lines in diverse cell types, offer valuable resources for systematically elucidating GPCR signaling regulation. Given the association of GPCR signaling with numerous diseases, uncovering the system-based signaling map is crucial for advancing the development of novel drugs targeting specific diseases.

Adrenoceptor Desensitization: Current Understanding of Mechanisms.

G-protein coupled receptors (GPCRs) transduce a wide range of extracellular signals. They are key players in the majority of biologic functions including vision, olfaction, chemotaxis, and immunity. However, as essential as most of them are to body function and homeostasis, overactivation of GPCRs has been implicated in many pathologic diseases such as cancer, asthma, and heart failure (HF). Therefore, an important feature of G protein signaling systems is the ability to control GPCR responsiveness, and one key process to control overstimulation involves initiating receptor desensitization. A number of steps are appreciated in the desensitization process, including cell surface receptor phosphorylation, internalization, and downregulation. Rapid or short-term desensitization occurs within minutes and involves receptor phosphorylation via the action of intracellular protein kinases, the binding of ß-arrestins, and the consequent uncoupling of GPCRs from their cognate heterotrimeric G proteins. On the other hand, long-term desensitization occurs over hours to days and involves receptor downregulation or a decrease in cell surface receptor protein level. Of the proteins involved in this biologic phenomenon, ß-arrestins play a particularly significant role in both short- and long-term desensitization mechanisms. In addition, ß-arrestins are involved in the phenomenon of biased agonism, where the biased ligand preferentially activates one of several downstream signaling pathways, leading to altered cellular responses. In this context, this review discusses the different patterns of desensitization of the α 1-, α 2- and the ß adrenoceptors and highlights the role of ß-arrestins in regulating physiologic responsiveness through desensitization and biased agonism. SIGNIFICANCE STATEMENT: A sophisticated network of proteins orchestrates the molecular regulation of GPCR activity. Adrenoceptors are GPCRs that play vast roles in many physiological processes. Without tightly controlled desensitization of these receptors, homeostatic imbalance may ensue, thus precipitating various diseases. Here, we critically appraise the mechanisms implicated in adrenoceptor desensitization. A better understanding of these mechanisms helps identify new druggable targets within the GPCR desensitization machinery and opens exciting therapeutic fronts in the treatment of several pathologies.

Therapeutic potentials of nonpeptidic V2R agonists for partial cNDI-causing V2R mutants.

Loss-of-function mutations in the type 2 vasopressin receptor (V2R) are a major cause of congenital nephrogenic diabetes insipidus (cNDI). In the context of partial cNDI, the response to desmopressin (dDAVP) is partially, but not entirely, diminished. For those with the partial cNDI, restoration of V2R function would offer a prospective therapeutic approach. In this study, we revealed that OPC-51803 (OPC5) and its structurally related V2R agonists could functionally restore V2R mutants causing partial cNDI by inducing prolonged signal activation. The OPC5-related agonists exhibited functional selectivity by inducing signaling through the Gs-cAMP pathway while not recruiting ß-arrestin1/2. We found that six cNDI-related V2R partial mutants (V882.53M, Y1283.41S, L1614.47P, T2736.37M, S3298.47R and S3338.51del) displayed varying degrees of plasma membrane expression levels and exhibited moderately impaired signaling function. Several OPC5-related agonists induced higher cAMP responses than AVP at V2R mutants after prolonged agonist stimulation, suggesting their potential effectiveness in compensating impaired V2R-mediated function. Furthermore, docking analysis revealed that the differential interaction of agonists with L3127.40 caused altered coordination of TM7, potentially contributing to the functional selectivity of signaling. These findings suggest that nonpeptide V2R agonists could hold promise as potential drug candidates for addressing partial cNDI.

Investigation of Strategies to Block Downstream Effectors of AT1R-Mediated Signalling to Prevent Aneurysm Formation in Marfan Syndrome.

Cardiovascular outcome in Marfan syndrome (MFS) patients most prominently depends on aortic aneurysm progression with subsequent aortic dissection. Angiotensin II receptor blockers (ARBs) prevent aneurysm formation in MFS mouse models. In patients, ARBs only slow down aortic dilation. Downstream signalling from the angiotensin II type 1 receptor (AT1R) is mediated by G proteins and ß-arrestin recruitment. AT1R also interacts with the monocyte chemoattractant protein-1 (MCP-1) receptor, resulting in inflammation. In this study, we explore the targeting of ß-arrestin signalling in MFS mice by administering TRV027. Furthermore, because high doses of the ARB losartan, which has been proven beneficial in MFS, cannot be achieved in humans, we investigate a potential additive effect by combining lower concentrations of losartan (25 mg/kg/day and 5 mg/kg/day) with barbadin, a ß-arrestin blocker, and DMX20, a C-C chemokine receptor type 2 (CCR2) blocker. A high dose of losartan (50 mg/kg/day) slowed down aneurysm progression compared to untreated MFS mice (1.73 ± 0.12 vs. 1.96 ± 0.08 mm, p = 0.0033). TRV027, the combination of barbadin with losartan (25 mg/kg/day), and DMX-200 (90 mg/kg/day) with a low dose of losartan (5 mg/kg/day) did not show a significant beneficial effect. Our results confirm that while losartan effectively halts aneurysm formation in Fbn1C1041G/+ MFS mice, neither TRV027 alone nor any of the other compounds combined with lower doses of losartan demonstrate a notable impact on aneurysm advancement. It appears that complete blockade of AT1R function, achieved by administrating a high dosage of losartan, may be necessary for inhibiting aneurysm progression in MFS.

Local anesthetics inhibit muscarinic acetylcholine receptor-mediated calcium responses and the recruitment of ß-arrestin in HSY human parotid cells.

OBJECTIVES: Local anesthetics act on G protein-coupled receptors (GPCRs); thus, their potential as allosteric modulators of GPCRs has attracted attention. Intracellular signaling via GPCRs involves both G-protein- and ß-arrestin-mediated pathways. To determine the effects of local anesthetics on muscarinic acetylcholine receptors (mAChR), a family of GPCRs, we analyzed the effects of local anesthetics on mAChR-mediated Ca2+ responses and formation of receptor-ß-arrestin complexes in the HSY human parotid cell line. METHODS: Ca2+ responses were monitored by fura-2 spectrofluorimetry. Ligand-induced interactions between mAChR and ß-arrestin were examined using a ß-arrestin GPCR assay kit. RESULTS: Lidocaine reduced mAChR-mediated Ca2+ responses but did not change the intracellular Ca2+ concentration in non-stimulated cells. The membrane-impermeant lidocaine analog QX314 and procaine inhibited mAChR-mediated Ca2+ responses, with EC50 values of 48.0 and 20.4 µM, respectively, for 50 µM carbachol-stimulated Ca2+ responses. In the absence of extracellular Ca2+, the pretreatment of cells with QX314 reduced carbachol-induced Ca2+ release, indicating that QX314 reduced Ca2+ release from intracellular stores. Lidocaine and QX314 did not affect store-operated Ca2+ entry as they did not alter the thapsigargin-induced Ca2+ response. QX314 and procaine reduced the carbachol-mediated recruitment of ß-arrestin, and administration of procaine suppressed pilocarpine-induced salivary secretion in mice. CONCLUSION: Local anesthetics, including QX314, act on mAChR to reduce carbachol-induced Ca2+ release from intracellular stores and the recruitment of ß-arrestin. These findings support the notion that local anesthetics and their derivatives are starting points for the development of functional allosteric modulators of mAChR.

Identification of 1,3,8-Triazaspiro[4.5]Decane-2,4-Dione Derivatives as a Novel δ Opioid Receptor-Selective Agonist Chemotype.

δ opioid receptors (DORs) hold potential as a target for neurologic and psychiatric disorders, yet no DOR agonist has proven efficacious in critical phase II clinical trials. The exact reasons for the failure to produce quality drug candidates for the DOR are unclear. However, it is known that certain DOR agonists can induce seizures and exhibit tachyphylaxis. Several studies have suggested that those adverse effects are more prevalent in delta agonists that share the (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)/4-[(αR*)-α-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-N,N-diethylbenzamide chemotype. There is a need to find novel lead candidates for drug development that have improved pharmacological properties to differentiate them from the current failed delta agonists. Our objective in this study was to identify novel DOR agonists. We used a ß-arrestin assay to screen a small G-protein coupled receptors (GPCR)-focused chemical library. We identified a novel chemotype of DOR agonists that appears to bind to the orthosteric site based of docking and molecular dynamic simulation. The most potent agonist hit compound is selective for the DOR over a panel of 167 other GPCRs, is slightly biased toward G-protein signaling and has anti-allodynic efficacy in a complete Freund's adjuvant model of inflammatory pain in C57BL/6 male and female mice. The newly discovered chemotype contrasts with molecules like SNC80 that are highly efficacious ß-arrestin recruiters and may suggest this novel class of DOR agonists could be expanded on to develop a clinical candidate drug. SIGNIFICANCE STATEMENT: δ opioid receptors are a clinical target for various neurological disorders, including migraine and chronic pain. Many of the clinically tested delta opioid agonists share a single chemotype, which carries risks during drug development. Through a small-scale high-throughput screening assay, this study identified a novel δ opioid receptor agonist chemotype, which may serve as alternative for the current analgesic clinical candidates.

Beneath the surface: endosomal GPCR signaling.

G protein-coupled receptors (GPCRs) located at the cell surface bind extracellular ligands and convey intracellular signals via activation of heterotrimeric G proteins. Traditionally, G protein signaling was viewed to occur exclusively at this subcellular region followed by rapid desensitization facilitated by ß-arrestin (ßarr)-mediated G protein uncoupling and receptor internalization. However, emerging evidence over the past 15 years suggests that these ßarr-mediated events do not necessarily terminate receptor signaling and that some GPCRs continue to activate G proteins after having been internalized into endosomes. Here, we review the recently elucidated mechanistic basis underlying endosomal GPCR signaling and discuss physiological implications and pharmacological targeting of this newly appreciated signaling mode.

The role of G protein-coupled receptor kinases in GLP-1R ß-arrestin recruitment and internalisation.

The glucagon-like peptide 1 receptor (GLP-1R) is a validated clinical target for the treatment of type 2 diabetes and obesity. Unlike most G protein-coupled receptors (GPCRs), the GLP-1R undergoes an atypical mode of internalisation that does not require ß-arrestins. While differences in GLP-1R trafficking and ß-arrestin recruitment have been observed between clinically used GLP-1R agonists, the role of G protein-coupled receptor kinases (GRKs) in affecting these pathways has not been comprehensively assessed. In this study, we quantified the contribution of GRKs to agonist-mediated GLP-1R internalisation and ß-arrestin recruitment profiles using cells where endogenous ß-arrestins, or non-visual GRKs were knocked out using CRISPR/Cas9 genome editing. Our results confirm the previously established atypical ß-arrestin-independent mode of GLP-1R internalisation and revealed that GLP-1R internalisation is dependent on the expression of GRKs. Interestingly, agonist-mediated GLP-1R ß-arrestin 1 and ß-arrestin 2 recruitment were differentially affected by endogenous GRK knockout with ß-arrestin 1 recruitment more sensitive to GRK knockout than ß-arrestin 2 recruitment. Moreover, individual overexpression of GRK2, GRK3, GRK5 or GRK6 in a newly generated GRK2/3/4/5/6 HEK293 cells, rescued agonist-mediated ß-arrestin 1 recruitment and internalisation profiles to similar levels, suggesting that there is no specific GRK isoform that drives these pathways. This study advances mechanistic understanding of agonist-mediated GLP-1R internalisation and provides novel insights into how GRKs may fine-tune GLP-1R signalling.

Exploring biased activation characteristics by molecular dynamics simulation and machine learning for the µ-opioid receptor.

Biased ligands selectively activating specific downstream signaling pathways (termed as biased activation) exhibit significant therapeutic potential. However, the conformational characteristics revealed are very limited for the biased activation, which is not conducive to biased drug development. Motivated by the issue, we combine extensive accelerated molecular dynamics simulations and an interpretable deep learning model to probe the biased activation features for two complex systems constructed by the inactive µOR and two different biased agonists (G-protein-biased agonist TRV130 and ß-arrestin-biased agonist endomorphin2). The results indicate that TRV130 binds deeper into the receptor core compared to endomorphin2, located between W2936.48 and D1142.50, and forms hydrogen bonding with D1142.50, while endomorphin2 binds above W2936.48. The G protein-biased agonist induces greater outward movements of the TM6 intracellular end, forming a typical active conformation, while the ß-arrestin-biased agonist leads to a smaller extent of outward movements of TM6. Compared with TRV130, endomorphin2 causes more pronounced inward movements of the TM7 intracellular end and more complex conformational changes of H8 and ICL1. In addition, important residues determining the two different biased activation states were further identified by using an interpretable deep learning classification model, including some common biased activation residues across Class A GPCRs like some key residues on the TM2 extracellular end, ECL2, TM5 intracellular end, TM6 intracellular end, and TM7 intracellular end, and some specific important residues of ICL3 for µOR. The observations will provide valuable information for understanding the biased activation mechanism for GPCRs.

Histamine H1 Receptor-Mediated JNK Phosphorylation Is Regulated by Gq Protein-Dependent but Arrestin-Independent Pathways.

Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H1 receptor-mediated activation of ERK is dually regulated by Gq proteins and arrestins. In this study, we investigated the roles of Gq proteins and arrestins in the H1 receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H1 receptors, the Gq protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H1 receptors (ketotifen and diphenhydramine), Gq proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca2+ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H1 receptor-mediated phosphorylation of JNK is regulated by Gq-protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways.

Muscarinic acetylcholine receptor-mediated phosphorylation of extracellular signal-regulated kinase in HSY salivary ductal cells involves distinct signaling pathways.

OBJECTIVES: Typical agonists of G protein-coupled receptors (GPCRs), including muscarinic acetylcholine receptors (mAChRs), activate both G-protein and ß-arrestin signaling systems, and are termed balanced agonists. In contrast, biased agonists selectively activate a single pathway, thereby offering therapeutic potential for the specific activation of that pathway. The mAChR agonists carbachol and pilocarpine are known to induce phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) via G-protein-dependent and -independent pathways, respectively. We investigated the involvement of ß-arrestin and its downstream mechanisms in the ERK1/2 phosphorylation induced by carbachol and pilocarpine in the human salivary ductal cell line, HSY cells. METHODS: HSY cells were stimulated with pilocarpine or carbachol, with or without various inhibitors. The cell lysates were analyzed by western blotting using the antibodies p44/p42MAPK and phosphor-p44/p42MAPK. RESULTS: Western blot analysis revealed that carbachol elicited greater stimulation of ERK1/2 phosphorylation compared to pilocarpine. ERK1/2 phosphorylation was inhibited by atropine and gefitinib, suggesting that mAChR activation induces transactivation of epidermal growth factor receptors (EGFR). Moreover, inhibition of carbachol-mediated ERK1/2 phosphorylation was achieved by GF-109203X (a PKC inhibitor), a ßARK1/GRK2 inhibitor, barbadin (a ß-arrestin inhibitor), pitstop 2 (a clathrin inhibitor), and dynole 34-2 (a dynamin inhibitor). In contrast, pilocarpine-mediated ERK1/2 phosphorylation was only inhibited by barbadin (a ß-arrestin inhibitor) and PP2 (a Src inhibitor). CONCLUSION: Carbachol activates both G-protein and ß-arrestin pathways, whereas pilocarpine exclusively activates the ß-arrestin pathway. Additionally, downstream of ß-arrestin, carbachol activates clathrin-dependent internalization, while pilocarpine activates Src.

Identification of an Antagonist Targeting G Protein and ß-Arrestin Signaling Pathways of 5-HT7R.

In consideration of the limited number of FDA-approved drugs for autism spectrum disorder (ASD), significant efforts have been devoted to identifying novel drug candidates. Among these, 5-HT7R modulators have garnered considerable attention due to their potential in alleviating autism-like behaviors in ASD animal models. In this study, we designed and synthesized biphenyl-3-ylmethylpyrrolidines 3 and biphenyl-3-yl-dihydroimidazoles 4 as 5-HT7R modulators. Through extensive biological tests of 3 and 4 in G protein and ß-arrestin signaling pathways of 5-HT7R, it was determined that 2-(2'-methoxy-[1,1'-biphenyl]-3-yl)-4,5-dihydro-1H-imidazole 4h acted as a 5-HT7R antagonist in both signaling pathways. In in vivo study with Shank3-/- transgenic (TG) mice, the self-grooming behavior test was performed with 4h, resulting in a significant reduction in the duration of self-grooming. In addition, an immunohistochemical experiment with 4h restored reduced neurogenesis in Shank3-/- TG mice, which is confirmed by the quantification of doublecortin (DCX) positive neurons, suggesting the promising therapeutic potential of 4h.

GPCR kinases differentially modulate biased signaling downstream of CXCR3 depending on their subcellular localization.

Some G protein-coupled receptors (GPCRs) demonstrate biased signaling such that ligands of the same receptor exclusively or preferentially activate certain downstream signaling pathways over others. This phenomenon may result from ligand-specific receptor phosphorylation by GPCR kinases (GRKs). GPCR signaling can also exhibit location bias because GPCRs traffic to and signal from subcellular compartments in addition to the plasma membrane. Here, we investigated whether GRKs contributed to location bias in GPCR signaling. GRKs translocated to endosomes after stimulation of the chemokine receptor CXCR3 or other GPCRs in cultured cells. GRK2, GRK3, GRK5, and GRK6 showed distinct patterns of recruitment to the plasma membrane and to endosomes depending on the identity of the biased ligand used to activate CXCR3. Analysis of engineered forms of GRKs that localized to either the plasma membrane or endosomes demonstrated that biased CXCR3 ligands elicited different signaling profiles that depended on the subcellular location of the GRK. Each GRK exerted a distinct effect on the regulation of CXCR3 engagement of ß-arrestin, internalization, and activation of the downstream effector kinase ERK. Our work highlights a role for GRKs in location-biased GPCR signaling and demonstrates the complex interactions between ligands, GRKs, and cellular location that contribute to biased signaling.

Novel 1-(1-Arylimiazolin-2-Yl)-3-Arylalkilurea Derivatives with Modulatory Activity on Opioid MOP Receptors.

µ-opioid receptor ligands such as morphine and fentanyl are the most known and potent painkillers. However, the severe side effects seen with their use significantly limit their widespread use. The continuous broadening of knowledge about the properties of the interactions of the MOP receptor (human mu opioid receptor, OP3) with ligands and specific intracellular signaling pathways allows for the designation of new directions of research with respect to compounds with analgesic effects in a mechanism different from classical ligands. Allosteric modulation is an extremely promising line of research. Compounds with modulator properties may provide a safer alternative to the currently used opioids. The aim of our research was to obtain a series of urea derivatives of 1-aryl-2-aminoimidazoline and to determine their activity, mechanism of biological action and selectivity toward the MOP receptor. The obtained compounds were subjected to functional tests (cAMP accumulation and ß-arrestin recruitment) in vitro. One of the obtained compounds, when administered alone, did not show any biological activity, while when co-administered with DAMGO, it inhibited ß-arrestin recruitment. These results indicate that this compound is a negative allosteric modulator (NAM) of the human MOP receptor.

TF-FVIIa PAR2-ß-Arrestin Signaling Sustains Organ Dysfunction in Coxsackievirus B3 Infection of Mice.

BACKGROUND: Accumulating evidence implicates the activation of G-protein-coupled PARs (protease-activated receptors) by coagulation proteases in the regulation of innate immune responses. METHODS: Using mouse models with genetic alterations of the PAR2 signaling platform, we have explored contributions of PAR2 signaling to infection with coxsackievirus B3, a single-stranded RNA virus provoking multiorgan tissue damage, including the heart. RESULTS: We show that PAR2 activation sustains correlates of severe morbidity-hemodynamic compromise, aggravated hypothermia, and hypoglycemia-despite intact control of the virus. Following acute viral liver injury, canonical PAR2 signaling impairs the restoration process associated with exaggerated type I IFN (interferon) signatures in response to viral RNA recognition. Metabolic profiling in combination with proteomics of liver tissue shows PAR2-dependent reprogramming of liver metabolism, increased lipid droplet storage, and gluconeogenesis. PAR2-sustained hypodynamic compromise, reprograming of liver metabolism, as well as imbalanced IFN responses are prevented in ß-arrestin coupling-deficient PAR2 C-terminal phosphorylation mutant mice. Thus, wiring between upstream proteases and immune-metabolic responses results from biased PAR2 signaling mediated by intracellular recruitment of ß-arrestin. Importantly, blockade of the TF (tissue factor)-FVIIa (coagulation factor VIIa) complex capable of PAR2 proteolysis with the NAPc2 (nematode anticoagulant protein c2) mitigated virus-triggered pathology, recapitulating effects seen in protease cleavage-resistant PAR2 mice. CONCLUSIONS: These data provide insights into a TF-FVIIa signaling axis through PAR2-ß-arrestin coupling that is a regulator of inflammation-triggered tissue repair and hemodynamic compromise in coxsackievirus B3 infection and can potentially be targeted with selective coagulation inhibitors.

Distinct roles of the extracellular surface residues of glucagon-like peptide-1 receptor in ß-arrestin 1/2 signaling.

Glucagon-like peptide-1 receptor (GLP-1R) is a prime drug target for type 2 diabetes and obesity. The ligand initiated GLP-1R interaction with G protein has been well studied, but not with ß-arrestin 1/2. Therefore, bioluminescence resonance energy transfer (BRET), mutagenesis and an operational model were used to evaluate the roles of 85 extracellular surface residues on GLP-1R in ß-arrestin 1/2 recruitment triggered by three representative GLP-1R agonists (GLP-1, exendin-4 and oxyntomodulin). Residues selectively regulated ß-arrestin 1/2 recruitment for diverse ligands, and ß-arrestin isoforms were identified. Mutation of residues K130-S136, L142 and Y145 on the transmembrane helix 1 (TM1)-extracellular domain (ECD) linker decreased ß-arrestin 1 recruitment but increased ß-arrestin 2 recruitment. Other extracellular loop (ECL) mutations, including P137A, Q211A, D222A and M303A selectively affected ß-arrestin 1 recruitment while D215A, L217A, Q221A, S223A, Y289A, S301A, F381A and I382A involved more in ß-arrestin 2 recruitment for the ligands. Oxyntomodulin engaged more broadly with GLP-1R extracellular surface to drive ß-arrestin 1/2 recruitment than GLP-1 and exendin-4; I147, W214 and L218 involved in ß-arrestin 1 recruitment, while L141, D215, L218, D293 and F381 in ß-arrestin 2 recruitment for oxyntomodulin particularly. Additionally, the non-conserved residues on ß-arrestin 1/2 C-domains contributed to interaction with GLP-1R. Further proteomic profiling of GLP-1R stably expressed cell line upon ligand stimulation with or without ß-arrestin 1/2 overexpression demonstrated both commonly and biasedly regulated proteins and pathways associated with cognate ligands and ß-arrestins. Our study offers valuable information about ligand induced ß-arrestin recruitment mediated by GLP-1R and consequent intracellular signaling events.

Investigating selectivity and bias for G protein subtypes and ß-arrestins by synthetic cannabinoid receptor agonists at the cannabinoid CB1 receptor.

The cannabinoid CB1 receptor (CB1) is a G protein-coupled receptor (GPCR) with widespread expression in the central nervous system. This canonically G⍺i/o-coupled receptor mediates the effects of Δ9-tetrahydrocannabinol (THC) and synthetic cannabinoid receptor agonists (SCRAs). Recreational use of SCRAs is associated with serious adverse health effects, making pharmacological research into these compounds a priority. Several studies have hypothesised that signalling bias may explain the different toxicological profiles between SCRAs and THC. Previous studies have focused on bias between G protein activation measured by cyclic adenosine monophosphate (cAMP) inhibition and ß-arrestin translocation. In contrast, the current study characterises bias between G⍺ subtypes of the G⍺i/o family and ß-arrestins; this method facilitates a more accurate assessment of ligand bias by assessing signals that have not undergone major amplification. We have characterised G protein dissociation and translocation of ß-arrestin 1 and 2 using real-time BRET reporters. The responses produced by each SCRA across the G protein subtypes tested were consistent with the responses produced by the reference ligand AMB-FUBINACA. Ligand bias was probed by applying the operational analysis to determine biases within the G⍺i/o family, and between G protein subtypes and ß-arrestins. Overall, these results confirm SCRAs to be balanced, high-efficacy ligands compared to the low efficacy ligand THC, with only one SCRA, 4CN-MPP-BUT7IACA, demonstrating statistically significant bias in one pathway comparison (towards ß-arrestin 1 when compared with G⍺oA/oB). This suggests that the adverse effects caused by SCRAs are due to high potency and efficacy at CB1, rather than biased agonism.

Mechanisms of biased agonism by Gαi/o-biased stapled peptide agonists of the relaxin-3 receptor.

The neuropeptide relaxin-3 is composed of an A chain and a B chain held together by disulfide bonds, and it modulates functions such as anxiety and food intake by binding to and activating its cognate receptor RXFP3, mainly through the B chain. Biased ligands of RXFP3 would help to determine the molecular mechanisms underlying the activation of G proteins and ß-arrestins downstream of RXFP3 that lead to such diverse functions. We showed that the i, i+4 stapled relaxin-3 B chains, 14s18 and d(1-7)14s18, were Gαi/o-biased agonists of RXFP3. These peptides did not induce recruitment of ß-arrestin1/2 to RXFP3 by GPCR kinases (GRKs), in contrast to relaxin-3, which enabled the GRK2/3-mediated recruitment of ß-arrestin1/2 to RXFP3. Relaxin-3 and the previously reported peptide 4 (an i, i+4 stapled relaxin-3 B chain) did not exhibit biased signaling. The staple linker of peptide 4 and parts of both the A chain and B chain of relaxin-3 interacted with extracellular loop 3 (ECL3) of RXFP3, moving it away from the binding pocket, suggesting that unbiased ligands promote a more open conformation of RXFP3. These findings highlight roles for the A chain and the N-terminal residues of the B chain of relaxin-3 in inducing conformational changes in RXFP3, which will help in designing selective biased ligands with improved therapeutic efficacy.