APLNR Regulates IFN-γ signaling via ß-arrestin 1 mediated JAK-STAT1 pathway in melanoma cells.

The apelin receptor (APLNR) regulates many biological processes including metabolism, angiogenesis, circulating blood volume and cardiovascular function. Additionally, APLNR is overexpressed in various types of cancer and influences cancer progression. APLNR is reported to regulate tumor recognition during immune surveillance by modulating the IFN-γ response. However, the mechanism of APLNR cross-talk with intratumoral IFN-γ signaling remains unknown. Here, we show that activation of APLNR up-regulates IFN-γ signaling in melanoma cells through APLNR mediated ß-arrestin 1 but not ß-arrestin 2 recruitment. Our data suggests that ß-arrestin 1 directly interacts with STAT1 to inhibit STAT1 phosphorylation to attenuate IFN-γ signaling. The APLNR mutant receptor, I109A, which is deficient in ß-arrestins recruitment, is unable to enhance intratumoral IFN-γ signaling. While APLNR N112G, a constitutively active mutant receptor, increases intratumoral sensitivity to IFN-γ signaling by enhancing STAT1 phosphorylation upon IFN-γ exposure. We also demonstrate in a co-culture system that APLNR regulates tumor survival rate. Taken together, our findings reveal that APLNR modulates IFN-γ signaling in melanoma cells and suggest that APLNR may be a potential target to enhance the efficacy of immunotherapy.

Endothelin-1 axis fosters YAP-induced chemotherapy escape in ovarian cancer.

The majority of ovarian cancer (OC) patients recur with a platinum-resistant disease. OC cells activate adaptive resistance mechanisms that are only partially described. Here we show that OC cells can adapt to chemotherapy through a positive-feedback loop that favors chemoresistance. In platinum-resistant OC cells we document that the endothelin-1 (ET-1)/endothelin A receptor axis intercepts the YAP pathway. This cross-talk occurs through the LATS/RhoA/actin-dependent pathway and contributes to prevent the chemotherapy-induced apoptosis. Mechanistically, ß-arrestin1 (ß-arr1) and YAP form a complex shaping TEAD-dependent transcriptional activity on the promoters of YAP target genes, including EDN1, which fuels a feed-forward signaling circuit that sustains a platinum-tolerant state. The FDA approved dual ET-1 receptor antagonist macitentan in co-therapy with cisplatin sensitizes resistant cells to the platinum-based therapy, reducing their metastatic potential. Furthermore, high ETAR/YAP gene expression signature is associated with a poor platinum-response in OC patients. Collectively, our findings identify in the networking between ET-1 and YAP pathways an escape strategy from chemotherapy. ET-1 receptor blockade interferes with such adaptive network and enhances platinum-induced apoptosis, representing a promising therapeutic opportunity to restore drug sensitivity in OC patients.

Tirzepatide is an imbalanced and biased dual GIP and GLP-1 receptor agonist.

Tirzepatide (LY3298176) is a dual GIP and GLP-1 receptor agonist under development for the treatment of type 2 diabetes mellitus (T2DM), obesity, and nonalcoholic steatohepatitis. Early phase trials in T2DM indicate that tirzepatide improves clinical outcomes beyond those achieved by a selective GLP-1 receptor agonist. Therefore, we hypothesized that the integrated potency and signaling properties of tirzepatide provide a unique pharmacological profile tailored for improving broad metabolic control. Here, we establish methodology for calculating occupancy of each receptor for clinically efficacious doses of the drug. This analysis reveals a greater degree of engagement of tirzepatide for the GIP receptor than the GLP-1 receptor, corroborating an imbalanced mechanism of action. Pharmacologically, signaling studies demonstrate that tirzepatide mimics the actions of native GIP at the GIP receptor but shows bias at the GLP-1 receptor to favor cAMP generation over ß-arrestin recruitment, coincident with a weaker ability to drive GLP-1 receptor internalization compared with GLP-1. Experiments in primary islets reveal ß-arrestin1 limits the insulin response to GLP-1, but not GIP or tirzepatide, suggesting that the biased agonism of tirzepatide enhances insulin secretion. Imbalance toward GIP receptor, combined with distinct signaling properties at the GLP-1 receptor, together may account for the promising efficacy of this investigational agent.

ß-Arrestin1 is involved in hepatocellular carcinoma metastasis via extracellular signal-regulated kinase-mediated epithelial-mesenchymal transition.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a malignant disease worldwide. It is implicated in high cancer-related mortality rates in humans. ß-Arrestin1 (ARRB1) has been demonstrated to be related to the development of several cancers, while the relationship between ARRB1 and metastasis in HCC is unknown. METHODS: A tissue microarray of 68 tissues from HCC patients with or without metastasis was collected. Wild-type and ARRB1 knockout mice were used to examine the role of ARRB1 in metastasis in vivo. The level of ARRB1 in HCC tissues, mouse liver tissues, and cell lines was determined by quantitative reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry. Migration, invasion, and motility capacities of HCC cells were determined by transwell assay and wound healing assay. Vein injection of nude mice model was used to reveal the metastatic abilities of HCC cell lines. For the mechanism study, we investigated the effects of ARRB1 on the phosphorylation of ERK1/2 and the expression of epithelial-mesenchymal transition (EMT) markers in HCC. RESULTS: We reveal that ARRB1 accelerates metastasis in HCC and that ARRB1 deficiency inhibits hepatocarcinogenesis and reverses EMT in mice. ARRB1 regulates HCC cell migration and invasion and suppresses HCC metastasis in vivo. Furthermore, we show that ARRB1 promotes EMT through the phosphorylation of ERK1/2. CONCLUSIONS: Our data suggest that ARRB1 promotes HCC invasion and metastasis through p-ERK1/2-mediated EMT and that suppression of ARRB1 or p-ERK1/2 may offer potential therapeutic targets for HCC therapy.

ß-Arrestin1-mediated decrease in endoplasmic reticulum stress impairs intestinal stem cell proliferation following radiation.

Gastrointestinal toxicity limits the clinical application of abdominal and pelvic radiotherapy and currently has no effective treatment. Intestinal leucine-rich-repeat-containing GPCR 5 (Lgr5)-positive stem cell depletion and loss of proliferative ability due to radiation may be the primary factors causing intestinal injury following radiation. Here, we report the critical role of ß-arrestin1 (ßarr1) in radiation-induced intestinal injury. Intestinal ßarr1 was highly expressed in radiation enteritis and in a radiation model. ßarr1 knockout (KO) or knockdown mice exhibited increased proliferation in intestinal Lgr5+ stem cell, crypt reproduction, and survival following radiation. Unexpectedly, the beneficial effects of ßarr1 deficiency on intestinal stem cells in response to radiation were compromised when the endoplasmic reticulum stress-related protein kinase RNA-like ER kinase (PERK)/eukaryotic initiation factor-2α (eIF2α) pathway was inhibited, and this result was further supported in vitro. Furthermore, we found that ßarr1 knockdown with small interfering RNA significantly enhanced intestinal Lgr5+ stem cell proliferation after radiation via directly targeting PERK. ßarr1 offers a promising target for mitigating radiation-induced intestinal injury.-Liu, Z., Jiang, J., He, Q., Liu, Z., Yang, Z., Xu, J., Huang, Z., Wu, B. ß-Arrestin1-mediated decrease in endoplasmic reticulum stress impairs intestinal stem cell proliferation following radiation.

PGE2 /EP4 receptor attenuated mucosal injury via ß-arrestin1/Src/EGFR-mediated proliferation in portal hypertensive gastropathy.

BACKGROUND AND PURPOSE: Portal hypertensive gastropathy (PHG) is a serious complication of liver cirrhosis and a potential cause of bleeding in patients with cirrhosis. Suppressed mucosal epithelial proliferation is a crucial pathological characteristic of PHG. Our studies demonstrated an important role for PGE2 and its EP4 receptor in the promotion of mucosal proliferation. However, whether ß-arrestin1 (ß-arr1), a well-established mediator of GPCRs, is involved in the PGE2 /EP4 receptor-mediated mucosal proliferation complex in PHG remains unclear. The aim of the study was to investigate whether ß-arr1 participated in PGE2 /EP4 receptor-mediated mucosal proliferation by recruiting the Src/EGF receptor (EGFR) complex to activate Akt/proliferating cell nuclear antigen (PCNA) signalling in PHG. EXPERIMENTAL APPROACH: Gastric mucosal proliferation was examined in patients with PHG and the PHG model of ß-arr1-knockout (ß-arr1-KO) and ß-arr1-wild type (ß-arr1-WT) mice. The induction of ß-arr1 and EP4 receptor expression and the Src/EGFR signalling elements was investigated, and the mechanisms underlying PGE2 -regulated gastric mucosal proliferation were analysed. KEY RESULTS: Portal hypertension suppressed COX-1 but not COX-2, which was accompanied by a down-regulation of PGE2 generation and EP4 receptor levels in the mucosa of patients with PHG. PGE2 administration markedly promoted mucosal proliferation in a mouse model of PHG. Targeted deletion of ß-arr1 abolished PGE2 /EP4 receptor-mediated gastric proliferation in PHG by repressing the Src/EGFR/Akt/PCNA signalling network. CONCLUSIONS AND IMPLICATIONS: These results indicate that ß-arr1 regulates PGE2 /EP4 receptor-mediated mucosal proliferation by promoting activation of the Src/EGFR/Akt/PCNA signalling pathway, and thus, this network is a potential therapeutic target for PHG.

RalA employs GRK2 and ß-arrestins for the filamin A-mediated regulation of trafficking and signaling of dopamine D2 and D3 receptor.

Filamin A (FLNA) is known to act as platform for the signaling and intracellular trafficking of various GPCRs including dopamine D2 and D3 receptors (D2R, D3R). To understand molecular mechanisms involved in the FLNA-mediated regulation of D2R and D3R, comparative studies were conducted on the signaling and intracellular trafficking of the D2R and D3R in FLNA-knockdown cells, with a specific focus on the roles of the proteins that interact with FLNA and the D2R and D3R. Lowering the level of cellular FLNA caused an elevation in RalA activity and resulted in selective interference with the normal intracellular trafficking and signaling of the D2R and D3R, through GRK2 and ß-arrestins, respectively. Knockdown of FLNA or coexpression of active RalA interfered with the recycling of the internalized D2R and resulted in the development of receptor tolerance. Active RalA was found to interact with GRK2 to sequester it from D2R. Knockdown of FLNA or coexpression of active RalA prevented D3R from coupling with G protein. The selective involvement of GRK2- and ß-arrestins in the RalA-mediated cellular processes of the D2R and D3R was achieved via their different modes of interactions with the receptor and their distinct functional roles in receptor regulation. Our results show that FLNA is a multi-functional protein that acts as a platform on which D2R and D3R can interact with various proteins, through which selective regulation of these receptors occurs in combination with GRK2 and ß-arrestins.