Protein-coupled Receptor Signaling Research Articles

Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers.

β‐arrestins (βarr1 and βarr2) are ubiquitous regulators of G protein‐coupled receptor (GPCR) signaling. Available data suggest that β‐arrestins dock to different receptors in different ways. However, the structural characterization of GPCR‐arrestin complexes is challenging and alternative approaches to study GPCR‐arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non‐canonical amino acids for photo‐ and chemical crosslinking into βarr1 and βarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin‐like), the corticotropin‐releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin‐like). We show that each receptor leaves a unique footprint on arrestins, whereas the two β‐arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell.

Angiotensin II Stimulates the Proliferation and Migration of Lymphatic Endothelial Cells Through Angiotensin Type 1 Receptors.

Background/AimThe proliferation and migration of lymphatic endothelial cells (LECs) is essential for lymphatic vessel growth (also known as lymphangiogenesis), which plays a crucial role in regulating the tissue fluid balance and immune cell trafficking under physiological and pathological conditions. Several growth factors, such as VEGF-C, can stimulate lymphangiogenesis. However, the effects of angiotensin II (Ang II) on the proliferation and migration of mouse LECs and the underlying potential mechanisms remain unknown.MethodsWild-type mice were infused with Ang II (1,000 ng/kg/min) for 1–2 weeks. Murine LECs were stimulated with Ang II (500 nM) or saline for 12–48 h. Cell proliferation was determined with 5-bromo-2-deoxyuridine (BrdU) incorporation assays, while cell migration was assessed by scratch wound healing and transwell chamber assays. The gene expression profiles were obtained by time series microarray and real-time PCR analyses.ResultsAng II treatment significantly induced lymphangiogenesis in the hearts of mice and the proliferation and migration of cultured LECs in a time-dependent manner. This effect was completely blocked by losartan, an angiotensin II type 1 receptor (AT1R) antagonist. The microarray results identified 1,385 differentially expressed genes (DEGs) at one or more time points in the Ang II-treated cells compared with the control saline-treated cells. These DEGs were primarily involved in biological processes and pathways, including sensory perception of smell, the G protein coupled receptor signaling pathway, cell adhesion, olfactory transduction, Jak-STAT, alcoholism, RIG-I-like receptor and ECM-receptor interaction. Furthermore, these DEGs were classified into 16 clusters, 7 of which (Nos. 13, 2, 8, 15, 7, 3, and 12, containing 586 genes) were statistically significant. Importantly, the Ang II-induced alterations the expression of lymphangiogenesis-related genes were reversed by losartan.ConclusionThe results of the present indicate that Ang II can directly regulate the proliferation and migration of LECs through AT1R in vivo and in vitro, which may provide new potential treatments for Ang II-induced hypertension and cardiac remodeling.

Utilizing Designed Receptors Exclusively Activated by Designer Drug Chemogenetic Tools to Identify Beneficial G Protein-Coupled Receptor Signaling for Fibrosis.

Fibrosis or accumulation of extracellular matrix is an evolutionarily conserved mechanism adopted by an organism as a response to chronic injury. Excessive fibrosis, however, leads to disruption of organ homeostasis and is a common feature of many chronic diseases. G protein-coupled receptors (GPCRs) are important cell signaling mediators and represent molecular targets for many Food and Drug Administration-approved drugs. To identify new targets for fibrosis, we used a synthetic GPCR system named designed receptors exclusively activated by designer drugs (DREADDs) to probe signaling pathways essential for fibrotic response. We found that upon expression in human lung fibroblasts, activation of Gq- and Gs-DREADDs abrogated the induction of TGFβ-induced fibrosis marker genes. Genome-wide transcriptome analysis identified dysregulation of multiple GPCRs in lung fibroblasts treated with TGFβ To investigate endogenous GPCR modulating TGFβ signaling, we selected 13 GPCRs that signal through Gq or Gs and activated them by using specific agonists. We examined the impact of each agonist and how activation of endogenous GPCR affects TGFβ signaling. Among the agonists examined, prostaglandin receptor agonists demonstrated the strongest inhibitory effect on fibrosis. Together, we have demonstrated that the DREADDs system is a valuable tool to identify beneficial GPCR signaling for fibrosis. This study in fibroblasts has served as a proof of concept and allowed us to further develop in vivo models for fibrosis GPCR discovery. SIGNIFICANCE STATEMENT: Fibrosis is the hallmark of many end-stage cardiometabolic diseases, and there is an unmet medical need to discover new antifibrotic therapies, reduce disease progression, and bring clinically meaningful efficacy to patients. Our work utilizes designed receptors exclusively activated by designer drug chemogenetic tools to identify beneficial GPCR signaling for fibrosis, providing new insights into GPCR drug discovery.

Genetic deletion of Rgs12 in mice affects serotonin transporter expression and function in vivo and ex vivo.

Regulator of G protein Signaling (RGS) proteins inhibit G protein-coupled receptor (GPCR) signaling, including the signals that arise from neurotransmitter release. We have shown that RGS12 loss diminishes locomotor responses of C57BL/6J mice to dopamine transporter (DAT)-targeting psychostimulants. This diminution resulted from a brain region-specific upregulation of DAT expression and function in RGS12-null mice. This effect on DAT prompted us to investigate whether the serotonin transporter (SERT) exhibits similar alterations upon RGS12 loss in C57BL/6J mice. Does RGS12 loss affect (a) hyperlocomotion to the preferentially SERT-targeting psychostimulant 3,4-methylenedioxymethamphetamine (MDMA), (b) SERT expression and function in relevant brain regions, and/or (c) serotonergically modulated behaviors? Open-field and spontaneous home-cage locomotor activities were quantified. 5-HT, 5-HIAA, and SERT levels in brain-region homogenates, as well as SERT expression and function in brain-region tissue preparations, were measured using appropriate biochemical assays. Serotonergically modulated behaviors were assessed using forced swim and tail suspension paradigms, elevated plus and elevated zero maze tests, and social interaction assays. RGS12-null mice displayed no hyperlocomotion to 10 mg/kg MDMA. There were brain region-specific alterations in SERT expression and function associated with RGS12 loss. Drug-naïve RGS12-null mice displayed increases in both anxiety-like and anti-depressive-like behaviors. RGS12 is a critical modulator of serotonergic neurotransmission and serotonergically modulated behavior in mice; lack of hyperlocomotion to low dose MDMA in RGS12-null mice is related to an alteration of steady-state SERT expression and 5-HT uptake.

β-arrestin 1 transfection induced cell death in high grade glioma in vitro

ABSTRACT The best known functions of β-arrestins (β-arr) are to regulate G protein-coupled receptors (GPCR) signaling through receptor desensitization and internalization. Many reports also suggest that β-arrs play important role in immune regulation and inflammatory responses, under physiological and pathological conditions. Recent studies have shown that β-arr 1 silencing halts proliferation and increases temozolomide (TMZ) response in glioblastoma (GBM) cells. The focus of this paper is to analyze the role of β-arr 1 overexpression in the 18 high grade glioma (HGG) cell line in terms of viability and their response to TMZ treatment. For this reason, the cell line was transfected with β-arr 1 and the effect was analyzed after 24 h, 48 h and 72 h in terms of proliferation and treatment response. We observed that β-arr 1 overexpression induced a time and dose dependant inhibition in the HGG cells. Unexpectedly, β-arr transfection resulted in a very mild increase in TMZ toxicity after 24 h, becoming non-statistically significant at 72 h. In conclusion, we showed that β-arr 1 overexpression inhibits cell proliferation in the 18 cell line but only has a very modest effect on treatment response with the alkylating agent TMZ.

Synthetic nanobodies as angiotensin receptor blockers

There is considerable interest in developing antibodies as functional modulators of G protein-coupled receptor (GPCR) signaling for both therapeutic and research applications. However, there are few antibody ligands targeting GPCRs outside of the chemokine receptor group. GPCRs are challenging targets for conventional antibody discovery methods, as many are highly conserved across species, are biochemically unstable upon purification, and possess deeply buried ligand-binding sites. Here, we describe a selection methodology to enrich for functionally modulatory antibodies using a yeast-displayed library of synthetic camelid antibody fragments called "nanobodies." Using this platform, we discovered multiple nanobodies that act as antagonists of the angiotensin II type 1 receptor (AT1R). Following angiotensin II infusion in mice, we found that an affinity matured nanobody antagonist has comparable antihypertensive activity to the angiotensin receptor blocker (ARB) losartan. The unique pharmacology and restricted biodistribution of nanobody antagonists may provide a path for treating hypertensive disorders when small-molecule drugs targeting the AT1R are contraindicated, for example, in pregnancy.

Serum-borne lipids amplify TLR-activated inflammatory responses.

TLRs recognize conserved pathogen associated molecular patterns and generate innate immune responses. Several circulating and cell membrane associated proteins have been shown to collaborate with TLRs in sensing microbial ligands and promoting inflammatory responses. Here, we show that serum and serum-borne lipids including lysophosphatidylcholine (LPC) amplify inflammatory responses from intestinal epithelial cells and mononuclear phagocytes primed with microbial TLR ligands. Treatment with the inhibitors of G protein-coupled receptor (GPCR) signaling, suramin, or pertussis toxin (PT), the inhibitor of JNK-MAPK, or knockdown of LPC response-regulating GPCR, G2A, decreases the augmentation brought about by serum or LPC in TLR-induced inflammatory response. In vivo administration of PT or anti-G2A antibody reduces TLR2-activated cytokine secretion. The ability of host lipids to costimulate TLR-generated cellular responses represents a novel pathway for the amplification of innate immunity and inflammation.

Key phosphorylation sites in GPCR s orchestrate the contribution of β‐Arrestin 1 in ERK 1/2 activation

β-arrestins (βarrs) are key regulators of G protein-coupled receptor (GPCR) signaling and trafficking, and their knockdown typically leads to a decrease in agonist-induced ERK1/2 MAP kinase activation. Interestingly, for some GPCRs, knockdown of βarr1 augments agonist-induced ERK1/2 phosphorylation although a mechanistic basis for this intriguing phenomenon is unclear. Here, we use selected GPCRs to explore a possible correlation between the spatial positioning of receptor phosphorylation sites and the contribution of βarr1 in ERK1/2 activation. We discover that engineering a spatially positioned double-phosphorylation-site cluster in the bradykinin receptor (B2 R), analogous to that present in the vasopressin receptor (V2 R), reverses the contribution of βarr1 in ERK1/2 activation from inhibitory to promotive. An intrabody sensor suggests a conformational mechanism for this role reversal of βarr1, and molecular dynamics simulation reveals a bifurcated salt bridge between this double-phosphorylation site cluster and Lys294 in the lariat loop of βarr1, which directs the orientation of the lariat loop. Our findings provide novel insights into the opposite roles of βarr1 in ERK1/2 activation for different GPCRs with a direct relevance to biased agonism and novel therapeutics.

Conformational Basis of G Protein-Coupled Receptor Signaling Versatility.

G protein-coupled receptors (GPCRs) are privileged structural scaffolds in biology that have the versatility to regulate diverse physiological processes. Interestingly, many GPCR ligands exhibit significant 'bias' - the ability to preferentially activate subsets of the many cellular pathways downstream of these receptors. Recently, complementary information from structural and spectroscopic approaches has made significant inroads into understanding the mechanisms of these biased ligands. The consistently emerging theme is that GPCRs are highly dynamic proteins, and ligands with varying pharmacological properties differentially modulate the equilibrium among multiple conformations. Biased signaling and other recently appreciated complexities of GPCR signaling thus appear to be a natural consequence of the conformational heterogeneity of GPCRs and GPCR-transducer complexes.

The CC-chemokine receptor 2 is involved in the control of ovarian folliculogenesis and fertility lifespan in mice

The chemokine receptor 2 (CCR2) was first described as a chemotactic factor involved in immune responses, but it also plays an essential function in several biological processes. The chemokine (C–C motif) ligand 2 (CCL2) binds to CCR2 triggering G protein-coupled receptor (GPCR) signaling in leukocytes, including activation of PI3K/Akt/mTOR, a key pathway that is also related to follicular activation and survival. However, the potential role of CCR2 in ovarian follicular physiology remain unexplored. Thus, we investigated the role of CCR2 on follicular growth during adult life and aging. Ovaries and oocytes were collected from wild type (WT) mice at 1.5 months old (mo), and CCR2 expression was observed predominantly in oocytes included in growing follicles, as well as after ovulation. Follicle populations were assessed in WT and CCR2-/- mice at 1.5 mo, and CCR2-/- mice had more primordial and less primary and secondary follicles, while there were no differences in antral follicle numbers. Pro-apoptotic genes Bax and Casp3 were downregulated, while anti-apoptotic Bcl2 was upregulated in CCR2-/- mice. To further characterize the role of CCR2 in ovarian aging, follicle populations were assessed in WT and CCR2-/- mice at 1.5, 2.5, 6, 10, and 12 mo. A larger ovarian follicular reserve at 1.5–6 mo was observed in CCR2-/- mice. Finally, CCR2-/- aged mice (6–12 mo) ovulated more oocytes than WT mice. Altogether, these data suggest that CCR2 plays an important role in the regulation of murine folliculogenesis, potentially affecting the reproductive lifespan.

Genetics of kidney stone disease.

Kidney stone disease (nephrolithiasis) is a common problem that can be associated with alterations in urinary solute composition including hypercalciuria. Studies suggest that the prevalence of monogenic kidney stone disorders, including renal tubular acidosis with deafness, Bartter syndrome, primary hyperoxaluria and cystinuria, in patients attending kidney stone clinics is ∼15%. However, for the majority of individuals, nephrolithiasis has a multifactorial aetiology involving genetic and environmental factors. Nonetheless, the genetic influence on stone formation in these idiopathic stone formers remains considerable and twin studies estimate a heritability of >45% for nephrolithiasis and >50% for hypercalciuria. The contribution of polygenic influences from multiple loci have been investigated by genome-wide association and candidate gene studies, which indicate that a number of genes and molecular pathways contribute to the risk of stone formation. Genetic approaches, studying both monogenic and polygenic factors in nephrolithiasis, have revealed that the following have important roles in the aetiology of kidney stones: transporters and channels; ions, protons and amino acids; the calcium-sensing receptor (a G protein-coupled receptor) signalling pathway; and the metabolic pathways for vitamin D, oxalate, cysteine, purines and uric acid. These advances, which have increased our understanding of the pathogenesis of nephrolithiasis, will hopefully facilitate the future development of targeted therapies for precision medicine approaches in patients with nephrolithiasis.

Biasing Neurotensin Receptor Signaling to Arrest Psychostimulant Abuse

Biasing G protein-coupled receptor signaling with ligands that promote select pathways is emerging as a powerful approach for therapeutic drug discovery. In this issue of Cell, Slosky etal. report a β-arrestin-biased neurotensin receptor ligand that may curtail drug abuse without the side effects induced by G protein signaling.

M180. SUSCEPTIBILITY AND RESILIENCE IN A MOUSE MODEL OF MATERNAL IMMUNE ACTIVATION

BackgroundEpidemiological studies over the past decades have repeatedly implicated maternal immune activation (MIA) in the etiology of psychiatric illnesses, including schizophrenia and related psychotic disorders. Not all offspring exposed to MIA, however, develop overt pathologies, suggesting that some are susceptible while others are resilient to MIA. To elucidate susceptibility and resilience in MIA, we used a mouse model that is based on prenatal exposure to the viral mimic poly(I:C).MethodsPoly(I:C)-based MIA was induced in C57BL6/N mice on gestation day 12. Control dams received vehicle solution only. Offspring of poly(I:C)- or vehicle-exposed dams were subjected to a comprehensive behavioral testing battery when they reached adulthood (12 weeks of age onwards). Next-generation mRNA sequencing and gene pathway analyses were conducted after behavioral testing to explore the molecular correlates of resilience and susceptibility to MIA.ResultsBehavioral characterization coupled with unbiased TwoStep cluster analysis of a large number offspring (N >150) revealed that offspring exposed to MIA could be stratified into susceptible and resilient subgroups. While the former was characterized by deficits in social interaction, sensorimotor gating, and working memory, the behavioral profile of the latter was indistinguishable from control offspring. Susceptible and resilient MIA offspring were also dissociable by the presence of distinct molecular profiles in cortical and subcortical brain areas. In the medial prefrontal cortex, susceptible MIA offspring displayed a more profound deregulation of genes relevant for oxidative phosphorylation and mitochondrial functions than resilient MIA offspring. In the amygdala, the susceptible and resilient offspring differed in gene transcription pertinent to opioid signaling, DARPP-32 signaling, and G protein-coupled receptor signaling.DiscussionOur data show that MIA can result in substantial phenotypic and transcriptomic variability even in the context of genetic homogeneity and under identical experimental conditions. If extended further, our model system may help to explain why only a subgroup of offspring exposed to MIA develops overt neurodevelopmental sequelae.

Whole exome sequencing in patients with Williams–Beuren syndrome followed by disease modeling in mice points to four novel pathways that may modify stenosis risk

Supravalvular aortic stenosis (SVAS) is a narrowing of the aorta caused by elastin (ELN) haploinsufficiency. SVAS severity varies among patients with Williams-Beuren syndrome (WBS), a rare disorder that removes one copy of ELN and 25-27 other genes. Twenty percent of children with WBS require one or more invasive and often risky procedures to correct the defect while 30% have no appreciable stenosis, despite sharing the same basic genetic lesion. There is no known medical therapy. Consequently, identifying genes that modify SVAS offers the potential for novel modifier-based therapeutics. To improve statistical power in our rare-disease cohort (N = 104 exomes), we utilized extreme-phenotype cohorting, functional variant filtration and pathway-based analysis. Gene set enrichment analysis of exome-wide association data identified increased adaptive immune system variant burden among genes associated with SVAS severity. Additional enrichment, using only potentially pathogenic variants known to differ in frequency between the extreme phenotype subsets, identified significant association of SVAS severity with not only immune pathway genes, but also genes involved with the extracellular matrix, G protein-coupled receptor signaling and lipid metabolism using both SKAT-O and RQTest. Complementary studies in Eln+/-; Rag1-/- mice, which lack a functional adaptive immune system, showed improvement in cardiovascular features of ELN insufficiency. Similarly, studies in mixed background Eln+/- mice confirmed that variations in genes that increase elastic fiber deposition also had positive impact on aortic caliber. By using tools to improve statistical power in combination with orthogonal analyses in mice, we detected four main pathways that contribute to SVAS risk.

SAT-313 Synaptojanin 2 Binding Protein Is Required for Efficient Somatostatin Receptor 2A Phosphorylation by G Protein Coupled Receptor Kinases and Signaling to the ERK/MAPK Pathway

Somatostatin receptor 2A (Sst2A) agonists are the primary pharmacological treatment for growth hormone (GH) secreting pituitary tumors to inhibit GH secretion and limit the destructive effects of these tumors. Sst2A agonists are also widely used as imaging agents for neuroendocrine tumors, and are under investigation for treatment of these cancers. However, despite the clinical importance of Sst2A agonists, regulatory mechanisms controlling Sst2A internalization and signaling are not fully understood. Sst2A contains a C-terminal PDZ binding site that serves as a docking platform for PDZ domain containing proteins. In an unbiased screen for PDZ domain proteins that can interact with Sst2A, we identified Synaptojanin 2 binding protein (SYNJ2BP) / Outer mitochondrial protein 25 (OMP25). SYNJ2BP is an outer mitochondrial protein that has been shown previously to promote the internalization of the Activin Type II receptor. We find that SYNJ2BP interacts with Sst2A in a ligand-dependent manner. Importantly, we show that SYNJ2BP promotes interaction of Sst2A with the G protein couple receptor kinase 2 (GRK2), and that siRNA-mediated knockdown of SYNJ2BP dramatically inhibits Sst2A phosphorylation by endogenous GRKs. Moreover, overexpression of SYNJ2BP strongly potentiates Sst2A phosphorylation on the GRK phosphorylation sites. Modulation of SYNJ2BP expression also regulates somatostatin-stimulated β-arrestin recruitment to the plasma membrane. Interestingly, in contrast to the large effects on Sst2A phosphorylation and β-arrestin recruitment, modulation of SYNJ2BP expression only caused a small change in ligand-stimulated receptor internalization. When we assessed downstream signaling, we found that SYNJ2BP overexpression potently stimulated receptor-dependent activation of ERK1/2, but had little effect on the ability of Sst2A to repress forskolin-stimulated cAMP production. Taken together, these data demonstrate for the first time that interaction of Sst2A with a PDZ domain protein affects receptor regulation and signaling. Because multiple PDZ domain containing proteins have been shown to interact with Sst2A, these data also suggest that interaction of Sst2A with this class of proteins may be an important regulatory mechanism to bias its function within the cell.

SUN-368 Wnt Inhibition Decreases Trabecular Bone in a Mouse Model of Fibrous Dysplasia

Background: G protein-coupled receptor (GPCR) signaling mediates a wide spectrum of physiological functions, including bone development and remodeling. Fibrous dysplasia (FD) is a common skeletal dysplasia where normal bone and bone marrow are replaced by fibrous tissue and expansile trabecular bone lesions. The craniofacial bones are often involved, leading to pain and facial deformities. FD is a mosaic disease caused by a somatic mutation in the GNAS gene encoding the G-protein alpha subunit (Gsα) that leads to constitutive activation of the Gs signaling pathway. Unfortunately, FD has no effective medical treatments.Major challenges have hampered the development of pharmacologic strategies that specifically target GNAS or the Gsα protein. We previously developed the Col1(2.3)/Rs1 mouse model (Rs1) in which the Gs signaling pathway is activated specifically in bone by an engineered GPCR protein. These mice showed increased trabecular bone formation with loss of marrow space and cortical bone, which strongly resembles human FD (1–4). There was also a dramatic increase in the number of immature osteoblasts present in the FD lesions, suggesting that activation of Gs signaling caused an accumulation of these cells. Our prior studies showed increased Wnt signaling, which may be a major driver of this effect. Furthermore, blocking the Gs signaling could reverse the bone phenotype, providing proof-of-concept for finding drugs that could reverse the phenotype. Therefore, we administered the Wnt inhibitor LGK974, currently used in human clinical trials, to the Rs1 mice to test if the FD lesions could be pharmacologically reversed. Methods: We administered LGK974 in 4-week-old Rs1 and non-Rs1 mice. We used a low dose (5mg/kg) for 8 weeks or high dose (30mg/kg) for 4 weeks. The mice were evaluated by histology and micro computed tomography (micro-CT) for mineral density (mg/cm3), bone volume (mm3), and trabecular thickness (um). Results: LGK974 decreased β-catenin levels in bone on western blots. In the low-dose group, the histology and micro-CT showed no statistically significant differences between drug and control groups. In the high-dose group, the micro-CT showed significantly decreased trabecular bone thickness (p=0.0364, n=3) in the drug-treated group (22±2µm) compared with controls (17±2µm). Furthermore, histology showed resorption of the abnormal bone; however, the fibrocellular infiltrate in the Rs1 mice was still present. Conclusions: Wnt inhibition can lead to decreased fibrous dysplastic bone, but separates abnormal bone formation from the fibrocellular infiltrate. These results provide new insight into understanding interactions between the Wnt and Gs signaling pathways in FD pathogenesis and bone formation.

Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

G protein-coupled receptor signaling is required for the navigation of immune cells along chemoattractant gradients. However, chemoattractant receptors may couple to more than one type of heterotrimeric G protein, each of which consists of a Gα, Gβ, and Gγ subunit, making it difficult to delineate the critical signaling pathways. Here, we used knockout mouse models and time-lapse microscopy to elucidate Gα and Gβ subunits contributing to complement C5a receptor-mediated chemotaxis. Complement C5a-mediated chemokinesis and chemotaxis were almost completely abolished in macrophages lacking Gnai2 (encoding Gαi2), consistent with a reduced leukocyte recruitment previously observed in Gnai2-/- mice, whereas cells lacking Gnai3 (Gαi3) exhibited only a slight decrease in cell velocity. Surprisingly, C5a-induced Ca2+ transients and lamellipodial membrane spreading were persistent in Gnai2-/- macrophages. Macrophages lacking both Gnaq (Gαq) and Gna11 (Gα11) or both Gna12 (Gα12) and Gna13 (Gα13) had essentially normal chemotaxis, Ca2+ signaling, and cell spreading, except Gna12/Gna13-deficient macrophages had increased cell velocity and elongated trailing ends. Moreover, Gnaq/Gna11-deficient cells did not respond to purinergic receptor P2Y2 stimulation. Genetic deletion of Gna15 (Gα15) virtually abolished C5a-induced Ca2+ transients, but chemotaxis and cell spreading were preserved. Homozygous Gnb1 (Gβ1) deletion was lethal, but mice lacking Gnb2 (Gβ2) were viable. Gnb2-/- macrophages exhibited robust Ca2+ transients and cell spreading, albeit decreased cell velocity and impaired chemotaxis. In summary, complement C5a-mediated chemotaxis requires Gαi2 and Gβ2, but not Ca2+ signaling, and membrane protrusive activity is promoted by G proteins that deplete phosphatidylinositol 4,5-bisphosphate.

Arresting Developments in Biased Signaling

The ability to design 'biased' drugs that selectively activate G protein-coupled receptor (GPCR) signaling pathways beneficial in treating a disease, while limiting their side effects, is of broad significance. Lee et al. move us a step closer to this important goal by identifying structural differences in the β1-adrenoceptor in complex with β-arrestin 1 versus a G protein-mimicking nanobody.

Investigation of cardiovascular protective effect of Shenmai injection by network pharmacology and pharmacological evaluation

BackgroundShenmai injection (SMI) has been used in the treatment of cardiovascular disease (CVD), such as heart failure, myocardial ischemia and coronary heart disease. It has been found to have efficacy on doxorubicin (DOX)-induced cardiomyopathy. The aims of this study were to explore the underlying molecular mechanisms of SMI treatment on CVD by using network pharmacology and its protective effect on DOX-induced cardiotoxicity by in vitro and in vivo experiment based on network pharmacology prediction.MethodsNetwork pharmacology method was used to reveal the relationship between ingredient-target-disease and function-pathway of SMI on the treatment of CVD. Chemical ingredients of SMI were collected form TCMSP, BATMAN-TCM and HIT Database. Drugbank, DisGeNET and OMIM Database were used to obtain potential targets for CVD. Networks were visualized utilizing Cytoscape software, and the enrichment analysis was performed using IPA system. Finally, cardioprotective effects and predictive mechanism confirmation of SMI were investigated in H9c2 rat cardiomyocytes and DOX-injured C57BL/6 mice.ResultsAn ingredient-target-disease & function-pathway network demonstrated that 28 ingredients derived from SMI modulated 132 common targets shared by SMI and CVD. The analysis of diseases & functions, top pathways and upstream regulators indicated that the cardioprotective effects of SMI might be associated with 28 potential ingredients, which regulated the 132 targets in cardiovascular disease through regulation of G protein-coupled receptor signaling. In DOX-injured H9c2 cardiomyocytes, SMI increased cardiomyocytes viability, prevented cell apoptosis and increased PI3K and p-Akt expression. This protective effect was markedly weakened by PI3K inhibitor LY294002. In DOX-treated mice, SMI treatment improved cardiac function, including enhancement of ejection fraction and fractional shortening.ConclusionsCollectively, the protective effects of SMI on DOX-induced cardiotoxicity are possibly related to the activation of the PI3K/Akt pathway, as the downstream of G protein-coupled receptor signaling pathway.

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 µm/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.