Effect of Yishen Daluo Prescription on Rho/ROCK Signaling Pathway in EAE Mice Based on Silencing of β-arrestin1 / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Yishen Daluo prescription （YSDL） on Ras homolog（Rho）/Rho-associated coiled-coil containing protein kinase（ROCK）signaling pathway in mice with experimental autoimmune encephalomyelitis （EAE） based on the silencing of β-arrestin1 gene. MethodSixty C57BL/6 female mice were randomly divided into a blank group， a model group， a virus group， a YSDL group， a virus + YSDL group， and a prednisone acetate group （hormone group）. The EAE model was induced in mice except for those in the normal group. Adeno-associated virus（AAV）solution （150 μL， 1×1011 vg·mL-1） was injected into the tail vein of each mouse in the virus group and the virus + YSDL group on the 4th day of immunization. Drugs were administered on the 8th day of modeling. Specifically， normal saline was given to the mice in the normal group，the model group，and the virus group at 10 mL∙kg-1， prednisone acetate suspension to those in the hormone group at 3.9 g∙kg-1，and YSDL to those in other groups at 20 g∙kg-1 for 14 consecutive days. The mice were weighed and scored every day. The neurological function scores of mice in each group were recorded every day after immunization. Hematoxylin-eosin （HE） staining was used to determine the inflammatory response and lesion location in the brain tissues and spinal cord tissues of mice. The protein expression of β-arrestin1，Ras homolog gene family member A（RhoA）， and Rho-associated coiled-coil forming protein kinase Ⅰ（ROCK Ⅰ） in spinal cord and brain tissues of EAE mice was determined by Western blot. ResultCompared with the model group， the virus group and the virus + YSDL group showed decreased neurological function scores （P<0.01），and the YSDL group also showed decreased neurological function scores（P<0.05）. HE results showed that there was obvious inflammatory reaction in the central nervous system （CNS） of the model group， which was alleviated to varying degrees in other groups compared with the model group. Western blot results showed that compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the spinal cord tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression levels of β-arrestin1， RhoA， and ROCKⅠ in the spinal cord tissues （P<0.01）. Compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the brain tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression level of β-arrestin1 in the brain tissues （P<0.01）， and the virus group and the YSDL group showed decreased protein expression levels of RhoA， and ROCKⅠ in the brain tissues （P<0.05）. Additionally， the virus + YSDL group and the hormone group showed decreased protein expression levels of RhoA and ROCKⅠ in the brain tissues （P<0.01）. ConclusionYSDL can improve the clinical symptoms of EAE mice and improve the inflammatory response of CNS. The mechanism is presumably attributed to the fact that YSDL inhibits the expression of β-arrestin1 in CNS，thereby reducing the expression of Rho/ROCK signaling pathway. Furthermore， YSDL may have a synergistic effect with the inhibition of β-arrestin1 gene expression.

Study on the mechanism of Yishen daluo decoction in alleviating nervous system inflammation in EAE model mice / 中国药房

OBJECTIVE To study the effects of Yishen daluo decoction on inflammatory factors and cyclic adenosine monophosphate（cAMP）/protein kinase A （PKA）/cAMP response element binding protein （CREB） signal pathway in experimental autoimmune encephalomyelitis （EAE） model mice by inhibiting the expressions of β-arrestin1， and to explore the mechanism of Yishen daluo decoction in the treatment of EAE. METHODS Sixty mice were randomly divided into normal group， model group， TCM group （Yishen daluo decoction 20 g/kg）， positive control group （prednisone acetate 3.9 mg/kg）， β-arrestin1 siRNA adeno- associated virus （AAV-β） group， AAV-β+TCM group， with 10 mice in each group. Except for normal group， EAE model was made in other groups. AAV-β group and AAV-β+TCM group were injected with AAV-β via tail vein to interfere with the expression of β -arrestin1 protein. Starting from the 8th day of modeling， they were given corresponding drug solution/normal saline intragastrically， once a day， for consecutive 14 days. The neurological function score of mice was detected； the pathological and morphological changes were observed in the brain and spinal cord tissues of mice； the serum levels of inflammatory factors [interleukin-2 （IL-2）， IL-23， interferon-γ （IFN-γ）] in mice were determined； the expressions of β-arrestin1， cAMP， PKA and CREB in brain and spinal cord were detected. RESULTS Compared with normal group， neurological function scores， serum levels of inflammatory factors， and protein expressions of β-arrestin1 in brain and spinal cord were significantly increased （P＜0.05 or P＜ 0.01）； protein expressions of PKA， CREB and cAMP in brain and spinal cord were decreased significantly（P＜0.05 or P＜0.01）. The deep staining of cellular shrinkage and aggregation of inflammatory cells were observed in most neurons of the brain and spinal cord， with varying degrees of demyelinating. Compared with model group， the neurological function scores， pathological changes in brain and spinal cord tissues， and most indicators （except for CREB and cAMP proteins in the brain tissue of AAV-β group） were significantly reversed （P＜0.05 or P＜0.01）.Compared with AAV- β group， the neurological function scores， the levels of IFN-γ in serum and β-arrestin1 in spinal cord were significantly decreased （P＜0.05 or P＜0.01）， PKA and cAMP in brain and spinal cord tissues were significantly increased in AAV- β +TCM group （P＜0.05 or P＜0.01）. CONCLUSIONS Yishen daluo decoction can inhibit the expression of β-arrestin1 in the central nervous system thus activating the cAMP/PKA/CREB signaling pathway， relieving nervous system inflammation， and ultimately alleviates the symptoms of EAE.

Activation of insulin-like growth factor-1 receptor (IGF-1R) promotes growth of colorectal cancer through triggering the MEX3A-mediated degradation of RIG-I

Insulin-like growth factor-1 receptor (IGF-1R) has been made an attractive anticancer target due to its overexpression in cancers. However, targeting it has often produced the disappointing results as the role played by cross talk with numerous downstream signalings. Here, we report a disobliging IGF-1R signaling which promotes growth of cancer through triggering the E3 ubiquitin ligase MEX3A-mediated degradation of RIG-I. The active β-arrestin-2 scaffolds this disobliging signaling to talk with MEX3A. In response to ligands, IGF-1Rβ activated the basal βarr2 into its active state by phosphorylating the interdomain domain on Tyr64 and Tyr250, opening the middle loop (Leu130‒Cys141) to the RING domain of MEX3A through the conformational changes of βarr2. The models of βarr2/IGF-1Rβ and βarr2/MEX3A could interpret the mechanism of the activated-IGF-1R in triggering degradation of RIG-I. The assay of the mutants βarr2Y64A and βarr2Y250A further confirmed the role of these two Tyr residues of the interlobe in mediating the talk between IGF-1Rβ and the RING domain of MEX3A. The truncated-βarr2 and the peptide ATQAIRIF, which mimicked the RING domain of MEX3A could prevent the formation of βarr2/IGF-1Rβ and βarr2/MEX3A complexes, thus blocking the IGF-1R-triggered RIG-I degradation. Degradation of RIG-I resulted in the suppression of the IFN-I-associated immune cells in the TME due to the blockade of the RIG-I-MAVS-IFN-I pathway. Poly(I:C) could reverse anti-PD-L1 insensitivity by recovery of RIG-I. In summary, we revealed a disobliging IGF-1R signaling by which IGF-1Rβ promoted cancer growth through triggering the MEX3A-mediated degradation of RIG-I.

Amlexanox Exhibits Cardioprotective Effects in 5/6 Nephrectomized Rats

Abstract Cardiorenal syndrome is a life-threatening condition. The aim of the current study was to determine the cardioprotective effects of amlexanox in 5/6 nephrectomized rats. Rats were randomly assigned to three groups: sham, 5/6 nephrectomized rats, and amlexanox-treated 5/6 nephrectomized group. Amlexanox (25 mg/kg/day, i.p.) administration was started just after surgery and continued for 10 weeks. After treatment, kidney function (serum creatinine and urea) and blood pressure (systolic and diastolic) were measured. Heart weight (normalized to tibial length) and fibrosis area percentage were measured. Serum brain natriuretic peptide (BNP, heart failure marker) and cardiac levels of ß1-adrenergic receptor (ß1AR), ß-arrestin-2, phosphatidylinositol-4,5-bisphosphate (PIP2), diacylglycerol (DAG), pS473 Akt (a survival marker), and caspase-3 activity (an apoptosis marker) were also measured. The 5/6 nephrectomy caused renal impairment, cardiac fibrosis, apoptosis, and heart failure indicated by down- regulation of cardiac ß1AR down-stream signals compared with those in the sham group. Interestingly, amlexanox significantly reduced all cardiopathological changes induced after 10 weeks of 5/6 nephrectomy. Amlexanox showed potent cardiac antifibrotic and antiapoptotic effects in 5/6 nephrectomized rats, which were associated with reduced heart failure. To our knowledge, this is the first study that addresses the potent in vivo cardioprotective effects of amlexanox

Advances in signaling pathway of 5-HT / 中国药理学通报

Classic serotonergic hallucinogens(also known as psychedelics)are powerful psychoactive substances that can induce profound alterations of human consciousness,emotion,and cognition. It is generally believed that the main target of psychedelics for their hallucinogenic effect is 5-hydroxytryptamine 2A receptor(5-HT

Oliceridine - Breakthrough in the Management of Pain

Oliceridine an intravenous opioid approved in 2020 by the Food and Drug Administration (FDA) to treat moderate-to-severe pain. Oliceridin developed with a novel mechanism that is biased agonism toward G-protein-coupled receptors pathway. Being biased agonist, it does not activate beta arrestin pathway responsible for opioid-related adverse events (ORAE), especially respiratory depression. Because of the novel mechanism, oliceridine has paved a pathway to decrease ORAE. Oliceridine has received breakthrough status by FDA. However, FDA denied oliceridine approval and withdrew breakthrough status by 2019. FDA made this decision because of the inadequacy of the safety data. Abuse potential and QT prolongation studies are conducted as per FDA recommendation in the year 2019; oliceridine was approved for moderate to severe pain in adults. This review will briefly summarize the pharmacological properties and study results of oliceridine in the management of pain. Thorough literature search was done for the efficacy and safety of oliceridine, search was done in electronic database of PubMed and Cochrane from inception till June 2021. Oliceridine was found to be effective in acute severe pain with less OREA when compared to morphine. Oliceridine has many drawbacks than what is hypothesized earlier, but this approach has opened new options for patients suffering from severe pain. Long?term effect of oliceridine has to be monitored to assess the effects of biased agonism.

Inhibitory Effect of CCK-8 on Methamphetamine-Induced Apoptosis / 法医学杂志

OBJECTIVES@#To investigate the inhibitory effect of cholecystokinin octapeptide (CCK-8) binding to cholecystokinin 2 receptor (CCK2R) on methamphetamine (METH)-induced neuronal apoptosis, and to explore the signal transduction mechanism of β-arrestin 2 in CCK-8 inhibiting METH-induced neuronal apoptosis.@*METHODS@#SH-SY5Y cell line was cultured, and HEK293-CCK1R and HEK293-CCK2R cell line were constructed by lentivirus transfection. Small interfering RNA (siRNA) was used to knockdown the expression of β-arrestin 2. Annexin Ⅴ-FITC/PI staining and flow cytometry were used to detect the apoptotic rate of cells, and Western blotting was used to detect the expression of apoptosis-related proteins.@*RESULTS@#The apoptosis of SH-SY5Y cells was induced by 1 mmol/L and 2 mmol/L METH treatment, the number of nuclear fragmentation and pyknotic cells was significantly increased, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were increased. CCK-8 pre-treatment at the dose of 0.1 mmol/L and 1 mmol/L significantly reversed METH-induced apoptosis in SH-SY5Y cells, and inhibited cell nuclear fragmentation, pyknosis and the changes of apoptosis-related proteins induced by METH. In lentivirus transfected HEK293-CCK1R and HEK293-CCK2R cells, the results revealed that CCK-8 had no significant effect on METH-induced changes of apoptosis-related proteins in HEK293-CCK1R cells, but it could inhibit the expression level of apoptosis-related proteins in HEK293-CCK2R cells induced by METH. The inhibitory effect of CCK-8 on METH-induced apoptosis was blocked by the knockdown of β-arrestin 2 expression in SH-SY5Y cells.@*CONCLUSIONS@#CCK-8 can bind to CCK2R and exert an inhibitory effect on METH-induced apoptosis by activating the β-arrestin 2 signal.

β-arrestin-2 inhibits autophagy by up-regulating PI3K/Akt signal to alleviate liver ischemia-reperfusion injury in mice / 器官移植

Objective To verify whether β-arrestin-2 inhibits autophagy by up-regulating PI3K/Akt signal to protect the liver from ischemia-reperfusion injury (IRI) in mice. Methods Twelve β-arrestin-2 knockout (KO) and twelve wild-type (WT) C57BL/6 mice were randomly divided into the KO+sham group, KO+IRI group, WT+sham group and WT+IRI group, six mice in each group. The mouse models with 70% liver IRI were established or sham operation was performed. Relevant experiments were carried out at 6 h after liver reperfusion or operation. The expression levels of apoptosis signal protein cleaved Caspase-3, proliferation signal protein Ki-67 and the PI3K/Akt signal protein p-Akt were detected by immunohistochemical staining. Results Immunohistochemical staining demonstrated that compared with the corresponding sham group, the positive cell count for cleaved Caspase-3, Ki-67 and p-Akt in liver tissues of mice was significantly increased in the KO+IRI and WT+IRI groups (all P < 0.01). Compared with the WT+IRI group, the positive cell count for cleaved Caspase-3 in liver tissues of mice was significantly increased, whereas the positive cell count forKi-67 and p-Akt was significantly decreased in the KO+IRI group (both P < 0.05). Conclusions β-arrestin-2 can mitigate the liver cell apoptosis and promote the repair of injury after IRI in mice. Moreover, β-arrestin-2 inhibits autophagy by up-regulating the PI3K/Akt signal to alleviate liver IRI in mice.

β-arrestin1 overexpression suppresses progression of human T-cell acute lymphatic leukemia Molt-4 cell xenograft in mice / 南方医科大学学报

OBJECTIVE@#To investigate the effect of β-arrestin1 overexpression on tumor progression in a NCG mouse model bearing T-cell acute lymphocytic leukemia (T-ALL) Molt-4 cell xenograft.@*METHODS@#Molt-4 cells were tagged with firefly-luciferase (F-Luc) by lentiviral infection, and fluorescence intensity of the cells was detected using a luminescence detector. Molt-4 cell lines with β-arrestin1 overexpression or knockdown were constructed by lentivirus infection and injected the tail vein in sub-lethal irradiated NCG mice. Body weight changes and survival time of the xenografted mice were observed, and the progression of T-ALL in the mice was evaluated using an fluorescence imaging system. Sixteen days after xenografting, the mice were euthanatized and tumor cell infiltration was observed in the slices of the liver and spleen.@*RESULTS@#We successfully tagged Molt-4 cells with F-Luc and overexpressed or knocked down β-arrestin1 in the tagged cells. Bioluminescent imaging showed obvious luminescence catalyzed by F-Luc in Molt-4 cells. After injection of Molt-4-Luc cells into irradiated NCG mice, a gradual enhancement of luminescence in the xenografted mice was observed over time, while the body weight of the mice decreased. Compared with the control mice, the mice xenografted with β-arrestin1-overexpressing Molt-4 cells had significantly prolonged survival time ( < 0.001), while the survival time of the mice xenografted with Molt-4 cells with β- arrestin1 knockdown was significantly shortened ( < 0.001). Histological examination revealed fewer infiltrating tumor cells in the liver and spleen of the mice xenografted with β-arrestin1-overexpressing Molt-4 cells in comparison with the mice bearing parental Molt-4 cell xenografts.@*CONCLUSIONS@#β-arrestin1 overexpression suppresses tumor progression in mice bearing Molt-4 cell xenograft.

β-arrestin 1 facilitates acetylation of IL-6 gene promoter to affect proliferation and apoptosis of non-small cell lung cancer cells / 肿瘤

Objective: To investigate the effects of β-arrestin 1 (ARRB1) on apoptosis and proliferation of non-small cell lung cancer (NSCLC) cells, and to explore the underlying molecular mechanism. Methods: In NSCLC cell lines A549 and H1650, the expression of ARRB1 was knocked down by transfection of siRNA or over-expressed by transfection of Flag-ARRB1 recombinant plasmids, which was verified by real-time fluorescent quantitative PCR and Western blotting. The effects of down-regulating and up-regulating ARRB1 expression on the transcription and secretion of interleukin-6 (IL-6) were detected by real-time fluorescent quantitative PCR and ELISA method, respectively. The interaction between ARRB1 and p300 was detected by protein immunocoprecipitation. The recruitment of p300 and the acetylation of histone in IL-6 promoter region after ARRB1 knock-down or overexpression were detected by chromatin immunocoprecipitation. The proliferation and apoptosis of ARRB1 silencing A549 cells were detected by CCK-8 assay and FCM, respectively. The expression and activation of IL-6/signal transducer and activator of transcription 3 (STAT3) signaling pathway-related molecules in ARRB1 silencing or overexpression NSCLC cells were investigated by Western blotting. Results: In ARRB1-silenced or overexpressed NSCLC cell lines A549 and H1650, ARRB1 enhanced the transcription and production of IL-6 (all P < 0.05). The interaction of ARRB1 and p300 was confirmed by forward and reverse immunocoprecipitation. After ARRB1 knockdown or overexpression, it was found that ARRB1 enhanced the recruitment of p300 in IL-6 promoter region (both P < 0.01) and increased the acetylating of IL-6 promoter (both P < 0.05). Moreover, ARRB1 could facilitate the growth (P < 0.01) and apoptosis inhibition of NSCLC cells. ARRB1 could promote the phosphorylation of STAT3 and the expressions of c-Myc and Bcl-2 proteins. Conclusion: In NSCLC cells, ARRB1 interacts with p300, facilitates the recruitment of p300 to IL-6 promoter, and up-regulates the acetylation of histone H3 and H4 in IL-6 promoter, leading to transcriptional activation of IL-6. So that ARRB1 positively regulates the activation of IL-6/STAT3 signaling through promoting the phosphorylation of STAT3 and the expressions of c-Myc and Bcl-2 proteins, contributing to the growth and anti-apoptosis of NSCLC cells.

Bone-edge electroacupuncture relieves pain by regulating expression of GRK5, β-arrestin 2 and PKCα proteins in locus coeruleus in bone cancer pain rats with morphine tolerance / 针刺研究

OBJECTIVE: To observe the effect of bone-edge electroacupuncture (EA) intervention on mechanical pain threshold (PT) and expression of G protein-coupled receptor kinase (GRK5), β-arrestin 2, total and phosphorylated PKC alpha (p-PKCα) proteins in the locus coeruleus (LC) of rats with bone cancer pain induced morphine tolerance, so as to reveal its partial central mechanisms underlying pain relief. METHODS: Forty SD rats were randomly divided into 5 groups, namely sham bone cancer, bone cancer pain, morphine tolerance, bone-edge EA, and sham EA (n＝ 8 rats in each group). The bone cancer with morphine tolerance model was established by intramedullary injection of MRMT-1 cells into the tibial cavity, and then intraperitoneal injection of morphine hydrochloride injection. After successful establishment of morphine tolerance model, the bone-edge EA (2 Hz/100 Hz，0.5－1.5 mA) was applied to bilateral "Zusanli" (ST36) and "Kunlun" (BL60) for 30 min, once a day for 7 days, after inserting the needle-tip to the tibial bone surface. The ipsilateral mechanical paw withdrawal thresholds (PWTs) were detected dynamically. The expression levels of GRK5, β-arrestin 2, PKCα and p-PKCα in the LC area were measured by Western blot. RESULTS: The PWTs of bone cancer pain rats were decreased on day 10 after inoculation of cancer cells (P0.05). The PWTs were significantly increased in the bone-edge EA intervention group (P0.05). In comparison with the sham bone cancer group, the expression of GRK5 protein in morphine tolerance group was significantly decreased (P<0.01); compared with morphine tolerance group, the expression of GRK5 protein in bone-edge EA group was increased(P<0.01). In comparison with the sham bone cancer group, the expression of β-arrestin 2 and p-PKCα in bone cancer group significantly increased (P<0.01). After the intervention, the increased β-arrestin 2 and p-PKCα expressions were reversed in the bone-edge EA group (P<0.01); compared with morphine tolerance group and sham EA group, the expression of PKCα protein was decreased(P<0.01). CONCLUSION: Bone-edge EA can effectively relieve morphine tolerance in bone cancer pain rats, which may be related to its functions in up-regulating GRK5 protein and down-regulating β-arrestin 2, PKCα and p-PKCα proteins in LC.　.

Role of Helix 8 in Dopamine Receptor Signaling

G protein-coupled receptors (GPCRs) are membrane receptors whose agonist-induced dynamic conformational changes trigger heterotrimeric G protein activation, followed by GRK-mediated phosphorylation and arrestin-mediated desensitization. Cytosolic regions of GPCRs have been studied extensively because they are direct contact sites with G proteins, GRKs, and arrestins. Among various cytosolic regions, the role of helix 8 is least understood, although a few studies have suggested that it is involved in G protein activation, receptor localization, and/or internalization. In the present study, we investigated the role of helix 8 in dopamine receptor signaling focusing on dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R). D1R couples exclusively to Gs, whereas D2R couples exclusively to Gi. Bioinformatic analysis implied that the sequences of helix 8 may affect GPCR-G protein coupling selectivity; therefore, we evaluated if swapping helix 8 between D1R and D2R changed G protein selectivity. Our results suggest that helix 8 is not involved in D1R-Gs or D2R-Gi coupling selectivity. Instead, we observed that D1R with D2R helix 8 or D1R with an increased number of hydrophobic residues in helix 8 relative to wild-type showed diminished β-arrestin-mediated desensitization, resulting in increased Gs signaling.

Effect of Modified Erchentang on β2AR/β-arrestin2 Signaling Pathway in Rats with Chronic Obstructive Pulmonary Disease / 中国实验方剂学杂志

Objective: To explore the effect of modified Erchentang on the signal pathway of β2 adrenergicreceptor(β2AR)/arrestin beta 2(β-arrestin2) in rats with chronic obstructive pulmonary disease (COPD), and the expression of interleukin-17(IL-17) in serum, lung homogenate and bronchoalveolar lavage fluid. Method: Seventy SD rats were randomly divided into seven groups:normal group, model group, modified Erchentang with high, medium and low doses (40, 20, 10 g · kg-1 · d-1), Xiaokechuan group (5 g · kg-1 · d-1), modified Erchentang group (5 g · kg-1 · d-1), 10 rats in each group. The rat model of COPD was established by smoking and lipopolysaccharide (LPS) intratracheal drip. After successful modeling, the treatment group was given intragastric administration, while the normal group and the model group were given the same amount of saline. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of IL-17 in serum, lung homogenate and bronchoalveolar lavage fluid of rats. Real-time fluorescence quantitative PCR (Real-time PCR) was used to detect the expression of β2AR gene. Western blot was used to detect the expression of β2AR protein in lung tissue. The expression of β2AR and β-arrestin2 in lung tissue was detected by immunohistochemistry. Result: Compared with the normal group, the expression of β2AR protein in lung tissue of model group was significantly decreased(Pβ2AR protein in lung tissue was significantly increased(PPβ2AR in model group was significantly lower(Pβ2AR in high, medium and low dose group, Xiaokechuan group and modified Erchentang group was significantly higher(PPPPConclusion: Modified Erchentang may increase the expression of β2AR and β-arrestin2 and decrease the content of IL-17 in order to resist inflammation and improve pulmonary function in COPD rats.

Mechanisms of β-arrestin-biased GPCR signal transduction and advances in drug research / 药学学报

G protein-coupled receptors (GPCR) are a class of receptor superfamily that exist on the surface of cell membrane. With the intensive studies on the GPCR desensitization regulator—β-arrestins, it is found that activated GPCR can not only conduct signal transduction through G protein-dependent pathway, but also mediate via non-G protein-dependent pathway. In addition to mediate endocytosis and desensitization, β-arrestins also initiate a new series of signal transduction events. Therefore, the concept of "biased transduction" was put forward: the receptor activated by a specific ligand could selectively activate a specific signaling pathway, leading the signal to be transmitted downstream along a "preferential" pathway. We call the ligand that binds to the receptor and causes biased activation "biased ligand". It is generally believed that the phenomenon of bias results from different binding modes of ligands and receptors, including multiple receptor conformations, diverse sites that downstream signal proteins bind, and signal proteins’ own conformations, etc. Here we give a brief review focusing on the mechanisms of β-arrestin-biased GPCR signal transduction and the advances in the drug development on β-arrestin biased ligands.

Arrestins: structural disorder creates rich functionality

Arrestins are soluble relatively small 44-46 kDa proteins that specifically bind hundreds of active phosphorylated GPCRs and dozens of non-receptor partners. There are binding partners that demonstrate preference for each of the known arrestin conformations: free, receptor-bound, and microtubule-bound. Recent evidence suggests that conformational flexibility in every functional state is the defining characteristic of arrestins. Flexibility, or plasticity, of proteins is often described as structural disorder, in contrast to the fixed conformational order observed in high-resolution crystal structures. However, protein-protein interactions often involve highly flexible elements that can assume many distinct conformations upon binding to different partners. Existing evidence suggests that arrestins are no exception to this rule: their flexibility is necessary for functional versatility. The data on arrestins and many other multi-functional proteins indicate that in many cases, "order" might be artificially imposed by highly non-physiological crystallization conditions and/or crystal packing forces. In contrast, conformational flexibility (and its extreme case, intrinsic disorder) is a more natural state of proteins, representing true biological order that underlies their physiologically relevant functions.

Can oliceridine (TRV130), an ideal novel µ receptor G protein pathway selective (µ-GPS) modulator, provide analgesia without opioid-related adverse reactions?

All drugs have both favorable therapeutic and untoward adverse effects. Conventional opioid analgesics possess both analgesia and adverse reactions, such as nausea, vomiting, and respiratory depression. The opioid ligand binds to µ opioid receptor and non-selectively activates two intracellular signaling pathways: the G protein pathway induce analgesia, while the β-arrestin pathway is responsible for the opioid-related adverse reactions. An ideal opioid should activate the G protein pathway while deactivating the β-arrestin pathway. Oliceridine (TRV130) has a novel characteristic mechanism on the action of the µ receptor G protein pathway selective (µ-GPS) modulation. Even though adverse reactions (ADRs) are significantly attenuated, while the analgesic effect is augmented, the some residual ADRs persist. Consequently, a G protein biased µ opioid ligand, oliceridine, improves the therapeutic index owing to increased analgesia with decreased adverse events. This review article provides a brief history, mechanism of action, pharmacokinetics, pharmacodynamics, and ADRs of oliceridine.

The changes of β-arrestin 2 and microtubule-associated protein light chain 3 in renal tissue of rats with renal ischemia reperfusion injury / 中国小儿急救医学

Objective To investigate the expression of β-arrestin2 and microtubule-associated pro-tein light chain(LC)3 in renal of rat with acute renal ischemia reperfusion injury,and to analyze the relation-ship between them and renal injury. Methods Fifty-four male SD rat(3-4 weeks old) were randomly divid-ed into three groups:control group,sham group,acute ischemic reperfusion injury group. We established the acute renal ischemia reperfusion injury model through removing the right kidney and clamping the left renal for 45 minutes with noninvasive arterial clip. We obtained the kidney and blood samples respectively at 12 h, 24 h,36 h,48 h,72 h,96 h after the surgery. Expressions ofβ-arrestin2 and LC3 protein were detected by the immunohistochemistry method and Western blot method. The renal function and morphological changes were assessed. Results Compared with control group and sham group,the serum creatinine and kidney pathologi-cal grading of acute ischemia reperfusion injury group obviously rised. The kidney injury was the most serious at the 24 h after acute ischemic reperfusion injury. The expressions of β-arrestin2 and LC3 were little in the control group and sham group. However,the expressions of these two indicators were obviously higher and reached the peak at the 12 h after acute ischemia reperfusion injury. All these results suggested that the chan-ges of these two indicators were anterior to the histopathological changes. The expressions ofβ-arrestin 2 and LC3 protein were in positive correlation with the kidney injury(r=0. 821,P<0. 05;r=0. 913,P<0. 05). Conclusion In the acute renal ischemia-reperfusion injury,β-arrestin2 may be as a kind of upstream regula-tory protein involving in the kidney pathological process through the regulation of the autophagy.

β-arrestin2 promoting mice colitis through impairment of epithelial barrier function / 实用医学杂志

Objective To investigate the role of β-arrestin2 in intestinal inflammation and illustrate the mechanisms from the perspective of epithelial barrier function. Methods Dextran sodium sulfate(DSS)is used to induce acute intestinal colitis in mice. The experiment groups are designed as the wild type control(WT),the wild type colitis (WT+DSS) and the β-arrestin2- knockout colitis (KO+DSS). The expression of β-arrestin2 gene by mRNA and protein level is compared between the WT and WT + DSS groups. The difference of weight loss , disease activity index(DAI),spleen weight,colon length,histological score,intestinal permeability and important tight junction proteins (occludin ,claudin1 and ZO-1) were detected in the WT+DSS and KO+DSS groups. Results Compared with the WT group,the expression of β-arrestin2 was significantly higher in the colon of the WT+DSS group. Compared with the WT+DSS group,the KO+DSS group had less weight loss(P < 0.05),lower DAI(P<0.05),smaller spleen,longer colon and lower histological score(P=0.002). The KO+DSS group had a lower intestinal permeability(P = 0.009)and higher protein level of occludin and claudin1.There was no signifi-cant difference of ZO-1 in the two groups. Conclusion β-arrestin2 may promote mouse colitis through impairment of epithelial barrier function.

Arrestin mediated GPCR biased signaling and its application in new drug discovery / 中国药理学与毒理学杂志

G protein coupled receptors (GPCR) are important transmembrane proteins which account for more than 30% of direct clinical drug targets. Two main signaling pathways, either mediated by different G protein subtype or arrestins, underlies most of 800 GPCR functions in human genome. Selective ligands targeting to one of the G protein or arrestin signaling through specific receptor, which is also called biased ligands, may have beneficial effects and delete the unwanted side effects compared with traditional full agonists or antagonists. However, the mechanism governing the arrestin mediated GPCR biased signaling is still unclear. In recent years, our research group have combined animal models, cell biology and biophysical approaches to address the arrestin mediated GPCR function and its underlying mechanism, which is a key issue for GPCR targeted drug discovery. We have identified that downstream of β2 adrenergic receptor, arrestin mediated signaling plays critical roles in maintaining the pancreatic islet homeostasis and promotes the learning and memory through regulation of astrocyte-neuron lactate transportation cycle. Targeting β- arrestin- 1 signaling rather than Gq signaling down?stream of CCK1R receptor may provide a better therapy for diabetes. Although arrestin mediated signaling was traditionally recognized as the second wave of GPCR signaling, our recent results indicated that β-arrestin-1 was able to induce the first wave signaling in to regulate the catecholamine secretion from adrenal gland, by directly mediating AT1R/TRPC3 coupling. This result provided new mode for the connection of GPCR to activation of ion channels. Moreover, all above arrestin mediated signaling are regulated by receptor phosphorylation barcode, a hypothesis brought up by Prof. Lefkowitz and Prof. Andrew Tobin. Using biophysical and cellular approaches, we have identified that the 10 distinct phosphorylation interacting sites along the N-terminal of arrestin is the ″phospho-code″ reader of the arrestin, which recognized the information passed by GPCR, then translated to more than 1000 distinct arrestin conformations, and recruit distinct downstream signaling molecule. We therefore proposed″a flute model″ working mechanism for arrestin mediated GPCR signaling. Using this flute model combined with GPCR ligand identification, we were able to regulate specific signaling and therefore arrestin mediated physiological functions by activation of the receptor and operation of the receptor phosphorylation barcode simultaneously (unpublished results). These knowledge advances in arrestin mediated GPCR signaling may facilitate further drug development targeting to GPCR family members.

Role of M3 receptor in the effect of penehyclidine hydrochloride up-regulating β-arrestin-1 expression / 重庆医学

Objective To investigate the role of M3 receptor in the effect of penehyclidine hydrochloride(PHC) upregulating β-arrestin-1 expression in lipopolysaccharide(LPS)-induced human pulmonary microvascular endothelial cell(HPMVEC) injury.Methods.M3 shRNA transfected HPMVEC and normal HPMVEC cells were randomly divided into LPS group(A),LPS+pHC group(B),LPS+ M3 shRNA transfection group(C) and PHC+ LPS+ M3 shRNA transfection group(D).The cytoskeleton change was observed by laser scanning confocal.The LDH level in cellular supernate was detected.The VCAM 1 protein expression was examined by immunofluorescence chemistry.β-arrestin-1 protein expression was determined by Western blot and β-arrestin-1mRNA expression was measured by real-time PCR.Results Compared with the group A or C,F-actin cytoskeleton arrangement in the group B or D was neat,the LDH level and VCAM-1 protein expression were decreased,and β-arrestin-1 expression was increased;compared with group A or B,F-actin cytoskeleton arrangement in the group C or D was neat,the LDH level and VCAM-1 protein expression were decreased,while the β-arrestin-1 expression had no obvious change.Conclusion Silence M3 receptor is conducive to reduce LPS-induced HPMVEC injury.But the role of PHC up-regulating β-arrestin-1 expression has no necessary connection with M3 receptor.