Interactions between AT1R and GRKs: the determinants for activation of signaling pathways involved in blood pressure regulation.

The success of Angiotensin II receptor blockers, specifically Angiotensin II type 1 receptor (AT1R) antagonists as antihypertensive drug emphasizes the involvement of AT1R in Essential hypertension. The structural insights and mutational studies of Ang II-AT1R have brought about the vision to design Ang II analogs that selectively activate the pathways with beneficial and cardioprotective effects such as cell survival and hinder the deleterious effects such as hypertrophy and cell death. AT1R belongs to G-protein coupled receptors and is regulated by G-protein coupled receptor kinases (GRKs) that either uncouples Gq protein for receptor desensitization or phosphorylate C-terminus to recruit ß-arrestin for internalization of the receptor. The interaction of GRKs with ligand activated AT1R induces conformational changes and signal either Gq dependent or Gq independent pathways. These interactions might explain the complex regulatory mechanisms and offer promising ideas for hypertension therapeutics. This article reviews the functional role of AT1R, organization of GRK genes and regulation of AT1R by GRKs that play significant role in desensitization and internalization of the receptors.

Characterization of behavioral changes in T-maze alternation from dopamine D1 agonists with different receptor coupling mechanisms.

RATIONALE: Dopamine D1 receptor agonists have been shown to improve working memory, but often have a non-monotonic (inverted-U) dose-response curve. One hypothesis is that this may reflect dose-dependent differential engagement of D1 signaling pathways, a mechanism termed functional selectivity or signaling bias. OBJECTIVES AND METHODS: To test this hypothesis, we compared two D1 ligands with different signaling biases in a rodent T-maze alternation task. Both tested ligands (2-methyldihydrexidine and CY208243) have high intrinsic activity at cAMP signaling, but the former also has markedly higher intrinsic activity at D1-mediated recruitment of ß-arrestin. The spatial working memory was assessed via the alternation behavior in the T-maze where the alternate choice rate quantified the quality of the memory and the duration prior to making a choice represented the decision latency. RESULTS: Both D1 drugs changed the alternate rate and the choice latency in a dose-dependent manner, albeit with important differences. 2-Methyldihydrexidine was somewhat less potent but caused a more homogeneous improvement than CY208243 in spatial working memory. The maximum changes in the alternate rate and the choice latency tended to occur at different doses for both drugs. CONCLUSIONS: These data suggest that D1 signaling bias in these two pathways (cAMP vs ß-arrestin) has complex effects on cognitive processes as assessed by T-maze alternation. Understanding these mechanisms should allow the identification or discovery of D1 agonists that can provide superior cognitive enhancement.

Glyphosate potentiates insulin resistance in skeletal muscle through the modulation of IRS-1/PI3K/Akt mediated mechanisms: An in vivo and in silico analysis.

Herbicides have been linked to a higher risk of developing diabetes. Certain herbicides also operate as environmental toxins. Glyphosate is a popular and extremely effective herbicide for weed control in grain crops that inhibits the shikimate pathway. It has been shown to negatively influence endocrine function. Few studies have demonstrated that glyphosate exposure results in hyperglycemic and insulin resistance; but the molecular mechanism underlying the diabetogenic potential of glyphosate on skeletal muscle, a primary organ that includes insulin-mediated glucose disposal, is unknown. In this study, we aimed to evaluate the impact of glyphosate on the detrimental changes in the insulin metabolic signaling in the gastrocnemius muscle. In vivo results showed that glyphosate exposure caused hyperglycemia, dyslipidemia, increased glycosylated hemoglobin (HbA1c), liver function, kidney function profile, and oxidative stress markers in a dose-dependent fashion. Conversely, hemoglobin and antioxidant enzymes were significantly reduced in glyphosate-induced animals indicating its toxicity is linked to induce insulin resistance. The histopathology of the gastrocnemius muscle and RT-PCR analysis of insulin signaling molecules revealed glyphosate-induced alteration in the expression of IR, IRS-1, PI3K, Akt, ß-arrestin-2, and GLUT4 mRNA. Lastly, molecular docking and dynamics simulations confirmed that glyphosate showed a high binding affinity with target molecules such as Akt, IRS-1, c-Src, ß-arrestin-2, PI3K, and GLUT4. The current work provides experimental proof that glyphosate exposure has a deleterious effect on the IRS-1/PI3K/Akt signaling pathways, which in turn causes the skeletal muscle to become insulin resistant and eventually develop type 2 diabetes mellitus.

C781, a ß-Arrestin Biased Antagonist at Protease-Activated Receptor-2 (PAR2), Displays in vivo Efficacy Against Protease-Induced Pain in Mice.

Given the limited options and often harmful side effects of current analgesics and the suffering caused by the opioid crisis, new classes of pain therapeutics are needed. Protease-activated receptors (PARs), particularly PAR2, are implicated in a variety of pathologies, including pain. Since the discovery of the role of PAR2 in pain, development of potent and specific antagonists has been slow. In this study, we describe the in vivo characterization of a novel small molecule/peptidomimetic hybrid compound, C781, as a ß-arrestin-biased PAR2 antagonist. In vivo behavioral studies were done in mice using von Frey filaments and the Mouse Grimace Scale. Pharmacokinetic studies were done to assess pharmacokinetic/pharmacodynamic relationship in vivo. We used both prevention and reversal paradigms with protease treatment to determine whether C781 could attenuate protease-evoked pain. C781 effectively prevented and reversed mechanical and spontaneous nociceptive behaviors in response to small molecule PAR2 agonists, mast cell activators, and neutrophil elastase. The ED50 of C781 (intraperitoneal dosing) for inhibition of PAR2 agonist (20.9 ng 2-AT)-evoked nociception was 6.3 mg/kg. C781 was not efficacious in the carrageenan inflammation model. Pharmacokinetic studies indicated limited long-term systemic bioavailability for C781 suggesting that optimizing pharmacokinetic properties could improve in vivo efficacy. Our work demonstrates in vivo efficacy of a biased PAR2 antagonist that selectively inhibits ß-arrestin/MAPK signaling downstream of PAR2. Given the importance of this signaling pathway in PAR2-evoked nociception, C781 exemplifies a key pharmacophore for PAR2 that can be optimized for clinical development. PERSPECTIVE: Our work provides evidence that PAR2 antagonists that only block certain aspects of signaling by the receptor can be effective for blocking protease-evoked pain in mice. This is important because it creates a rationale for developing safer PAR2-targeting approaches for pain treatment.

Inhibition of MRGPRX2 but not FcεRI or MrgprB2-mediated mast cell degranulation by a small molecule inverse receptor agonist.

Mas-related G protein-coupled receptor-X2 (MRGPRX2) expressed on mast cells (MCs) contributes to hypersensitivity reactions to cationic US-Food and Drug Administration (FDA) approved drugs such as the neuromuscular blocking agent, rocuronium. In addition, activation of MRGPRX2 by the neuropeptide substance P (SP) and the pro-adrenomedullin peptide (PAMP-12) is associated with a variety of cutaneous conditions such as neurogenic inflammation, pain, atopic dermatitis, urticaria, and itch. Thus, small molecules aimed at blocking MRGPRX2 constitute potential options for modulating IgE-independent MC-mediated disorders. Two inverse MRGPRX2 agonists, named C9 and C9-6, have recently been identified, which inhibit basal G protein activation and agonist-induced calcium mobilization in transfected HEK293 cells. Substance P serves as a balanced agonist for MRGPRX2 whereby it activates both G protein-mediated degranulation and ß-arrestin-mediated receptor internalization. The purpose of this study was to determine if C9 blocks MRGPRX2's G protein and ß-arrestin-mediated signaling and to determine its specificity. We found that C9, but not its inactive analog C7, inhibited degranulation in RBL-2H3 cells stably expressing MRGPRX2 in response to SP, PAMP-12 and rocuronium with an IC50 value of ~300 nM. C9 also inhibited degranulation as measured by cell surface expression of CD63, CD107a and ß-hexosaminidase release in LAD2 cells and human skin-derived MCs in response to SP but not the anaphylatoxin, C3a or FcϵRI-aggregation. Furthermore, C9 inhibited ß-arrestin recruitment and MRGPRX2 internalization in response to SP and PAMP-12. We found that a G protein-coupling defective missense MRGPRX2 variant (V282M) displays constitutive activity for ß-arrestin recruitment, and that this response was significantly inhibited by C9. Rocuronium, SP and PAMP-12 caused degranulation in mouse peritoneal MCs and these responses were abolished in the absence of MrgprB2 or cells treated with pertussis toxin but C9 had no effect. These findings suggest that C9 could provide an important framework for developing novel therapeutic approaches for the treatment of IgE-independent MC-mediated drug hypersensitivity and cutaneous disorders.

Morphine-induced changes in the function of microglia and macrophages after acute spinal cord injury.

BACKGROUND: Opioids are among the most effective and commonly prescribed analgesics for the treatment of acute pain after spinal cord injury (SCI). However, morphine administration in the early phase of SCI undermines locomotor recovery, increases cell death, and decreases overall health in a rodent contusion model. Based on our previous studies we hypothesize that morphine acts on classic opioid receptors to alter the immune response. Indeed, we found that a single dose of intrathecal morphine increases the expression of activated microglia and macrophages at the injury site. Whether similar effects of morphine would be seen with repeated intravenous administration, more closely simulating clinical treatment, is not known. METHODS: To address this, we used flow cytometry to examine changes in the temporal expression of microglia and macrophages after SCI and intravenous morphine. Next, we explored whether morphine changed the function of these cells through the engagement of cell-signaling pathways linked to neurotoxicity using Western blot analysis. RESULTS: Our flow cytometry studies showed that 3 consecutive days of morphine administration after an SCI significantly increased the number of microglia and macrophages around the lesion. Using Western blot analysis, we also found that repeated administration of morphine increases ß-arrestin, ERK-1 and dynorphin (an endogenous kappa opioid receptor agonist) production by microglia and macrophages. CONCLUSIONS: These results suggest that morphine administered immediately after an SCI changes the innate immune response by increasing the number of immune cells and altering neuropeptide synthesis by these cells.

ß2 -Adrenoceptor agonist profiling reveals biased signalling phenotypes for the ß2 -adrenoceptor with possible implications for the treatment of asthma.

BACKGROUND AND PURPOSE: ß-Adrenoceptor agonists relieve airflow obstruction by activating ß2 -adrenoceptors, which are G protein-coupled receptors (GPCRs) expressed on human airway smooth muscle (HASM) cells. The currently available ß-adrenoceptor agonists are balanced agonists, however, and signal through both the stimulatory G protein (Gs )- and ß-arrestin-mediated pathways. While Gs signalling is beneficial and promotes HASM relaxation, ß-arrestin activation is associated with reduced Gs efficacy. In this context, biased ligands that selectively promote ß2 -adrenoceptor coupling to Gs signalling represent a promising strategy to treat asthma. Here, we examined several ß-adrenoceptor agonists to identify Gs -biased ligands devoid of ß-arrestin-mediated effects. EXPERIMENTAL APPROACH: Gs -biased ligands for the ß2 -adrenoceptor were identified by high-throughput screening and then evaluated for Gs interaction, Gi interaction, cAMP production, ß-arrestin interaction, GPCR kinase (GRK) phosphorylation of the receptor, receptor trafficking, ERK activation, and functional desensitization of the ß2 -adrenoceptor. KEY RESULTS: We identified ractopamine, dobutamine, and higenamine as Gs -biased agonists that activate the Gs /cAMP pathway upon ß2 -adrenoceptor stimulation while showing minimal Gi or ß-arrestin interaction. Furthermore, these compounds did not induce any receptor trafficking and had reduced GRK5-mediated phosphorylation of the ß2 -adrenoceptor. Finally, we observed minimal physiological desensitization of the ß2 -adrenoceptor in primary HASM cells upon treatment with biased agonists. CONCLUSION AND IMPLICATIONS: Our work demonstrates that Gs -biased signalling through the ß2 -adrenoceptor may prove to be an effective strategy to promote HASM relaxation in the treatment of asthma. Such biased compounds may also be useful in identifying the molecular mechanisms that determine biased signalling and in design of safer drugs.

α and ß catalytic subunits of cAMP-dependent protein kinase regulate formoterol-induced inflammatory gene expression changes in human bronchial epithelial cells.

BACKGROUND AND PURPOSE: It has been proposed that genomic mechanisms contribute to adverse effects often experienced by asthmatic subjects who take regular, inhaled ß2 -adrenoceptor agonists as a monotherapy. Moreover, data from preclinical models of asthma suggest that these gene expression changes are mediated by ß-arrestin-2 rather than PKA. Herein, we tested this hypothesis by comparing the genomic effects of formoterol, a ß2 -adrenoceptor agonist, with forskolin in human primary bronchial epithelial cells (HBEC). EXPERIMENTAL APPROACH: Gene expression changes were determined by RNA-sequencing. Gene silencing and genome editing were employed to explore the roles of ß-arrestin-2 and PKA. KEY RESULTS: The formoterol-regulated transcriptome in HBEC treated concurrently with TNFα was defined by 1480 unique gene expression changes. TNFα-induced transcripts modulated by formoterol were annotated with enriched gene ontology terms related to inflammation and proliferation, notably "GO:0070374~positive regulation of ERK1 and ERK2 cascade," which is an apparent ß-arrestin-2 target. However, expression of the formoterol- and forskolin-regulated transcriptomes were highly rank-order correlated and the effects of formoterol on TNFα-induced inflammatory genes were abolished by an inhibitor of PKA. Furthermore, formoterol-induced gene expression changes in BEAS-2B bronchial epithelial cell clones deficient in ß-arrestin-2 were comparable with those expressed by their parental counterparts. Contrariwise, gene expression was partially inhibited in clones lacking the α-catalytic subunit (Cα) of PKA and abolished following the additional knockdown of the ß-catalytic subunit (Cß) paralogue. CONCLUSIONS: The effects of formoterol on inflammatory gene expression in airway epithelia are mediated by PKA and involve the cooperation of PKA-Cα and PKA-Cß.

The Extended N-Terminal Domain Confers Atypical Chemokine Receptor Properties to CXCR3-B.

The chemokine receptor CXCR3 plays a critical role in immune cell recruitment and activation. CXCR3 exists as two main isoforms, CXCR3-A and CXCR3-B, resulting from alternative splicing. Although the two isoforms differ only by the presence of an N-terminal extension in CXCR3-B, they have been attributed divergent functional effects on cell migration and proliferation. CXCR3-B is the more enigmatic isoform and the mechanisms underlying its function and signaling remain elusive. We therefore undertook an in-depth cellular and molecular comparative study of CXCR3-A and CXCR3-B, investigating their activation at different levels of the signaling cascades, including G protein coupling, ß-arrestin recruitment and modulation of secondary messengers as well as their downstream gene response elements. We also compared the subcellular localization of the two isoforms and their trafficking under resting and stimulated conditions along with their ability to internalize CXCR3-related chemokines. Here, we show that the N-terminal extension of CXCR3-B drastically affects receptor features, modifying its cellular localization and preventing G protein coupling, while preserving ß-arrestin recruitment and chemokine uptake capacities. Moreover, we demonstrate that gradual truncation of the N terminus leads to progressive recovery of surface expression and G protein coupling. Our study clarifies the molecular basis underlying the divergent effects of CXCR3 isoforms, and emphasizes the ß-arrestin-bias and the atypical nature of CXCR3-B.

Structure-Based Evolution of G Protein-Biased µ-Opioid Receptor Agonists.

The µ-opioid receptor (µOR) is the major target for opioid analgesics. Activation of µOR initiates signaling through G protein pathways as well as through ß-arrestin recruitment. µOR agonists that are biased towards G protein signaling pathways demonstrate diminished side effects. PZM21, discovered by computational docking, is a G protein biased µOR agonist. Here we report the cryoEM structure of PZM21 bound µOR in complex with Gi protein. Structure-based evolution led to multiple PZM21 analogs with more pronounced Gi protein bias and increased lipophilicity to improve CNS penetration. Among them, FH210 shows extremely low potency and efficacy for arrestin recruitment. We further determined the cryoEM structure of FH210 bound to µOR in complex with Gi protein and confirmed its expected binding pose. The structural and pharmacological studies reveal a potential mechanism to reduce ß-arrestin recruitment by the µOR, and hold promise for developing next-generation analgesics with fewer adverse effects.

Molecular insights into the allosteric coupling mechanism between an agonist and two different transducers for µ-opioid receptors.

G protein-coupled receptors (GPCRs) as the most important class of pharmacological targets regulate G-protein and ß-arrestin-mediated signaling through allosteric interplay, which are responsible for different biochemical and physiological actions like therapeutic efficacy and side effects. However, the allosteric mechanism underlying preferentially recruiting one transducer versus the other has been poorly understood, limiting drug design. Motivated by this issue, we utilize accelerated molecular dynamics simulation coupled with potential of mean force (PMF), molecular mechanics Poisson Boltzmann surface area (MM/PBSA) and protein structure network (PSN) to study two ternary complex systems of a representative class A GPCR (µ-opioid receptor (µOR)) bound by an agonist and one specific transducer (G-protein and ß-arrestin). The results show that no significant difference exists in the whole structure of µOR between two transducer couplings, but displays transducer-dependent changes in the intracellular binding region of µOR, where the ß-arrestin coupling results in a narrower crevice with TM7 inward movement compared with the G-protein. In addition, both the G-protein and ß-arrestin coupling can increase the binding affinity of the agonist to the receptor. However, the interactions between the agonist and µOR also exhibit transducer-specific changes, in particular for the interaction with ECL2 that plays an important role in recruiting ß-arrestin. The allosteric network analysis further indicates that Y1483.33, F1523.37, F1563.41, N1914.49, T1603.45, Y1062.42, W2936.48, F2896.44, I2485.54 and Y2525.58 play important roles in equally activating G-protein and ß-arrestin. In contrast, M1613.46 and R1653.50 devote important contributions to preferentially recruit G-protein while D1643.49 and R179ICL2 are revealed to be important for selectively activating ß-arrestin. The observations provide useful information for understanding the biased activation mechanism.

Antinociceptive Effects of Kappa-Opioid Receptor Agonists.

Preclinical models that assess "pain" in rodents typically measure increases in behaviors produced by a "pain stimulus." A large literature exists showing that kappa opioid receptor (KOR) agonists can decrease these "pain-stimulated behaviors" following many different pain stimuli. Despite showing apparent antinociceptive properties in these preclinical models, KOR agonists failed as analgesics in clinical trials. Recent studies that assessed decreases in behavior due to a pain stimulus show that KOR agonists are not effective in restoring these "pain-depressed behaviors" to normal levels, which agrees with the lack of effectiveness for KOR agonists in clinical trials. One current explanation for the failure of previous KOR agonists in clinical trials is that those agonists activated beta-arrestin signaling and that KOR agonists with a greater bias for G protein signaling will be more successful. However, neither G protein-biased agonists nor beta-arrestin-biased agonists are very effective in assays of pain-depressed behavior, which suggests that novel biased agonists may still not be effective analgesics. This review provides a concise account of the effectiveness of KOR agonists in preclinical models of pain-stimulated and pain-depressed behaviors following the administration of different pain stimuli. Based on the previous results, it may be appropriate to include both behaviors when testing the analgesic potential of KOR agonists.

A novel ß2-AR agonist, Higenamine, induces ß-arrestin-biased signaling.

The biased ligands in G protein-coupled receptors (GPCRs) have opened new avenues for developing safer and more effective drugs. However, the identification of such biased ligands as drug candidates is highly desirable. Here, we report that Higenamine, a compound isolated from a Chinese herb, functions as a novel ß-arrestin-biased ligand of the ß2-adrenergic receptor (ß2-AR). The radioligand binding assays demonstrated that Higenamine was the ligand of ß2-AR. Higenamine induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), which can be blocked by propranolol, an inhibitor of ß2-AR. The Gi protein inhibitor, pertussis toxin, had no effect on the phosphorylation of ERK1/2 induced by Higenamine. Furthermore, Higenamine induced ERK1/2 phosphorylation through transactivation of Epithelial growth factor receptor (EGFR). We also found that Higenamine-induced-ERK1/2 phosphorylation is dependent on ß-arrestin1/2, and HG inhibits Doxorubicin-induced cardiomyocyte apoptosis. Our results identify Higenamine as a novel biased ligand via the ß-arrestin-dependent pathway. These findings give us a better understanding of Higenamine's potential role in designing diagnostic and therapeutic strategies.

Unique Positive Cooperativity Between the ß-Arrestin-Biased ß-Blocker Carvedilol and a Small Molecule Positive Allosteric Modulator of the ß2-Adrenergic Receptor.

Among ß-blockers that are clinically prescribed for heart failure, carvedilol is a first-choice agent with unique pharmacological properties. Carvedilol is distinct from other ß-blockers in its ability to elicit ß-arrestin-biased agonism, which has been suggested to underlie its cardioprotective effects. Augmenting the pharmacologic properties of carvedilol thus holds the promise of developing more efficacious and/or biased ß-blockers. We recently identified compound-6 (cmpd-6), the first small molecule positive allosteric modulator of the ß2-adrenergic receptor (ß2AR). Cmpd-6 is positively cooperative with orthosteric agonists at the ß2AR and enhances agonist-mediated transducer (G-protein and ß-arrestin) signaling in an unbiased manner. Here, we report that cmpd-6, quite unexpectedly, displays strong positive cooperativity only with carvedilol among a panel of structurally diverse ß-blockers. Cmpd-6 enhances the binding affinity of carvedilol for the ß2AR and augments its ability to competitively antagonize agonist-induced cAMP generation. Cmpd-6 potentiates ß-arrestin1- but not Gs-protein-mediated high-affinity binding of carvedilol at the ß2AR and ß-arrestin-mediated cellular functions in response to carvedilol including extracellular signal-regulated kinase phosphorylation, receptor endocytosis, and trafficking into lysosomes. Importantly, an analog of cmpd-6 that selectively retains positive cooperativity with carvedilol acts as a negative modulator of agonist-stimulated ß2AR signaling. These unprecedented cooperative properties of carvedilol and cmpd-6 have implications for fundamental understanding of G-protein-coupled receptor (GPCR) allosteric modulation, as well as for the development of more effective biased beta blockers and other GPCR therapeutics. SIGNIFICANCE STATEMENT: This study reports on the small molecule-mediated allosteric modulation of the ß-arrestin-biased ß-blocker, carvedilol. The small molecule, compound-6 (cmpd-6), displays an exclusive positive cooperativity with carvedilol among other ß-blockers and enhances the binding affinity of carvedilol for the ß2-adrenergic receptor. Cooperative effects of cmpd-6 augment the ß-blockade property of carvedilol while potentiating its ß-arrestin-mediated signaling functions. These findings have potential implications in advancing G-protein-coupled receptor allostery, developing biased therapeutics and remedying cardiovascular ailments.

Kappa opioid receptors internalization is protective against oxygen-glucose deprivation through ß-arrestin activation and Akt-mediated signaling pathway.

Hypoxia induces reversible κ-opioid receptor (KOR) internalization similar to the internalization that is induced by KOR agonists. In the current study, we demonstrate that this KOR internalization is a protective mechanism via the ß-arrestin specific pathway in an oxygen-glucose deprivation (OGD) model. Mouse neuroblastoma Neuro2A cells were stably transfected with mouse KOR-tdTomato fusion protein (N2A-mKOR-tdT cells). Various concentrations of salvinorin A (SA), a highly selective KOR agonist, were given in the presence and absence of norbinaltorphimine (norBNI), which is a KOR antagonist, or Dyngo-4a (internalization inhibitor) or API-2 (Akt/Protein kinase B signaling inhibitor-2). Various concentrations of SA and RB-64 (22-thiocyanatosalvinorin A, selective for the G protein signaling pathway) were administered both in normoxic and hypoxic conditions. Autophagosomes and ultrastructural components of cells were observed using transmission electron microscopy (TEM). Cell viability, severity of cell injury, and levels of proteins related to the Akt signaling pathway were evaluated using live cell counting (by Cell Counting Kit-8), the lactic acid dehydrogenase (LDH) release rate, and Western blot analysis, respectively. SA promoted cell survival and attenuated OGD-induced cell injury. The Akt signaling pathway is activated by SA. KOR internalization, when blocked by norBNI or Dyngo-4a, increased LDH release and decreased cell viability under OGD. Treatment with SA significantly inhibited autophagy, and the effects of SA on autophagy were reversed by API-2 pretreatment. RB-64 in a low concentration without ß-arrestin recruitment did not reduce LDH release and increase cell viability as observed with SA. KOR internalization through ß-arrestin activation is a protective mechanism against OGD. The Akt pathway might play a critical role in modulating these protective effects by inhibiting autophagy.

Discovery of ß-Arrestin Biased, Orally Bioavailable, and CNS Penetrant Neurotensin Receptor 1 (NTR1) Allosteric Modulators.

Neurotensin receptor 1 (NTR1) is a G protein coupled receptor that is widely expressed throughout the central nervous system where it acts as a neuromodulator. Neurotensin receptors have been implicated in a wide variety of CNS disorders, but despite extensive efforts to develop small molecule ligands there are few reports of such compounds. Herein we describe the optimization of a quinazoline based lead to give 18 (SBI-553), a potent and brain penetrant NTR1 allosteric modulator.

Differential Effects of a Full and Biased Ghrelin Receptor Agonist in a Mouse Kindling Model.

The ghrelin system has received substantial recognition as a potential target for novel anti-seizure drugs. Ghrelin receptor (ghrelin-R) signaling is complex, involving Gαq/11, Gαi/o, Gα12/13, and ß-arrestin pathways. In this study, we aimed to deepen our understanding regarding signaling pathways downstream the ghrelin-R responsible for mediating anticonvulsive effects in a kindling model. Mice were administered the proconvulsive dopamine 1 receptor-agonist, SKF81297, to gradually induce a kindled state. Prior to every SKF81297 injection, mice were treated with a ghrelin-R full agonist (JMV-1843), a Gαq and Gα12 biased ligand unable to recruit ß-arrestin (YIL781), a ghrelin-R antagonist (JMV-2959), or saline. Mice treated with JMV-1843 had fewer and less severe seizures compared to saline-treated controls, while mice treated with YIL781 experienced longer and more severe seizures. JMV-2959 treatment did not lead to differences in seizure severity and number. Altogether, these results indicate that the Gαq or Gα12 signaling pathways are not responsible for mediating JMV-1843's anticonvulsive effects and suggest a possible involvement of ß-arrestin signaling in the anticonvulsive effects mediated by ghrelin-R modulation.

Pharmacological Assays for Investigating the NOP Receptor.

The nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) is a G protein-coupled receptor involved in the regulation of several physiological functions and pathological conditions. Thus, researchers from academia and industry are pursuing NOP to discover and study novel pharmacological entities. In a multidisciplinary effort of pharmacologists, medicinal chemists, and molecular and structural biologists the mechanisms of NOP activation and inhibition have been, at least partially, disentangled. Here, we review the in vitro methodologies employed, which have contributed to our understanding of this target. We hope this chapter guides the reader through the mostly established assay platforms to investigate NOP pharmacology, and gives some hints taking advantage from what has already illuminated the function of other GPCRs. We analyzed the pharmacological results obtained with a large panel of NOP ligands investigated in several assays including receptor binding, stimulation of GTPγS binding, decrease of cAMP levels, calcium flux stimulation via chimeric G proteins, NOP/G protein and NOP/ß-arrestin interaction, label-free assays such as dynamic mass redistribution, and bioassays such as the electrically stimulated mouse vas deferens.

Effect of ß-arrestin on damage of human umbilical vein endothelial cell induced by angiotensin II.

OBJECTIVE: ß-arrestin (ARRB2) is a member of arrestin family and a negative regulatory protein of G-coupling receptor, which is closely associated with the pathogenesis of several autoimmune diseases. This study aimed to investigate the mechanism of the effect of ARRB2 on the damage of human umbilical vein endothelial cells (HUVECs), which is induced by angiotensin II (Ang II). MATERIALS AND METHODS: ARRB2 at different concentration was used to interfere with the damage of HUVECs induced by Ang II or RNA interference technology to interfere with the expression of HUVECs followed by addition of Ang II to culture for 24 hours. Nitrate reduction method was used to measure the content of nitric oxide (NO) and radioimmunoassay was used to measure endothelin-1; Western blot assay was used to detect the expression of B-cell lymphoma-2 (Bcl-2), and flow cytometry was used to detect the intracellular level of reactive oxygen (ROS) and apoptosis of HUVECs. RESULTS: Our study found that ARRB2 could significantly reduce the generation and release of ROS, endothelin-1 (ET-1), lactic dehydrogenase (LDH) of HUVECs induced by Ang II and promote the generation of NO, superoxide dismutase (SOD) and scavenging in a dose-dependent manner. On the contrary, when expression of ARRB2 was disturbed by siRNA, increased generation and release of ROS, ET-1, and LDH were observed with reduced generation of NO, SOD and scavenging. In addition, ARRB2 could reverse the apoptosis of HUVECs induced by Ang II and was related to upregulate the expression of Bax. CONCLUSIONS: ARRB2 could protect the damage of HUVECs induced by Ang II and the mechanism was associated with upregulation of the expression of apoptosis and anti-apoptosis protein of Bcl-2.

G Protein-Coupled Receptor Signaling Through ß-Arrestin-Dependent Mechanisms.

ß-arrestin1 (or arrestin2) and ß-arrestin2 (or arrestin3) are ubiquitously expressed cytosolic adaptor proteins that were originally discovered for their inhibitory role in G protein-coupled receptor (GPCR) signaling through heterotrimeric G proteins. However, further biochemical characterization revealed that ß-arrestins do not just "block" the activated GPCRs, but trigger endocytosis and kinase activation leading to specific signaling pathways that can be localized on endosomes. The signaling pathways initiated by ß-arrestins were also found to be independent of G protein activation by GPCRs. The discovery of ligands that blocked G protein activation but promoted ß-arrestin binding, or vice-versa, suggested the exciting possibility of selectively activating intracellular signaling pathways. In addition, it is becoming increasingly evident that ß-arrestin-dependent signaling is extremely diverse and provokes distinct cellular responses through different GPCRs even when the same effector kinase is involved. In this review, we summarize various signaling pathways mediated by ß-arrestins and highlight the physiologic effects of ß-arrestin-dependent signaling.