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Neutrophils are not only involved in immune defense against infection but also contribute to the exacerbation of tissue damage after ischemia and reperfusion. We have previously shown that genetic ablation of regulatory Gα i proteins in mice has both protective and deleterious effects on myocardial ischemia reperfusion injury (mIRI), depending on which isoform is deleted. To deepen and analyze these findings in more detail the contribution of Gα i2 proteins in resident cardiac vs circulating blood cells for mIRI was first studied in bone marrow chimeras. In fact, the absence of Gα i2 in all blood cells reduced the extent of mIRI (22,9% infarct size of area at risk (AAR) Gnai2 -/-  → wt vs 44.0% wt → wt; p < 0.001) whereas the absence of Gα i2 in non-hematopoietic cells increased the infarct damage (66.5% wt → Gnai2 -/- vs 44.0% wt → wt; p < 0.001). Previously we have reported the impact of platelet Gα i2 for mIRI. Here, we show that infarct size was substantially reduced when Gα i2 signaling was either genetically ablated in neutrophils/macrophages using LysM-driven Cre recombinase (AAR: 17.9% Gnai2 fl/fl LysM-Cre +/tg vs 42.0% Gnai2 fl/fl ; p < 0.01) or selectively blocked with specific antibodies directed against Gα i2 (AAR: 19.0% (anti-Gα i2 ) vs 49.0% (IgG); p < 0.001). In addition, the number of platelet-neutrophil complexes (PNCs) in the infarcted area were reduced in both, genetically modified (PNCs: 18 (Gnai2 fl/fl ; LysM-Cre +/tg ) vs 31 (Gnai2 fl/fl ); p < 0.001) and in anti-Gα i2 antibody-treated (PNCs: 9 (anti-Gα i2 ) vs 33 (IgG); p < 0.001) mice. Of note, significant infarct-limiting effects were achieved with a single anti-Gα i2 antibody challenge immediately prior to vessel reperfusion without affecting bleeding time, heart rate or cellular distribution of neutrophils. Finally, anti-Gα i2 antibody treatment also inhibited transendothelial migration of human neutrophils (25,885 (IgG) vs 13,225 (anti-Gα i2 ) neutrophils; p < 0.001), collectively suggesting that a therapeutic concept of functional Gα i2 inhibition during thrombolysis and reperfusion in patients with myocardial infarction should be further considered., (© 2024. The Author(s).)

MiRNAs are related to neuronal proliferation and apoptosis following cerebral ischemia-reperfusion injury (CIRI). This study focused on miR-30c-5p in the disease. An oxygen-glucose deprivation/re-oxygenation (OGD/R) model was prepared in HT22 cells and transfected to overexpress miR-30c-5p and G Protein Subunit Alpha I2 (GNAI2) respectively or co-transfected to silence miR-30c-5p and GNAI2. Meanwhile, a middle cerebral artery occlusion (MCAO) model was constructed in mice, and miR-30c-5p and GNAI2 were silenced in vivo simultaneously. The mice were evaluated for neurological damage, apoptosis, and inflammation. HT22 cells were tested for cytotoxicity, proliferation, apoptosis, and inflammatory factors. The interaction between miR-30c-5p and GNAI2 was predicted, analyzed, and confirmed. MiR-30c-5p was found to be downregulated in both experimental models. miR-30c-5p reduced lactate dehydrogenase production, inflammatory response, inhibit apoptosis, and enhanced neuronal proliferation, while GNAI2 overexpression showed the opposite results. Downregulated miR-30c-5p worsened neurological function, apoptosis, and inflammation of MCAO mice while silencing GNAI2 attenuated the influence of downregulated miR-30c-5p. MiR-30c-5p can improve neuronal apoptosis and inflammatory response caused by CIRI and is neuroprotective by targeting GNAI2, providing a new target for treating CIRI., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Highly homologous members of the Gα i family, Gα i1-3 , have distinct tissue distributions and physiological functions, yet their biochemical and functional properties are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gα i1 effector that is poorly activated by Gα i2 . In a proteomic proximity labeling screen we observed a strong preference for Gα i1 relative to Gα i2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gα i1 into Gα i2 or substitution of a single amino acid, A230 in Gα i2 with the corresponding D in Gα i1 , largely rescues PRG activation and interactions with other potential Gα i targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HD-Ras-like domain (RLD) interface is more pronounced in Gα i2 than Gα i1 . Mutation of A230 to D in Gα i2 stabilizes HD-RLD interactions via ionic interactions with R145 in the HD which in turn modify the conformation of Switch III. These data support a model where D229 in Gα i1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gα i2 is unable to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gα i1 or Gα i2 by G protein-coupled receptors., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3, and GNAO proteins, but may also induce unrelated defects. Here, we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner G protein signaling modulator 2 (GPSM2), whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the sub-cellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: (1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and (2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2., Competing Interests: AJ, AT, MD, BK, BL, MW, BT No competing interests declared, (© 2023, Jarysta et al.)

Here we explored the potential role of Gαi2 (G protein subunit alpha i2) in endothelial cell function and angiogenesis. Methods: Genetic methodologies such as shRNA, CRISPR/Cas9, dominant negative mutation, and overexpression were utilized to modify Gαi2 expression or regulate its function. Their effects on endothelial cell functions were assessed in vitro . In vivo , the endothelial-specific Gαi2 shRNA adeno-associated virus (AAV) was utilized to silence Gαi2 expression. The impact of this suppression on retinal angiogenesis in control mice and streptozotocin (STZ)-induced diabetic retinopathy (DR) mice was analyzed. Results: Analysis of single-cell RNA sequencing data revealed Gαi2 ( GNAI2 ) was predominantly expressed in retinal endothelial cells and expression was increased in retinal endothelial cells following oxygen-induced retinopathy (OIR) in mice. Moreover, transcriptome analysis linking Gαi2 to angiogenesis-related processes/pathways, supported by increased Gαi2 expression in experimental OIR mouse retinas, highlighted its possible role in angiogenesis. In various endothelial cell types, shRNA-induced silencing and CRISPR/Cas9-mediated knockout (KO) of Gαi2 resulted in substantial reductions in cell proliferation, migration, invasion, and capillary tube formation. Conversely, Gαi2 over-expression in endothelial cells induced pro-angiogenic activities, enhancing cell proliferation, migration, invasion, and capillary tube formation. Furthermore, our investigation revealed a crucial role of Gαi2 in NFAT (nuclear factor of activated T cells) activation, as evidenced by the down-regulation of NFAT-luciferase reporter activity and pro-angiogenesis NFAT-targeted genes ( Egr3 , CXCR7 , and RND1 ) in Gαi2-silenced or -KO HUVECs, which were up-regulated in Gαi2-overexpressing endothelial cells. Expression of a dominant negative Gαi2 mutation (S48C) also down-regulated NFAT-targeted genes, slowing proliferation, migration, invasion, and capillary tube formation in HUVECs. Importantly, in vivo experiments revealed that endothelial Gαi2 knockdown inhibited retinal angiogenesis in mice, with a concomitant down-regulation of NFAT-targeted genes in mouse retinal tissue. In contrast, Gαi2 over-expression in endothelial cells enhanced retinal angiogenesis in mice. Single-cell RNA sequencing data confirmed increased levels of Gαi2 specifically in retinal endothelial cells of mice with streptozotocin (STZ)-induced diabetic retinopathy (DR). Importantly, endothelial Gαi2 silencing ameliorated retinal pathological angiogenesis in DR mice. Conclusion: Our study highlights a critical role for Gαi2 in NFAT activation, endothelial cell activation and angiogenesis, offering valuable insights into potential therapeutic strategies for modulating these processes., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: Although the G protein subunit α i2 (GNAI2) is upregulated in multiple cancers, its prognostic value and exact role in the development of gastric cancer (GC) remain largely unknown., Methods: This study evaluated the effect of GNAI2 on the tumor microenvironment (TME) in GC, constructed an immune risk score (IRS) model based on differentially-expressed immune genes, and systematically correlated GNAI2 and epigenetic factor expression patterns with TME and IRS. Also, RT-qPCR, flow cytometry, Western blotting (WB), and transwell assays were carried out to explore the regulatory mechanism of GNAI2 in GC., Results: High GNAI2 expression was associated with poor prognosis. Cytokine activation, an increase in tumor-infiltrating immune cells (TIIC), and the accumulation of regulatory T cells in the tumor immune cycle were all promoted by the TME, which was significantly associated with GNAI2 expression. Two different differentially expressed mRNA (DER) modification patterns were determined. These two DERs-clusters had significantly different TME cell infiltrations and were classified as either noninflamed or immune-inflamed phenotypes. The IRS model constructed using differentially expressed genes (DEGs) had great potential in predicting GC prognosis. The IRS model was also used in assessing clinicopathological features, such as microsatellite instability (MSI) status, epithelial-mesenchymal transition (EMT) status, clinical stages, tumor mutational burden (TMB), and tumor immune dysfunction and exclusion (TIDE) scores. Low IRS scores were associated with high immune checkpoint gene expression. Cell and animal studies confirmed that GNAI2 activated PI3K/AKT pathway and promoted the growth and migration of GC cells., Conclusion: The IRS model can be used for survival prediction and GNAI2 serves as a candidate therapeutic target for GC patients., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Han Yu et al.)

Background : Intervertebral disc degeneration (IDD), the main cause of low back pain, is closely related to the inflammatory microenvironment in the nucleus pulposus (NP). Tumor necrosis factor-α (TNF-α) plays an important role in inflammation-related metabolic disturbance of NP cells. Melatonin has been proven to regulate the metabolism of NP cells, but whether it can protect NP cells from TNF-α-induced damage is still unclear. Therefore, this study aims to investigate the role and specific mechanism of melatonin on regulating the metabolism of NP cells in the inflammatory microenvironment. Methods: Western blotting, RT-qPCR and immunohistochemistry were used to detect the expression of melatonin membrane receptors (MTNR1A/B) and TNF-α in human NP tissues. In vitro , human primary NP cells were treated with or without vehicle, TNF-α and melatonin. And the metabolic markers were also detected by western blotting and RT-qPCR. The activity of NF-κB signaling and Hippo/YAP signaling were assessed by western blotting and immunofluorescence. Membrane receptors inhibitors, pathway inhibitors, lentiviral infection, plasmids transfection and immunoprecipitation were used to explore the specific mechanism of melatonin. In vivo , the rat IDD model was constructed and melatonin was injected intraperitoneally to evaluate its therapeutical effect on IDD. Results: The upregulation of TNF-α and downregulation of melatonin membrane receptors (MTNR1A/B) were observed in degenerative NP tissues. Then we demonstrated that melatonin could alleviate the development of IDD in a rat model and reverse TNF-α-impaired metabolism of NP cells in vitro . Further investigation revealed that the protective effects of melatonin on NP cells mainly rely on MTNR1B, which subsequently activates Gαi2 protein. The activation of Gαi2 could upregulate the yes-associated protein (YAP) level, resulting in anabolic enhancement of NP cells. In addition, melatonin-mediated YAP upregulation increased the expression of IκBα and suppressed the TNF-α-induced activation of the NF-κB pathway, thereby inhibiting the catabolism of NP cells. Conclusions: Our results revealed that melatonin can reverse TNF-α-impaired metabolism of NP cells via the MTNR1B/Gαi2/YAP axis and suggested that melatonin can be used as a potential therapeutic drug in the treatment of IDD., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Conductin/axin2 is a scaffold protein negatively regulating the pro-proliferative Wnt/β-catenin signaling pathway. Accumulation of scaffold proteins in condensates frequently increases their activity, but whether condensation contributes to Wnt pathway inhibition by conductin remains unclear. Here, we show that the Gαi2 subunit of trimeric G-proteins induces conductin condensation by targeting a polymerization-inhibiting aggregon in its RGS domain, thereby promoting conductin-mediated β-catenin degradation. Consistently, transient Gαi2 expression inhibited, whereas knockdown activated Wnt signaling via conductin. Colorectal cancers appear to evade Gαi2-induced Wnt pathway suppression by decreased Gαi2 expression and inactivating mutations, associated with shorter patient survival. Notably, the Gαi2-activating drug guanabenz inhibited Wnt signaling via conductin, consequently reducing colorectal cancer growth in vitro and in mouse models. In summary, we demonstrate Wnt pathway inhibition via Gαi2-triggered conductin condensation, suggesting a tumor suppressor function for Gαi2 in colorectal cancer, and pointing to the FDA-approved drug guanabenz for targeted cancer therapy., (© 2022. The Author(s).)

Background and Aims: NASH is an advanced stage of liver disease accompanied by lipid accumulation, inflammation, and liver fibrosis. Guanine nucleotide-binding protein G(i) subunit alpha-2 (GNAI2) is a member of the "inhibitory" class of α-subunits, and recent studies showed that Gnai2 deficiency is known to cause reduced weight in mice. However, the role of GNAI2 in hepatocytes, particularly in the context of liver inflammation and lipid metabolism, remains to be elucidated. Herein, we aim to ascertain the function of GNAI2 in hepatocytes and its impact on the development of NASH., Approach and Results: Human liver tissues were obtained from NASH patients and healthy persons to evaluate the expression and clinical relevance of GNAI2. In addition, hepatocyte-specific Gnai2-deficient mice (Gnai2 hep-/- ) were fed either a Western diet supplemented with fructose in drinking water (WDF) for 16 weeks or a methionine/choline-deficient diet (MCD) for 6 weeks to investigate the regulatory role and underlying mechanism of Gnai2 in NASH. GNAI2 was significantly up-regulated in liver tissues of patients with NASH. Following feeding with WDF or MCD diets, livers from Gnai2 hep-/- mice had reduced steatohepatitis with suppression of markers of inflammation and an increase in lipophagy compared to Gnai2 flox/flox mice. Toll-like receptor 4 signals through nuclear factor kappa B to trigger p65-dependent transcription of Gnai2. Intriguingly, immunoprecipitation, immunofluorescence, and mass spectrometry identified peroxiredoxin 1 (PRDX1) as a binding partner of GNAI2. Moreover, the function of PRDX1 in the suppression of TNF receptor-associated factor 6 ubiquitin-ligase activity and glycerophosphodiester phosphodiesterase domain-containing 5-related phosphatidylcholine metabolism was inhibited by GNAI2. Suppression of GNAI2 combined with overexpression of PRDX1 reversed the development of steatosis and fibrosis in vivo., Conclusions: GNAI2 is a major regulator that leads to the development of NASH. Thus, inhibition of GNAI2 could be an effective therapeutic target for the treatment of NASH., (© 2021 by the American Association for the Study of Liver Diseases.)

The adenosine A 1 receptor (A 1 R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain 1,2 . However, development of analgesic orthosteric A 1 R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects 3 . Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A 1 R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A 1 R co-bound to adenosine, MIPS521 and a G i2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also called satellite cells, SCs). SCs are normally quiescent; when stimulated by damage, they activate and expand to form new fibers. The mechanisms underlying SC proliferative progression remain poorly understood. Here we show that DHX36, a helicase that unwinds RNA G-quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. DHX36 (initially named RHAU) is barely expressed at quiescence but is highly induced during SC activation and proliferation. Inducible deletion of Dhx36 in adult SCs causes defective proliferation and muscle regeneration after damage. System-wide mapping in proliferating SCs reveals DHX36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling shows that DHX36 promotes mRNA translation via 5'-untranslated region (UTR) rG4 binding. Furthermore, we demonstrate that DHX36 specifically regulates the translation of Gnai2 mRNA by unwinding its 5' UTR rG4 structures and identify GNAI2 as a downstream effector of DHX36 for SC expansion. Altogether, our findings uncover DHX36 as an indispensable post-transcriptional regulator of SC function and muscle regeneration acting through binding and unwinding rG4 structures at 5' UTR of target mRNAs., (© 2021. The Author(s).)

Increased expression of GNAi2 , which encodes the α-subunit of G-protein i2, has been correlated with the late-stage progression of ovarian cancer. GNAi2 , also referred to as the proto-oncogene gip2 , transduces signals from lysophosphatidic acid (LPA)-activated LPA-receptors to oncogenic cellular responses in ovarian cancer cells. To identify the oncogenic program activated by gip2 , we carried out micro-array-based transcriptomic and bioinformatic analyses using the ovarian cancer cell-line SKOV3, in which the expression of GNAi2 / gip2 was silenced by specific shRNA. A cut-off value of 5-fold change in gene expression ( p < 0.05) indicated that a total of 264 genes were dependent upon gip2 -expression with 136 genes coding for functional proteins. Functional annotation of the transcriptome indicated the hitherto unknown role of gip2 in stimulating the expression of oncogenic/growth-promoting genes such as KDR/VEGFR2, CCL20, and VIP. The array results were further validated in a panel of High-Grade Serous Ovarian Carcinoma (HGSOC) cell lines that included Kuramochi, OVCAR3, and OVCAR8 cells. Gene set enrichment analyses using DAVID, STRING, and Cytoscape applications indicated the potential role of the gip2 -stimulated transcriptomic network involved in the upregulation of cell proliferation, adhesion, migration, cellular metabolism, and therapy resistance. The results unravel a multi-modular network in which the hub and bottleneck nodes are defined by ACKR3/CXCR7, IL6, VEGFA, CYCS, COX5B, UQCRC1, UQCRFS1, and FYN. The identification of these genes as the critical nodes in GNAi2/gip2 orchestrated onco-transcriptome establishes their role in ovarian cancer pathophysiology. In addition, these results also point to these nodes as potential targets for novel therapeutic strategies.

Background: Alpha 2A -adrenergic (α 2A -AR) and cannabinoid CB 1 (CB 1 -R) receptors exert their functions modulating multiple signaling pathways, including MEK-ERK (extracellular signal-regulated kinases) and FADD (Fas-associated protein with death domain) cascades. These molecules are relevant in finding biased agonists with fewer side effects, but the mechanisms involving their modulations by α 2A -AR- and CB 1 -R in vivo are unclear. This study investigated the roles of Gαi 2 and Gαz proteins in mediating α 2A -AR- and CB 1 -R-induced alterations of MEK-ERK and FADD phosphorylation (p-) in mouse brain cortex., Methods: Gαi 2 or Gαz protein knockdown was induced in mice with selective antisense oligodeoxinucleotides (ODNs; 3 nmol/day, 5 days) prior to UK-14,304 (UK or brimonidine; 1 mg/kg) or WIN55212-2 (WIN; 8 mg/kg) acute treatments. Inactivated (p-T 286 ) MEK1, activated (p-S 217/221 ) MEK1/2, activated (p-T 202 /Y 204 ) ERK1/2, p-S 191 FADD, and the corresponding total forms of these proteins were quantified by immunoblotting., Results: Increased (+ 88%) p-T 286 MEK1 cortical density, with a concomitant reduction (-43%) of activated ERK was observed in UK-treated mice. Both effects were attenuated by Gαi 2 or Gαz antisense ODNs. Contrastingly, WIN induced Gαi 2 - and Gαz-independent upregulations of p-T 286 MEK1 (+ 63%), p-S 217/221 MEK1/2 (+ 86%), and activated ERK (+ 111%) in brain. Pro-apoptotic FADD was downregulated (- 34 to 39%) following UK and WIN administration, whereas the neuroprotective p-S 191 FADD was increased (+ 74%) in WIN-treated mice only. None of these latter effects required from Gαi 2 or Gαz protein integrity., Conclusion: The results indicate that α 2A -AR (UK), but not CB 1 -R (WIN), agonists use Gαi 2 and Gαz proteins to modulate MEK-ERK, but not FADD, pathway in mouse brain cortex., (© 2021. Maj Institute of Pharmacology Polish Academy of Sciences.)

GNAI2 (G protein subunit alpha i2) is a signaling modulator or transducer, involved in several transmembrane signaling systems, that plays a vital role in the melanogenesis signaling pathway. However, whether GNAI2 regulates cell proliferation and apoptosis in rabbit melanocytes is not known. We found that GNAI2 was differentially expressed in rabbits with different coat colors using qRT-PCR and Wes assays. Furthermore, it was observed that the rabbits with black skin had the highest GNAI2 levels, and those with white skin had the lowest expression. The coding sequence of GNAI2 was successfully cloned and inserted into pcDNA3.1 and pcDNA3.1-Myc vectors. It was observed that the GNAI2 protein was mainly localized in the cytoplasm using the indirect immunofluorescence staining assay. Overexpression of GNAI2 significantly increased melanin content, promoted melanocyte proliferation, and inhibited melanocyte apoptosis. On the contrary, the knockdown of GNAI2 using siRNA had the opposite effect. In addition, GNAI2 significantly increased the mRNA expression levels of the melanin-related genes TYR, GPNMB, PMEL, and DCT in rabbit melanocytes. The results suggested that GNAI2 regulated melanocyte development by promoting melanocyte proliferation and inhibiting apoptosis.

Atherosclerosis-associated cardiovascular disease is one of the main causes of death and disability among patients with diabetes mellitus. However, little is known about the impact of S-nitrosylation in diabetes-accelerated atherosclerosis. Here, we show increased levels of S-nitrosylation of guanine nucleotide-binding protein G(i) subunit alpha-2 (SNO-GNAI2) at Cysteine 66 in coronary artery samples from diabetic patients with atherosclerosis, consistently with results from mice. Mechanistically, SNO-GNAI2 acted by coupling with CXCR5 to dephosphorylate the Hippo pathway kinase LATS1, thereby leading to nuclear translocation of YAP and promoting an inflammatory response in endothelial cells. Furthermore, Cys-mutant GNAI2 refractory to S-nitrosylation abrogated GNAI2-CXCR5 coupling, alleviated atherosclerosis in diabetic mice, restored Hippo activity, and reduced endothelial inflammation. In addition, we showed that melatonin treatment restored endothelial function and protected against diabetes-accelerated atherosclerosis by preventing GNAI2 S-nitrosylation. In conclusion, SNO-GNAI2 drives diabetes-accelerated atherosclerosis by coupling with CXCR5 and activating YAP-dependent endothelial inflammation, and reducing SNO-GNAI2 is an efficient strategy for alleviating diabetes-accelerated atherosclerosis., (© 2021. The Author(s).)

Neutrophil trafficking, homeostatic and pathogen elicited, depends upon chemoattractant receptors triggering heterotrimeric G-protein Gα i β γ signaling, whose magnitude and kinetics are governed by RGS protein/Gα i interactions. RGS proteins typically limit Gα i signaling by reducing the duration that Gα i subunits remain GTP bound and able to activate downstream effectors. Yet how in totality RGS proteins shape neutrophil chemoattractant receptor activated responses remains unclear. Here, we show that C57Bl/6 mouse neutrophils containing a genomic knock-in of a mutation that disables all RGS protein-Gα i2 interactions (G184S) cannot properly balance chemoattractant receptor signaling, nor appropriately respond to inflammatory insults. Mutant neutrophils accumulate in mouse bone marrow, spleen, lung, and liver; despite neutropenia and an intrinsic inability to properly mobilize from the bone marrow. In vitro they rapidly adhere to ICAM-1 coated plates, but in vivo they poorly adhere to blood vessel endothelium. Those few neutrophils that cross blood vessels and enter tissues migrate haphazardly. Following Concanavalin-A administration fragmented G184S neutrophils accumulate in liver sinusoids leading to thrombo-inflammation and perivasculitis. Thus, neutrophil Gα i2 /RGS protein interactions both limit and facilitate Gα i2 signaling thereby promoting normal neutrophil trafficking, aging, and clearance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yan, Hwang, Kamenyeva, Kabat, Kim, Park and Kehrl.)

The mu opioid receptor (MOR) is a G protein-coupled receptor that plays an essential role in reward and hedonic processes, and that has been implicated in disorders such as depression and addiction. Over the last decade, several brain imaging studies in depressed patients have consistently found that dysregulation of MOR function occurs in particular in the anterior insular cortex, an important brain site for the perception of internal states and emotional regulation. To investigate molecular mechanisms that may underlie these effects, here we assessed genetic polymorphisms, expression, and functional G-protein coupling of MOR in a large post-mortem cohort (N = 95) composed of depressed individuals who died by suicide, and healthy controls. Results indicated that depression, but not comorbid substance use disorder or acute opiate consumption, was associated with increased MOR activity. This effect was partly explained by a specific increase in expression of the inhibitory alpha G-protein subunit GNAI2. Consistent with previous neuroimaging studies, our findings support the notion that enhanced endogenous opioidergic tone in the anterior insula may buffer negative affective states in depressed individuals, a mechanism that could potentially contribute to the antidepressant efficacy of emerging opioid-based medications.

Mice carrying an RGS-insensitive Gαi2 mutation display growth retardation early after birth. Although the growth hormone (GH)-axis is a key endocrine modulator of postnatal growth, its functional state in these mice has not been characterized. The present study was undertaken to address this issue. Results revealed that pituitary mRNA levels for GH, prolactin (PRL), somatostatin (SST), GH-releasing-hormone receptor (GHRH-R) and GH secretagogue receptor (GHS-R) were decreased in mutants compared to controls. These changes were reflected by a significant decrease in plasma levels of GH, IGF-1 and IGF-binding protein-3 (IGFBP-3). Mutants were also less responsive to GHRH and ghrelin (GhL) on GH stimulation of release from pituitary primary cell cultures. In contrast, they were more sensitive to the inhibitory effect of SST. These data provide the first evidence for an alteration of the functional state of the GH-axis in Gαi2G184S mice that likely contributes to their growth retardation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hypertension is a global health burden. The hypothalamic paraventricular nucleus (PVN) is an essential component of the neuronal network that regulates sodium homeostasis and blood pressure (BP). Previously, we have shown PVN-specific G protein-coupled receptor-coupled Gαi 2 subunit proteins are essential to counter the development of salt-sensitive hypertension by mediating the sympathoinhibitory and natriuretic responses to increased dietary sodium intake to maintain sodium homeostasis and normotension. However, the cellular localization and identity of PVN Gαi 2 -expressing neurons are currently unknown. In this study using in situ hybridization, we determined the neuroanatomical characterization of Gαi 2 -expressing PVN neurons in 3-mo-old male and female Sprague-Dawley rats. We observed that Gαi 2 -expressing neurons containing Gnai2 mRNA are highly localized in the parvocellular region of the hypothalamic PVN. At level 2 of the hypothalamic PVN, Gnai2 mRNA colocalized with ∼ 85% of GABA-expressing neurons and ∼28% of glutamatergic neurons. Additionally, within level 2 Gnai2 mRNA colocalized with ∼75% of corticotrophin-releasing hormone PVN neurons. Gnai2 neurons had lower colocalization with tyrosine hydroxylase (∼33%)-, oxytocin (∼6%)-, and arginine vasopressin (∼10%)-expressing parvocellular neurons in level 2 PVN. Colocalization was similar among male and female rats. The high colocalization of Gnai2 mRNA with GABAergic neurons, in conjunction with our previous findings that PVN Gαi 2 proteins mediate sympathoinhibition, suggests that Gαi 2 proteins potentially modulate GABAergic signaling to impact sympathetic outflow and BP.

Objectives: Typically, obesity results from an inappropriate balance between energy uptake from nutrient consumption and burning of calories, which leads to a pathological increase in fat mass. Obesity is a major cause of insulin resistance and diabetes. Inhibitory G proteins (Gα i ) form a subfamily that is involved in the regulation of adipose tissue function. Among the three Gα i members, i.e. Gα i1 , Gα i2 , Gα i3 , the Gα i2 , protein is predominantly expressed in adipose tissue. However, the functions of the Gα i2 isoform in adipose tissue and its impact on the development of obesity are poorly understood., Methods: By using AdipoqCreER T2 mice, we generated adipocyte-specific Gnai2-deficient mice to study Gα i2 function, specifically in white and brown adipocytes. These mice were fed either a control diet (CD) or a high fat diet (HFD). Mice were examined for obesity development, insulin resistance and glucose intolerance. We examined adipocyte morphology and the development of inflammation in the white adipose tissue. Finally, intracellular cAMP levels as an indicator of Gα i signaling and glycerol release as an indicator of lipolysis rates were measured to verify the impact of Gα i2 on the signaling pathway in brown and white adipocytes., Results: An adipocyte-specific deficiency of Gα i2 significantly reduced diet-induced obesity, leading to decreased fat masses, smaller adipocytes and decreased inflammation in the white adipose tissue relative to littermate controls. Concurrently, oxygen consumption of brown adipocytes and in vivo measured energy expenditure were significantly enhanced. In addition, glucose tolerance and insulin sensitivity of HFD-fed adipocyte-specific Gnai2-deficient mice were improved compared to the respective controls. In the absence of Gα i2 , adrenergic stimulation of intracellular adipocyte cAMP levels was increased, which correlated with increased lipolysis and energy expenditure., Conclusion: We conclude that adipocyte Gα i2 is a major regulator of adipocyte lipid content in diet-induced obesity by inhibiting adipocyte lipolysis in a cAMP-dependent manner resulting in increased energy expenditure., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

We have previously reported that in salt-resistant rat phenotypes brain, Gαi 2 (guanine nucleotide-binding protein alpha inhibiting activity polypeptide 2) proteins are required to maintain blood pressure and sodium balance. However, the impact of hypothalamic paraventricular nucleus (PVN) Gαi 2 proteins on the salt sensitivity of blood pressure is unknown. Here, by the bilateral PVN administration of a targeted Gαi 2 oligodeoxynucleotide, we show that PVN-specific Gαi 2 proteins are required to facilitate the full natriuretic response to an acute volume expansion (peak natriuresis [μeq/min] scrambled (SCR) oligodeoxynucleotide 41±3 versus Gαi 2 oligodeoxynucleotide 18±4; P <0.05) via a renal nerve-dependent mechanism. Furthermore, in response to chronically elevated dietary sodium intake, PVN-specific Gαi 2 proteins are essential to counter renal nerve-dependent salt-sensitive hypertension (mean arterial pressure [mm Hg] 8% NaCl; SCR oligodeoxynucleotide 128±2 versus Gαi 2 oligodeoxynucleotide 147±3; P <0.05). This protective pathway involves activation of PVN Gαi 2 signaling pathways, which mediate sympathoinhibition to the blood vessels and kidneys (renal norepinephrine [pg/mg] 8% NaCl; SCR oligodeoxynucleotide 375±39 versus Gαi 2 oligodeoxynucleotide 850±27; P <0.05) and suppression of the activity of the sodium chloride cotransporter assessed as peak natriuresis to hydrochlorothiazide. Additionally, central oligodeoxynucleotide-mediated Gαi 2 protein downregulation prevented PVN parvocellular neuron activation, assessed by FosB immunohistochemistry, in response to increased dietary salt intake. In our analysis of the UK BioBank data set, it was observed that 2 GNAI2 single nucleotide polymorphism (SNP) (rs2298952, P =0.041; rs4547694, P =0.017) significantly correlate with essential hypertension. Collectively, our data suggest that selective targeting and activation of PVN Gαi 2 proteins is a novel therapeutic approach for the treatment of salt-sensitive hypertension.

Pheromone detection by the vomeronasal organ (VNO) mediates important social behaviors across different species, including aggression and sexual behavior. However, the relationship between vomeronasal function and social hierarchy has not been analyzed reliably. We evaluated the role of pheromone detection by receptors expressed in the apical layer of the VNO such as vomeronasal type 1 receptors (V1R) in dominance behavior by using a conditional knockout mouse for G protein subunit Gαi2, which is essential for V1R signaling. We used the tube test as a model to analyze the within-a-cage hierarchy in male mice, but also as a paradigm of novel territorial competition in animals from different cages. In absence of prior social experience, Gαi2 deletion promotes winning a novel social competition with an unfamiliar control mouse but had no effect on an established hierarchy in cages with mixed genotypes, both Gαi2 -/- and controls. To further dissect social behavior of Gαi2 -/- mice, we performed a 3-chamber sociability assay and found that mutants had a slightly altered social investigation. Finally, gene expression analysis in the medial prefrontal cortex (mPFC) for a subset of genes previously linked to social status revealed no differences between group-housed Gαi2 -/- and controls. Our results reveal a direct influence of pheromone detection on territorial dominance, indicating that olfactory communication involving apical VNO receptors like V1R is important for the outcome of an initial social competition between two unfamiliar male mice, whereas final social status acquired within a cage remains unaffected. These results support the idea that previous social context is relevant for the development of social hierarchy of a group. Overall, our data identify two context-dependent forms of dominance, acute and chronic, and that pheromone signaling through V1R receptors is involved in the first stages of a social competition but in the long term is not predictive for high social ranks on a hierarchy.

Cancer stem cells (CSCs) exist in colon cancer and exhibit characteristics of stem cells which are due to lineages of tissues where they arise. Epithelial to mesenchymal transition (EMT)-undergoing cancer cells display CSC properties and therapeutic resistance. Cancer and stromal cells comprise of a tumor microenvironment. One way the two populations communicate with each other is to secret CXC ligands (CXCLs). CXCLs are capable of causing chemotaxis of specific types of stromal cells and control angiogenesis. Double immunofluorescence, western blot analysis, and colony-formation assay were carried out to compare parental and CPT-11-resistant LoVo cells. CPT-11-R LoVo colon cancer cells showed increased expression of CXCL1, CXCL2, CXCL3, and CXCL8. They displayed significantly increased intracellular protein levels of CXCL2 and CXCR2. CPT-11-R LoVo cells showed significantly elevated expression in aldehyde dehydrogenase 1 (ALDH1), cluster of differentiation 24 (CD24), cluster of differentiation 44 (CD44), and epithelial cell adhesion molecule (EpCAM). CXCL2 knockdown by short hairpin RNA resulted in reduced expression of CSC proteins, cyclins, EMT markers, G proteins, and matrix metalloproteinases (MMPs). Finally, Gαi-2 was found to promote expression of CSC genes and tumorigenesis which were more apparent in the resistant cells. In addition, Gαq/11 showed a similar pattern with exceptions of EpCAM and MMP9. Therefore, CXCL2-CXCR2 axis mediates through Gαi-2 and Gαq/11 to promote tumorigenesis and contributes to CSC properties of CPT-11-R LoVo cells., (© 2018 Wiley Periodicals, Inc.)

Hepatic ischemia-reperfusion (I/R) injury is a major challenge in liver resection and transplantation surgeries. Previous studies have revealed that guanine nucleotide-binding protein G(i)α2 (GNAI2) was involved in the progression of myocardial and cerebral I/R injury, but the role and function of GNAI2 in hepatic I/R have not been elucidated. The hepatocyte-specific GNAI2 knockout (GNAI2 hep-/- ) mice were generated and subjected to hepatic I/R injury. Primary hepatocytes isolated from GNAI2 hep-/- and GNAI2 flox/flox mice were cultured and challenged to hypoxia-reoxygenation insult. The specific function of GNAI2 in I/R-triggered hepatic injury and the underlying molecular mechanism were explored by various phenotypic analyses and molecular biology methods. In this study, we demonstrated that hepatic GNAI2 expression was significantly increased in liver transplantation patients and wild-type mice after hepatic I/R. Interestingly, hepatocyte-specific GNAI2 deficiency attenuated I/R-induced liver damage, inflammation cytokine expression, macrophage/neutrophil infiltration, and hepatocyte apoptosis in vivo and in vitro . Mechanistically, up-regulation of GNAI2 phosphorylates mixed-lineage protein kinase 3 (MLK3) through direct binding, which exacerbated hepatic I/R damage via MAPK and NF-κB pathway activation. Furthermore, blocking MLK3 signaling reversed GNAI2-mediated hepatic I/R injury. Our study firstly identifies GNAI2 as a promising target for prevention of hepatic I/R-induced injury and related liver diseases.-Sun, Q., He, Q., Xu, J., Liu, Q., Lu, Y., Zhang, Z., Xu, X., Sun, B. Guanine nucleotide-binding protein G(i)α2 aggravates hepatic ischemia-reperfusion injury in mice by regulating MLK3 signaling.

The transduction compartment of inner ear hair cells, the hair bundle, is composed of stereocilia rows of graded height, a property essential for sensory function that remains poorly understood at the molecular level. We previously showed that GPSM2-GNAI is enriched at stereocilia distal tips and required for their postnatal elongation and bundle morphogenesis-two characteristics shared with MYO15A (short isoform), WHRN, and EPS8 proteins. Here we first performed a comprehensive genetic analysis of the mouse auditory epithelium to show that GPSM2, GNAI, MYO15A, and WHRN operate in series within the same pathway. To understand how these functionally disparate proteins act as an obligate complex, we then systematically analyzed their distribution in normal and mutant bundles over time. We discovered that WHRN-GPSM2-GNAI is an extra module recruited by and added to a pre-existing MYO15A-EPS8 stereocilia tip complex. This extended complex is only present in the first, tallest row, and is required to stabilize larger amounts of MYO15A-EPS8 than in shorter rows, which at tips harbor only MYO15A-EPS8. In the absence of GPSM2 or GNAI function, including in the epistatic Myo15a and Whrn mutants, bundles retain an embryonic-like organization that coincides with generic stereocilia at the molecular level. We propose that GPSM2-GNAI confers on the first row its unique tallest identity and participates in generating differential row identity across the hair bundle., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aggression is controlled by the olfactory system in many animal species. In male mice, territorial and infant-directed aggression are tightly regulated by the vomeronasal organ (VNO), but how diverse subsets of sensory neurons convey pheromonal information to limbic centers is not yet known. Here, we employ genetic strategies to show that mouse vomeronasal sensory neurons expressing the G protein subunit Gαi2 regulate male-male and infant-directed aggression through distinct circuit mechanisms. Conditional ablation of Gαi2 enhances male-male aggression and increases neural activity in the medial amygdala (MeA), bed nucleus of the stria terminalis, and lateral septum. By contrast, conditional Gαi2 ablation causes reduced infant-directed aggression and decreased activity in MeA neurons during male-infant interactions. Strikingly, these mice also display enhanced parental behavior and elevated neural activity in the medial preoptic area, whereas sexual behavior remains normal. These results identify Gαi2 as the primary G protein α-subunit mediating the detection of volatile chemosignals in the apical layer of the VNO, and they show that Gαi2 + VSNs and the brain circuits activated by these neurons play a central role in orchestrating and balancing territorial and infant-directed aggression of male mice through bidirectional activation and inhibition of different targets in the limbic system., Competing Interests: The authors declare no conflict of interest.

A single base mutation in the Gα i2 protein (G184S) renders this Gα subunit insensitive to the negative modulatory effects of Regulator of G-protein Signaling (RGS) proteins. Mice expressing this RGS insensitive (RGSi) variant of Gα i2 (RGSi Gα i2 ) display a spontaneous antidepressant-like phenotype that is reversible by treatment with the 5-HT1A receptor (5-HT1AR) antagonist WAY100635. Here we test the hypothesis that increased activity of 5-HT1ARs in the hippocampus of RGSi Gα i2 knock-in mice is responsible for the expression of the observed antidepressant-like behavior. We administered the 5-HT1AR antagonist WAY100635 or the agonist 8-OH-DPAT via bilateral intra-hippocampal infusion cannulae and evaluated antidepressant-like behavior using the tail suspension test (TST). WAY100635 reversed the antidepressant-like phenotype of the RGSi Gα i2 knock-in mice and 8-OH-DPAT produced an antidepressant-like response in wild type mice that was blocked by systemic WAY100635. Furthermore, intra-hippocampal infusion of the RGS19/4 inhibitor CCG-203769 produced an antidepressant-like effect in female mice. Ex-vivo slice recording confirmed the 5-HT1AR-mediated decrease in hippocampal CA1 pyramidal neuron excitability was enhanced in the RGSi Gα i2 knock-in mice. There was no change in hippocampal 5-HT1AR expression as measured by ligand binding but there was a compensatory reduction in Gαi proteins. The findings demonstrate that RGS protein control of hippocampal 5-HT1AR signaling is necessary and sufficient to account for the antidepressant-like phenotype in the RGSi Gα i2 knock-in mice and that RGS proteins highly expressed in the hippocampus should be investigated as targets for novel antidepressant therapies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Gα i2 , a heterotrimeric G-protein subunit, regulates various cell functions including ion channel activity, cell differentiation, proliferation and apoptosis. Platelet-expressed Gα i2 is decisive for the extent of tissue injury following ischemia/reperfusion. However, it is not known whether Gα i2 plays a role in the regulation of platelet apoptosis, which is characterized by caspase activation, cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) translocation to the platelet surface. Stimulators of platelet apoptosis include thrombin and collagen-related peptide (CoRP), which are further known to enhance degranulation and activation of α II b β3-integrin and caspases. Using FACS analysis, we examined the impact of agonist treatment on activation and apoptosis in platelets drawn from mice lacking Gα i2 and their wild-type (WT) littermates. As a result, treatment with either thrombin (0.01 U/mL) or CoRP (2 μg/mL or 5 μg/mL) significantly upregulated PS-exposure and significantly decreased forward scatter, reflecting cell size, in both genotypes. Exposure to CoRP triggered a significant increase in active caspase 3, ceramide formation, surface P-selectin, and α II b β3-integrin activation. These molecular alterations were significantly less pronounced in Gα i2 -deficient platelets as compared to WT platelets. In conclusion, our data highlight a previously unreported role of Gα i2 signaling in governing platelet activation and apoptosis., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

The heterotrimeric G-protein Go with its splice variants, Go1α and Go2α, seems to be involved in the regulation of motor function but isoform-specific effects are still unclear. We found that Go1α-/- knockouts performed worse on the rota-rod than Go2α-/- and wild-type (WT) mice. In Go1+2α-/- mice motor function was partially recovered. Furthermore, Go1+2α-/- mice showed an increased spontaneous motor activity. Compared to wild types or Go2α-/- mice, Go1+2α-/- mice developed increased behavioural sensitization following repetitive cocaine treatment, but failed to develop conditioned place preference. Analysis of dopamine concentration and expression of D1 and D2 receptors unravelled splice-variant-specific imbalances in the striatal dopaminergic system: In Go1α-/- mice dopamine concentration and vesicular monoamine uptake were increased compared to wild types. The expression of the D2 receptor was higher in Go1α-/- compared to wild type littermates, but unchanged in Go2α-/- mice. Deletion of both Go1α and Go2α re-established both dopamine and D2 receptor levels comparable to those in the wild-type. Cocaine treatment had no effect on the ratio of D1 receptor to D2 receptor in Go1+2α-/- mutants, but decreased this ratio in Go2α-/- mice. Finally, we observed that the deletion of Go1α led to a threefold higher striatal expression of Go2α. Taken together our data suggest that a balance in the expression of Go1α and Go2α sustains normal motor function. Deletion of either splice variant results in divergent behavioural and molecular alterations in the striatal dopaminergic system. Deletion of both splice variants partially restores the behavioural and molecular changes. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/., (© 2018 International Society for Neurochemistry.)

Dexmedetomidine (DMED) is a potent and highly selective α 2 -adrenergic receptor agonist and is widely used for short-term sedation. However, the mechanism of DMED-induced sedation has not been deciphered. In the present study, we investigated the mechanism of G αi and G βγ subunits on DMED-induced sedation. An ED 50 of DMED-induced loss of righting reflex (200.0nmol/kg) was increased to 375.0 or 433.3nmol/kg after pre-treatment with cAMP analog dbcAMP (50nmol/5 μl/mouse, i.c.v.) or the phosphodiesterase 4 inhibitor rolipram (100nmol/5 μl/mouse, i.c.v.). Conversely, the ED 50 of DMED-induced LORR decreased to 113.6 or 136.5 nmol/kg after pre-treated with G βγ subunit inhibitor M119 (100 mg/kg, i.p.) or gallein (100 mg/kg, i.p.) respectively. Administration of dbcAMP, rolipram, gallein or M119 alone had no effect on LORR. Gallein (10 μM) significantly inhibited forskolin-stimulated cAMP accumulation in α 2A -AR -CHO cells. Compared with G βγ subunit inhibitors or DMED alone, [Ca 2+ ]i and pERK1/2 was significantly increased after co-administration with G βγ subunit inhibitors and DMED. DbcAMP (5 μM) or rolipram (5 μM) alone had no effect on ERK1/2 phosphorylation, but decreased DMED-induced ERK1/2 phosphorylation after co-administration with DMED. G βγ subunit inhibitor treatment increased DMED-induced phosphorylation of CREB, whereas dbcAMP or rolipram had no effect on pCREB induced by DMED. From our results we conclude that, G βγ subunit may inhibit DMED-induced sedation through the cAMP and pERK1/2 pathway., (Copyright © 2018 Elsevier B.V. All rights reserved.)

G protein-gated inwardly rectifying K + (GIRK) channels are the major inwardly rectifying K + currents in cardiac atrial myocytes and an important determinant of atrial electrophysiology. Inhibitory G protein α-subunits can both mediate activation via acetylcholine but can also suppress basal currents in the absence of agonist. We studied this phenomenon using whole cell patch clamping in murine atria from mice with global genetic deletion of Gα i2 , combined deletion of Gα i1 /Gα i3 , and littermate controls. We found that mice with deletion of Gα i2 had increased basal and agonist-activated currents, particularly in the right atria while in contrast those with Gα i1 /Gα i3 deletion had reduced currents. Mice with global genetic deletion of Gα i2 had decreased action potential duration. Tissue preparations of the left atria studied with a multielectrode array from Gα i2 knockout mice showed a shorter effective refractory period, with no change in conduction velocity, than littermate controls. Transcriptional studies revealed increased expression of GIRK channel subunit genes in Gα i2 knockout mice. Thus different G protein isoforms have differential effects on GIRK channel behavior and paradoxically Gα i2 act to increase basal and agonist-activated GIRK currents. Deletion of Gα i2 is potentially proarrhythmic in the atria.

Stress-related alterations in brain-derived neurotrophic factor (BDNF) expression, a neurotrophin that plays a key role in synaptic plasticity, are believed to contribute to the pathophysiology of depression. Here, we show that in a chronic mild stress (CMS) model of depression the Gαi1 and Gαi3 subunits of heterotrimeric G proteins are down-regulated in the hippocampus, a key limbic structure associated with major depressive disorder. We provide evidence that Gαi1 and Gαi3 (Gαi1/3) are required for the activation of TrkB downstream signaling pathways. In mouse embryonic fibroblasts (MEFs) and CNS neurons, Gαi1/3 knockdown inhibited BDNF-induced tropomyosin-related kinase B (TrkB) endocytosis, adaptor protein activation, and Akt-mTORC1 and Erk-MAPK signaling. Functional studies show that Gαi1 and Gαi3 knockdown decreases the number of dendrites and dendritic spines in hippocampal neurons. In vivo, hippocampal Gαi1/3 knockdown after bilateral microinjection of lentiviral constructs containing Gαi1 and Gαi3 shRNA elicited depressive behaviors. Critically, exogenous expression of Gαi3 in the hippocampus reversed depressive behaviors in CMS mice. Similar results were observed in Gαi1/Gαi3 double-knockout mice, which exhibited severe depressive behaviors. These results demonstrate that heterotrimeric Gαi1 and Gαi3 proteins are essential for TrkB signaling and that disruption of Gαi1 or Gαi3 function could contribute to depressive behaviors., Competing Interests: The authors declare no conflict of interest.

Resveratrol, a natural polyphenolic compound has been reported to attenuate angiotensin II -induced vascular smooth muscle cell (VSMC) hypertrophy; however, whether resveratrol could also inhibit hyperproliferation of VSMC from spontaneously hypertensive rats (SHR) is unexplored. The present study investigates the effect of resveratrol on hyperproliferation of VSMC from SHR and the underlying molecular mechanisms responsible for this response. For these studies, aortic VSMC from SHR and Wistar-Kyoto (WKY) rats were used. The proliferation of VSMC was determined by [ 3 H] thymidine incorporation and the levels of proteins were determined by Western blotting. The enhanced proliferation exhibited by VSMC from SHR was attenuated by resveratrol. In addition, resveratrol attenuated the overexpression of cyclin D1, cyclin E, cyclin dependent kinase 4 (Cdk4), Cdk2, phosphorylated retinoblastoma protein (pRb), Giα proteins and enhanced phosphorylation of ERK1/2 and AKT in VSMC from SHR. Furthermore, the overproduction of superoxide anion, increased NADPH oxidase activity, overexpression of Nox2, Nox4 and p47 phox proteins, increased phosphorylation of EGFR, IGF-IR, and c-Src were all abrogated by resveratrol. These results suggest that resveratrol attenuates the hyperproliferation of VSMC from SHR through the inhibition of ROS, c-Src, growth factor receptor activation, MAPK/PI3K, Giα and cell cycle proteins that are implicated in VSMC hyperproliferation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background/aims: From invertebrates to mammals, Gαi proteins act together with their common binding partner Gpsm2 to govern cell polarization and planar organization in virtually any polarized cell. Recently, we demonstrated that Gαi3-deficiency in pre-hearing murine cochleae pointed to a role of Gαi3 for asymmetric migration of the kinocilium as well as the orientation and shape of the stereociliary ("hair") bundle, a requirement for the progression of mature hearing. We found that the lack of Gαi3 impairs stereociliary elongation and hair bundle shape in high-frequency cochlear regions, linked to elevated hearing thresholds for high-frequency sound. How these morphological defects translate into hearing phenotypes is not clear., Methods: Here, we studied global and conditional Gnai3 and Gnai2 mouse mutants deficient for either one or both Gαi proteins. Comparative analyses of global versus Foxg1-driven conditional mutants that mainly delete in the inner ear and telencephalon in combination with functional tests were applied to dissect essential and redundant functions of different Gαi isoforms and to assign specific defects to outer or inner hair cells, the auditory nerve, satellite cells or central auditory neurons., Results: Here we report that lack of Gαi3 but not of the ubiquitously expressed Gαi2 elevates hearing threshold, accompanied by impaired hair bundle elongation and shape in high-frequency cochlear regions. During the crucial reprogramming of the immature inner hair cell (IHC) synapse into a functional sensory synapse of the mature IHC deficiency for Gαi2 or Gαi3 had no impact. In contrast, double-deficiency for Gαi2 and Gαi3 isoforms results in abnormalities along the entire tonotopic axis including profound deafness associated with stereocilia defects. In these mice, postnatal IHC synapse maturation is also impaired. In addition, the analysis of conditional versus global Gαi3-deficient mice revealed that the amplitude of ABR wave IV was disproportionally elevated in comparison to ABR wave I indicating that Gαi3 is selectively involved in generation of neural gain during auditory processing., Conclusion: We propose a so far unrecognized complexity of isoform-specific and overlapping Gαi protein functions particular during final differentiation processes., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

In this study, we investigated the role of CB 1 palmitoylation in modulating the functional interaction with G proteins both in the absence and presence of agonist binding. Our data show that the nonpalmitoylated CB 1 receptor significantly reduced its association with Gα i2 . The agonist stimulation induced a partial dissociation of Gα i2 proteins from the wild-type receptor, while on the C415A mutant the agonist binding was not able to induce a significant dissociation of Gα i2 from the receptor. The lack of palmitoyl chain seems to hamper the ability of the receptor to functionally interact with the Gα i2 and indicate that the palmitoyl chain is responsible for the functional transmission of the agonist-induced conformational change in the receptor of the G protein. These data were further corroborated by molecular dynamics simulations. Overall these results suggest that palmitoylation of the CB 1 receptor finely tunes its interaction with G proteins and serves as a targeting signal for its functional regulation. Of note, the possibility to reversibly modulate the palmitoylation of CB 1 receptor may offer a coordinated process of regulation and could open new therapeutic approaches., (© 2017 International Union of Biochemistry and Molecular Biology, Inc.)

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The platelet ADP-receptor which has been denominated P2Y 12 receptor is an important target in pharmacotherapy. The receptor couples to G αi2 mediating an inhibition of cyclic AMP accumulation and additional downstream events including the activation of phosphatidylinositol-3-kinase and Rap1b proteins. The nucleoside analogue ticagrelor and active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel block P2Y 12 receptors and, thereby, inhibit ADP-induced platelet aggregation. These drugs are used for the prevention and therapy of cardiovascular events such as acute coronary syndromes or stroke. The recently published three-dimensional crystal structures of the human P2Y 12 receptor in complex with agonists and antagonists will facilitate the development of novel therapeutic agents with reduced adverse effects. P2Y 12 receptors are also expressed on vascular smooth muscle cells and may be involved in the pathophysiology of atherogenesis. P2Y 12 receptors on microglial cells operate as sensors for adenine nucleotides released during brain injury. A recent study indicated the involvement of microglial P2Y 12 receptors in the activity-dependent neuronal plasticity. Interestingly, there is evidence for changes in P2Y 12 receptor expression in CNS pathologies including Alzheimer's diseases and multiple sclerosis. P2Y 12 receptors may also be involved in systemic immune modulating responses and the susceptibility to develop bronchial asthma.

Ovarian carcinoma is the most lethal gynecologic tumor worldwide. Despite having developed molecular diagnostic tools and targeted therapies over the past few decades, patient survival is still quite poor. Numerous studies suggest that microRNAs are key regulators of many fundamental biological processes, including neoplasia and tumor progression. miR-222 is one of those miRNAs that has attracted much attention for its multiple roles in human diseases, especially cancer. The potential role of microRNAs in ovarian cancer has attracted much attention in recent years. Some of these microRNAs have been suggested as potential therapeutic targets for EOC patients. In this study, we sought to investigate the biologic functions of miR-222-3p in EOC carcinogenesis. Herein, we examined the expression of miR-222-3p in EOC patients, mouse models and cell lines, and found that higher expression of miR-222-3p was associated with better overall survival in EOC patients, and its level was negatively correlated with tumor growth in vivo. Furthermore, in-vitro experiments indicated that miR-222-3p inhibited EOC cell proliferation and migration, and decreased the phosphorylation of AKT. We identified GNAI2 as a target of miR-222-3p. We also found that GNAI2 promoted EOC cell proliferation, and is an activator of the PI3K/AKT pathway. We describe the characterization of a novel regulatory axis in ovarian cancer cells, miR-222-3p/GNAI2/AKT and its potential application as a therapeutic target for EOC patients.

Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson's disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [(35)S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced β-arrestin membrane recruitment; however, it prevents β-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time β-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of β-arrestin post-endocytic fate revealed that quinpirole treatment caused β-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of β-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired β-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells. Thus, RGS2 is integral for β-arrestin-mediated D2R internalization. The current study revealed a novel regulation of D2R signaling and internalization by RGS2 proteins., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Urbanization in Africa is associated with an increased incidence of non-communicable diseases, yet the cause and the mechanism remain poorly understood. Here, we propose a role for G protein-coupled receptor (GPCR) signaling in the biological changes that occur with urbanization and suggest a critical area of convergence in GPCR signaling might provide a molecular signature for exposure to environmental factors. As a first step in investing this hypothesis, we examined the expression of the G protein α, β and γ subunit, G protein related kinase, and β-arrestin genes in a rural and urban population living in Morocco (NCBI GSE8847). Three genes associated with the phosphatidylinositol signaling pathway (GNAQ, GNA11 and GNA15), and one gene controlling the cyclic adenosine monophosphate pathway (GNAI2) was altered by urbanization. Of note, the expression of ARRB1 gene, which encodes the β-arrestin 1 protein and dampens the cellular responses to extracellular signals, was greater in the rural compared to the urban population (P<0.00002). These preliminary findings support our hypothesis that urbanization fundamentally alters GPCR signaling, resulting in both a qualitative and quantitative change in the signaling process. Because GPCR signaling is involved in a broad spectrum of cellular functions, further research is needed confirm these preliminary findings and to investigate what role GPCRs might have in the biological changes that occur with urbanization., (Copyright © 2016. Published by Elsevier Ltd.)

Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca(2+) activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2(-/-)) and corresponding wild-type mice (Gαi2(+/+)). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2(-/-) and Gαi2(+/+) mice but the mean corpuscular volume was significantly larger in Gαi2(-/-) mice. Spontaneous PS exposure of circulating Gαi2(-/-) erythrocytes was significantly lower than that of circulating Gαi2(+/+) erythrocytes. PS exposure was significantly lower in Gαi2(-/-) than in Gαi2(+/+) erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca(2+) activity and cell shrinkage. Moreover, Gαi2(-/-) erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2(+/+) erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death.

Leukocyte recruitment to inflammation sites progresses in a multistep cascade. Chemokines regulate multiple steps of the cascade, including arrest, transmigration, and chemotaxis. The most important chemokine receptor in mouse neutrophils is CXCR2, which couples through Gαi2- and Gαi3-containing heterotrimeric G proteins. Neutrophils arrest in response to CXCR2 stimulation. This is defective in Gαi2-deficient neutrophils. In this study, we show that Gαi3-deficient neutrophils showed reduced transmigration but normal arrest in mice. We also tested Gαi2- or Gαi3-deficient neutrophils in a CXCL1 gradient generated by a microfluidic device. Gαi3-, but not Gαi2-, deficient neutrophils showed significantly reduced migration and directionality. This was confirmed in a model of sterile inflammation in vivo. Gαi2-, but not Gαi3-, deficient neutrophils showed decreased Ca(2+) flux in response to CXCR2 stimulation. Conversely, Gαi3-, but not Gαi2-, deficient neutrophils exhibited reduced AKT phosphorylation upon CXCR2 stimulation. We conclude that Gαi2 controls arrest and Gαi3 controls transmigration and chemotaxis in response to chemokine stimulation of neutrophils., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Anabolic β2-adrenoceptor (β2-AR) agonists have been proposed as therapeutics for treating muscle wasting but concerns regarding possible off-target effects have hampered their use. We investigated whether β2-AR-mediated signalling could be modulated in skeletal muscle via gene delivery to the target tissue, thereby avoiding the risks of β2-AR agonists. In mice, intramuscular administration of a recombinant adeno-associated virus-based vector (rAAV vector) expressing the β2-AR increased muscle mass by >20% within 4 weeks. This hypertrophic response was comparable to that of 4 weeks' treatment with the β2-AR agonist formoterol, and was not ablated by mTOR inhibition. Increasing expression of inhibitory (Gαi2) and stimulatory (GαsL) G-protein subunits produced minor atrophic and hypertrophic changes in muscle mass, respectively. Furthermore, Gαi2 over-expression prevented AAV:β2-AR mediated hypertrophy. Introduction of the non-muscle Gαs isoform, GαsXL elicited hypertrophy comparable to that achieved by AAV:β2-AR. Moreover, GαsXL gene delivery was found to be capable of inducing hypertrophy in the muscles of mice lacking functional β1- and β2-ARs. These findings demonstrate that gene therapy-based interventions targeting the β2-AR pathway can promote skeletal muscle hypertrophy independent of ligand administration, and highlight novel methods for potentially modulating muscle mass in settings of disease.

The cellular mechanisms underlying feedback signaling from horizontal cells to photoreceptors, which are important for the formation of receptive field surrounds of early visual neurons, remain unsettled. Mammalian horizontal cells express a complement of synaptic proteins that are necessary and sufficient for calcium-dependent exocytosis of inhibitory neurotransmitters at their contacts with photoreceptor terminals, suggesting that they are capable of releasing GABA via vesicular release. To test whether horizontal cell vesicular release is involved in feedback signaling, we perturbed inhibitory neurotransmission in these cells by targeted deletion of the vesicular GABA transporter (VGAT), the protein responsible for the uptake of inhibitory transmitter by synaptic vesicles. To manipulate horizontal cells selectively, an iCre mouse line with Cre recombinase expression controlled by connexin57 (Cx57) regulatory elements was generated. In Cx57-iCre mouse retina, only horizontal cells expressed Cre protein, and its expression occurred in all retinal regions. After crossing with a VGAT(flox/flox) mouse line, VGAT was selectively eliminated from horizontal cells, which was confirmed immunohistochemically. Voltage-gated ion channel currents in horizontal cells of Cx57-VGAT(-/-) mice were the same as Cx57-VGAT(+/+) controls, as were the cell responses to the ionotropic glutamate receptor agonist kainate, but the response to the GABAA receptor agonist muscimol in Cx57-VGAT(-/-) mice was larger. In contrast, the feedback inhibition of photoreceptor calcium channels, which in control animals is induced by horizontal cell depolarization, was completely absent in Cx57-VGAT(-/-) mice. The results suggest that vesicular release of GABA from horizontal cells is required for feedback inhibition of photoreceptors.

Activating transcription factor 5 (ATF5) is a member of the CREB/ATF family of transcription factors, which is highly expressed in olfactory chemosensory tissues, the main olfactory epithelium and vomeronasal epithelium (VNE) in mice. The vomeronasal sensory neurons in the VNE detect pheromones in order to regulate social behaviors such as mating and aggression; however, the physiological role of ATF5 in the vomeronasal sensory system remains unknown. In this study, we found that the differentiation of mature vomeronasal sensory neurons, assessed by olfactory marker protein expression, was inhibited in ATF5-deficient VNE. In addition, many apoptotic vomeronasal sensory neurons were evident in ATF5-deficient VNE. The vomeronasal sensory neurons consist of two major types of neuron expressing either vomeronasal 1 receptor (V1r)/Gαi2 or vomeronasal 2 receptor (V2r)/Gαo. We demonstrated that the differentiation, survival and axonal projection of V2r/Gαo-type rather than V1r/Gαi2-type vomeronasal sensory neurons were severely inhibited in ATF5-deficient VNE. These results suggest that ATF5 is one of the transcription factors crucial for the vomeronasal sensory formation.

Aim: We determined the role of brain Gαi2 proteins in mediating the neural and humoral responses of conscious male Sprague-Dawley rats to acute peripheral sodium challenge., Methods: Rats pre-treated (24-h) intracerebroventricularly with a targeted oligodeoxynucleotide (ODN) (25 μg per 5 μL) to downregulate brain Gαi2 protein expression or a scrambled (SCR) control ODN were challenged with an acute sodium load (intravenous bolus 3 m NaCl; 0.14 mL per 100 g), and cardiovascular parameters were monitored for 120 min. In additional groups, hypothalamic paraventricular nucleus (PVN) Fos immunoreactivity was examined at baseline, 40, and 100 min post-sodium challenge., Results: In response to intravenous hypertonic saline (HS), no difference was observed in peak change in mean arterial pressure between groups. In SCR ODN pre-treated rats, arterial pressure returned to baseline by 100 min, while it remained elevated in Gαi2 ODN pre-treated rats (P < 0.05). No difference between groups was observed in sodium-evoked increases in Fos-positive magnocellular neurons or vasopressin release. V1a receptor antagonism failed to block the prolonged elevation of arterial pressure in Gαi2 ODN pre-treated rats. A significantly greater number of Fos-positive ventrolateral parvocellular, lateral parvocellular, and medial parvocellular neurons were observed in SCR vs. Gαi2 ODN pre-treated rats at 40 and 100 min post-HS challenge (P < 0.05). In SCR, but not Gαi2 ODN pre-treated rats, HS evoked suppression of plasma norepinephrine (P < 0.05)., Conclusion: This highlights Gαi2 protein signal transduction as a novel central mechanism acting to differentially influence PVN parvocellular neuronal activation, sympathetic outflow, and arterial pressure in response to acute HS, independently of actions on magnocellular neurons and vasopressin release., (© 2015 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Study Objectives: This study tested the hypothesis that Regulators of G protein Signaling (RGS) proteins contribute to the regulation of wakefulness, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep, and to sleep disruption caused by volatile anesthetics., Methods: The three groups used in this study included wild-type (WT; n = 7) mice and knock-in mice that were heterozygous (+/GS; n = 7) or homozygous (GS/GS; n = 7) for an RGS-insensitive allele that causes prolonged Gαi2 signaling. Mice were implanted with electrodes for recording sleep and conditioned for 1 week or more to sleep in the laboratory. Using within and between groups designs, 24-h recordings of wakefulness, NREM sleep, and REM sleep were compared across three interventions: (1) baseline (control) and after 3 h of being anesthetized with (2) isoflurane or (3) sevoflurane., Results: Baseline recordings during the light phase revealed that relative to WT mice, homozygous RGS-insensitive (GS/GS) mice exhibit significantly increased wakefulness and decreased NREM and REM sleep. During the dark phase, these state-specific differences remained significant but reversed direction of change. After cessation of isoflurane and sevoflurane anesthesia there was a long-lasting and significant disruption of sleep and wakefulness. The durations of average episodes of wakefulness, NREM sleep, and REM sleep were significantly altered as a function of genotype and isoflurane and sevoflurane anesthesia., Conclusions: RGS proteins and Gαi2 play a significant role in regulating states of wakefulness, NREM sleep, and REM sleep. Genotype-specific differences demonstrate that RGS proteins modulate sleep disruption caused by isoflurane and sevoflurane anesthesia. The results also support the conclusion that isoflurane and sevoflurane anesthesia do not satisfy the homeostatic drive for sleep., (© 2016 Associated Professional Sleep Societies, LLC.)

The planar cell polarity (PCP) pathway is responsible for polarizing and orienting cochlear hair cells during development through movement of a primary cilium, the kinocilium. GPSM2/LGN, a mitotic spindle-orienting protein associated with deafness in humans, is a PCP effector involved in kinocilium migration. Here, we link human and mouse truncating mutations in the GPSM2/LGN gene, both leading to hearing loss. The human variant, p.(Trp326*), was identified by targeted genomic enrichment of genes associated with deafness, followed by massively parallel sequencing. Lgn (ΔC) mice, with a targeted deletion truncating the C-terminal GoLoco motifs, are profoundly deaf and show misorientation of the hair bundle and severe malformations in stereocilia shape that deteriorates over time. Full-length protein levels are greatly reduced in mutant mice, with upregulated mRNA levels. The truncated Lgn (ΔC) allele is translated in vitro, suggesting that mutant mice may have partially functioning Lgn. Gαi and aPKC, known to function in the same pathway as Lgn, are dependent on Lgn for proper localization. The polarization of core PCP proteins is not affected in Lgn mutants; however, Lgn and Gαi are misoriented in a PCP mutant, supporting the role of Lgn as a PCP effector. The kinocilium, previously shown to be dependent on Lgn for robust localization, is essential for proper localization of Lgn, as well as Gαi and aPKC, suggesting that cilium function plays a role in positioning of apical proteins. Taken together, our data provide a mechanism for the loss of hearing found in human patients with GPSM2/LGN variants.

Chemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the α and β subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstream signaling, relatively little is known about their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1, GNB2, GNB4, and GNB5) in HL60 cells and primary murine hematopoietic cells. Neutrophil function was assessed by using intravital microscopy, flow chamber assays, and chemotaxis and biochemistry studies. We unexpectedly discovered that all expressed GNB isoforms are required for LFA-1 activation. Their downregulation led to a significant impairment of LFA-1 activation, which was demonstrated in vitro and in vivo. Furthermore, we showed that GPCR activation leads to Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipase C β2 (Plcβ2) and Plcβ3. They act nonredundantly to produce inositol triphosphate-mediated intracellular Ca(2+) flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcβ2-, Plcβ3-, or Rac1-deficient mice were protected from lipopolysaccharide-induced lung injury. Taken together, we demonstrated that all Gnb isoforms are required for chemokine-induced downstream signaling, and Rac1, Plcβ2, and Plcβ3 are critically involved in integrin activation and leukocyte arrest., (© 2016 by The American Society of Hematology.)

In this issue of Science Signaling, Villanueva et al. report on the identification of heterotrimeric guanine nucleotide-binding protein (G protein) Gαi2 as an essential effector of Smoothened-mediated mammary epithelial cell proliferation. Through a series of in vivo experiments, the authors delineate a Smoothened-regulated Gli-independent Gαi signal that provides direct genetic evidence connecting Smoothened with Gαi in mice., (Copyright © 2015, American Association for the Advancement of Science.)