Background: Chronic diabetes accelerates vascular dysfunction often resulting in cardiomyopathy but underlying mechanisms remain unclear. Recent studies have shown that the deregulated unfolded protein response (UPR) dependent on highly conserved IRE1α-spliced X-box- binding protein (XBP1s) and the resulting endoplasmic reticulum stress (ER-Stress) plays a crucial role in the occurrence and development of diabetic cardiomyopathy (DCM). In the present study, we determined whether targeting MAPK/ERK pathway using MEK inhibitor U0126 could ameliorate DCM by regulating IRE1α-XBP1s pathway.Method: Three groups of 8-week-old C57/BL6J mice were studied: one group received saline injection as control (n=8) and two groups were made diabetic by streptozotocin (STZ) (n=10 each). 18 weeks after STZ injection and stable hyperglycemia, one group had saline treatment while the second group was treated with U0126 (1mg/kg/day), 8 weeks later, all groups were sacrificed. Cardiac function/histopathological changes were determined by echocardiogram examination, Millar catheter system, hematoxylin-eosin staining and western blot analysis. H9C2 cardiomyocytes were employed for in vitro studies.Results: Echocardiographic, hemodynamic and histological data showed overt myocardial hypertrophy and worsened cardiac function in diabetic mice. Chronic diabetic milieu enhanced SUMOylation and impaired nuclear translocation of XBP1s. Intriguingly, U0126 treatment significantly ameliorated progression of DCM, and this protective effect was achieved through enriching XBP1s' nuclear accumulation. Mechanistically, U0126 inhibited XBP1s' phosphorylation on S348 and SUMOylation on K276 promoting XBP1s' nuclear translocation. Collectively, these results identify that MEK inhibition restores XBP1s-dependent UPR and protects against diabetes-induced cardiac remodeling.Conclusion: The current study identifies previously unknown function of MEK/ERK pathway in regulation of ER-stress in DCM. U0126 could be a therapeutic target for the treatment of DCM. It has two major subtypes, type 1 (T1DM) and type 2 (T2DM). Cardiovascular disease (CVD) is a major cause of morbidity and mortality in diabetic patients. Patients with diabetes mellitus have a risk of cardiovascular events that is two to three times as high as the risk among those without diabetesWith improvements in clinical treatments, the relative risk of death declined by 29% over a 10-year period, however, mortality in T1DM still increased 2- to 8- fold. Early onset T1DM is a strong risk factor associated with serious CVD, moreover, with a significant higher risk level in women, they also die around 18 years earlier than their diabetes free counterpartsetc. However, the XBP1s' role in DCM remains largely unknownAs reported in the literature, increases in oxidative stress, reduced nitric oxide bioavailability, elevations in advanced glycation end products, mitochondrial dysfunction and collagen-based cardiomyocyte and extracellular matrix stiffness, inflammation, renin-angiotensin-aldosterone system activation, cardiac autonomic neuropathy, endoplasmic reticulum (ER) stress, microvascular dysfunction and a myriad of cardiac metabolic abnormalities have all been implicated in the development and progression of DCMet al. reported that XBP1 can be SUMOylated by PIAS2 (protein inhibitor of activated STAT2) at two lysine residues located in the C-terminal transactivation domain. These SUMOylation events significantly decrease the transcriptional activity of XBP1 towards UPR target genesFurthermore, post-translational modification of XBP1, especially its SUMOylation has been shown to be involved in UPR regulation. Chen H. Within the current study, we confirmed a correlation of XBP1s' nuclear translocation in cardiomyocyte and DCM phenotype in a streptozotocin induced type 1 mouse DM model. Furthermore, we found that the impaired XBP1s' nuclear translocation is regulated by its SUMOylation in the diabetic hearts. Subsequently, the molecular mechanisms underlying the regulation of the XBP1s' SUMOylation in cardiomyocyte were systematically investigated, and we found that high glucose induced activation of MAP kinase ERK1/2 can increase XBP1s' phosphorylation on its serine residue S348, which in turn can promote its SUMOylation on lysine residue K276 and result in its impaired nuclear translocation. Finally, we tested whether the ERK1/2 inhibitor U0126 treatment can protect against DCM, as hypothesized U0126 treatment could be of therapeutic potential for DCM.Animal experiments were conducted following standards and procedures approved by the local Animal Care and Use Committee . Diabetes was induced by intraperitoneal administration of streptozotocin  at 60 mg /kg, freshly dissolved in 0.05 M sterile sodium citrate (pH 4.5) on five successive days in 8-week-old male C57BL/6J mice Mice were anesthetized using light isoflurane (1-2%). Echocardiography was performed as described previously using a high-resolution imaging system with a 13-MHz linear ultrasound transducer .Measurements of LV function were performed using Millar catheter system as described before. In brief, mice were anaesthetized, and a pressure-volume catheter  was inserted into the right carotid artery and advanced into the left ventricle under pressure control to measure instantaneous intraventricular pressure and volume.Histology analysis was performed as previously describedH9C2 cells (ATCC) were cultured in RPMI 1640 medium  supplemented with 10% Fetal bovine serum  and Penicillin/Streptomycin (P/S) (100 IU/mL) in a humidified atmosphere of 95% air and 5% CO2 at 37°C. HEK293T cells (ATCC) were maintained in DMEM medium supplemented with 10% FBS. Cells were starved overnight before treatment with high concentrations of glucose  or mannitol (25 mM). Transfection was performed using transfection reagent FuGENE  according to the manufacturer's protocol. At desired time points post glucose and mannitol treatment, total lysates or cytosolic and nuclear fractions of cells were prepared for immunoblotting analysis.2, 1 mM EDTA, 0.6% NP-40, 0.5 mM DTT, protease inhibitor cocktail  and lysates were incubated for 10 min on ice. After brief vortexing the lysates were centrifuged for 10 min at 5,000 rpm at 4°C. Supernatants were collected as cytosolic fractions and the pellets were resuspended in 100 ml of buffer-B containing 10 mM HEPES-KOH (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT and protease inhibitors. Lysates were incubated for 20 min on ice followed by centrifugation at 12,000 g at 4°C for 10 min. Supernatants containing the nuclear extracts were collected and stored at - 80°C.Isolation of cytosolic and nuclear fractions was performed as previously described2, 0.5 mM DTT and protease inhibitors. Protein concentration was measured using Bradford reagent or BCA reagent , and purity of nuclear and cytoplasmic fractions was determined by Lamin A/C  and GAPDH  western blots, respectively.A similar procedure was used for isolation of cytosolic and nuclear fractions of cells where buffer-A contains 10 mM HEPES-KOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl3VO4, supplemented with protease inhibitor cocktail). Lysates were centrifuged  and supernatant was kept, while the pellet containing debris was discarded. The protein concentration in supernatants was quantified using BCA reagent. Equal amounts of protein were electrophoretically separated on 10% or 12.5% SDS polyacrylamide gels, transferred to PVDF membranes, and probed with the desired primary antibodies overnight at 4 °C. Membranes were then washed with TBST and incubated with anti-mouse or anti-rabbit IgG  horseradish peroxidase-conjugated antibodies as indicated. Blots were developed with the immobilon western chemiluminiscent HRP substrate. The density of each band was measured by using ImageJ software.Immunoblotting and Immunoprecipitation were performed as previously describedFor Immunoprecipitation of total cellular and heart tissue proteins, proteins were extracted with RIPA containing complete protease inhibitor cocktail. Lysates were combined with 1 μg of specific antibody and incubated overnight at 4 °C on rotating shaker. Immunoprecipitates were purified with protein A/G agarose beads  and washed with PBS containing protease inhibitor cocktail. Immunoprecipitates were fractionated by SDS-PAGE (10%), transferred to PVDF membranes, and subjected to immunoblotting with appropriate primary and secondary antibodies as described above. The following antibodies were used in the current study: ANP , Flag, XBP-1, BNP , SUMO-1, SUMO2/3, ERK1/2, P-ERK1/2, P38, p-P38, JNK, p-JNK , β-MHC .Plasmids encoding wildtype or lysine mutant XBP1s-GFP fusion  proteins were gifts from Dr. Ling Qi (Cornell University)To examine the downstream UPR genes in wild type and K276R mutant transfected H9C2 cells, we performed UPR pathway-specific expression analyses. Quantitative RT-PCR were conducted essentially as previously describedFluorescent microscopy was performed as previously describedImmunofluorescence microscopy was performed as previously describedt-test or ANOVA as appropriate and post-hoc comparisons of ANOVA were corrected with the method of Tukey. Prism 5 (www.graphpad.com) software was used for statistical analyses. All data presented involving cell culture is representative of at least three independent repeat experiments. Statistical significance was accepted at values of p < 0.05.The data are summarized as the means ± SEM (standard error of the mean). Statistical analyses were performed with Student's Figure AFigure B. As shown in Figure , the myocardial structure was examined by Hematoxylin and eosin (H&E) and WGA staining, diabetic hearts displayed structural abnormalities, including disrupted cardiac fibers, deranged cellular structures, obscured intercellular border and increased cardiomyocyte transverse cross-sectional areas . Immunoblots showed an increase in the expression of atrial natriuretic peptide (ANP), B type natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) in the heart tissues of diabetic cardiomyopathy mice . To evaluate the changes of left ventricular performance in diabetic mice, M-mode echocardiography was performed at 26 weeks after STZ injection Figure I. Compared to control, diabetic cardiomyopathy mice showed a significant reduction of ejection fraction and fraction shorting Figure J-K. We also detected the left ventricular hemodynamic alternation simultaneously. Diabetic mice showed aggravated cardiac dysfunction, which was mainly manifested in the decline in + dP/dt max and - dP/dt minFigure L-M. These data indicated that the type 1 DCM was successfully established, characterized by diastolic and systolic dysfunction and myocardial hypertrophy.As described in materials and methods, we used a mouse model of persistent hyperglycemia induced by streptozotocin (STZ model), reflecting insulinopenic type 1 DM Figure A, B. Besides, other brunches of the UPR were subsequently deregulated, such as ATF6 and CHOP . Furthermore, in vitro studies with neonatal cardiomyocytes H9C2 cells showed that high concentration glucose  but not mannitol impaired nuclear translocation of XBP1s Figure . These data suggest that akin to diabetic kidney, nuclear translocation of XBP1s is impaired in diabetic heart promoting maladaptive ER-stress and cardiomyopathy.Chronic hyperglycemia in diabetes impairs nuclear translocation of XBP1s compromising UPR resulting in maladaptive ER-stress in diabetic kidneyFigure . Taken together, these data suggest that its SUMOylation could regulate the impaired XBP1s nuclear translocation observed in STZ-induced diabetic cardiomyopathy mice.SUMOylation of proteins, post-translational modification by the small ubiquitin-like modifier (SUMO), is an important transient regulatory mechanism in many cellular processes, most notably transcriptional regulation, DNA damage, and signal transduction A. In order to exclude the possibility of non-specific modification events, mutation of K298 to R (K298R) which had no effect on SUMOylation of XBP1s protein that carried a negative charge. The scheme shown in  summarizes mutational loci of K276R, K297R and K298R. Next, we expressed XBP1s-GFP fusion proteins for wild type and lysine mutant XBP1s-GFP fusion  proteins in H9C2 cells B-C. Interestingly, mutation of K276 to R (K276R) obviously increased XBP1s' nuclear translocation, while other mutations had no significant effect Figure A, B. Consistent with this result, fluorescent microscopic images of XBP1s-GFP fusion adenovirus constructs upon 24 hours transfection into H9C2 cells following HG intervention for 6 hours showed that mutant K276R predominantly localized to the nucleus Figure C. To further prove its functional relevance in ER-stress, we examined the downstream UPR genes in wild type and K276R mutant transfected H9C2 cells simultaneously. In congruent with increased nuclear translocation of XBP1s, when compared to wild type, K276R mutant enhanced the expression of genes that modulate UPR Figure D-E. Thus, our data suggest that HG inhibited XBP1s' nuclear translocation through enhancing its SUMOylation at lysine residue 276.SUMOylation of transcription factors often leads to alterations in the intracellular localization of target proteinsin vivo and in vitro studies have demonstrated that chronic hyperglycemia can activate MAPKs, which play a pivotal role in the development of DCMFigure ). Therefore, these data indicated ERK1/2 could be one of the important effectors in response to consistent hyperglycemia.Mitogen-activated protein kinases (MAPKs) consist of three subfamilies, including extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK)http://gps.biocuckoo.org/userguide.php), we identified a series of potential ERK1/2 phosphorylation sites on XBP1s and the three highest-ranking sites  were selected. We performed site-directed mutagenesis with Alanine (Ala) to analyze the functional relevance of these phosphorylation sites.Given the specific increase in p-ERK1/2 in the diabetic heart, we next determined whether the phosphorylation of ERK1/2 modulates XBP1s function by modulating its phosphorylation. For this we investigated the ERK1/2 phosphorylation sites on XBP1s. By using the phosphorylation prediction software GPS 3.0 . Immunoprecipitation was performed using a Flag antibody. As shown in Figure , the SUMO1 and SUMO2/3 levels were significantly decreased, indicating that mutation of the ERK consensus site (Ser 348) in XBP1s has a substantial effect on XBP1s' phosphorylation and SUMOylation. However, The S69A and S161A mutants showed no decreases in XBP1s SUMOylation Figure C, indicating that the Ser348 site has a crucial role in XBP1s SUMOylation.Phosphorylation is an important protein posttranslational modificationFigure , consistent with the foregoing results, the SUMO1 and SUMO2/3 levels were significantly reduced in S348A mutant in compared with WT construct. These data indicated that ERK1/2 pathway regulates HG-induced XBP1s SUMOylation by phosphorylating Ser348.To ascertain whether the phosphorylation of XBP1s at the Ser348 site by ERK1/2 regulate its SUMOylation in H9C2 cells, we constructed adenoviruses carrying the XBP1s wild type and S348A mutant and expressed in H9C2 cells. As shown in Figure ) and ERK1/2 pathway regulated HG-induced XBP1s phosphorylation and SUMOylation . So, we used U0126, an inhibitor of MEK1/2, for subsequent experimental studies.ERK1/2 signaling pathway could promote cardiac differentiation and plays a role in myocardial protection in myocardial ischemia reperfusionFigure , U0126 markedly reduced p-ERK1/2 level induced by HG.We initially determined the efficacy of U0126 on the phosphorylation of ERK1/2 (p-ERK1/2). H9C2 cells pretreated with 10μM U0126 for 1 hour were incubated with HG (25mM) for additional 6 hours. As shown in Figure B. These data suggest that XBP1s SUMOylation is dependent on ERK phosphorylation in vitro.To determine if the reduction of p-ERK1/2 by U0126 can regulate XBP1s' SUMOylation, we initially determined XBP1s SUMOylation in H9C2 cells in response to U0126 treatment. The results demonstrate that U0126 significantly inhibit XBP1 SUMOylation induced by HG in H9C2 cells Figure C-D. Taken together, ERK1/2 inhibition with U0126 can increase XBP1s' nuclear translocation by inhibiting XBP1s' SUMOylation in HG-induced H9C2 cells in vitro.Similar to increasing XBP1s' SUMOylation, U0126 promotes XBP1s' nuclear translocation, which is inhibited by HG in H9C2 cells Figure A. 18 weeks after persistent hyperglycemia, 8 weeks U0126 (1mg/kg) or vehicle administration did not alter blood glucose in DCM mice Figure B. To investigate the possible protective effects of U0126 on cardiac hypertrophy, we conducted Hematoxylin and eosin (H&E) staining and WGA staining of heart tissue. We found that aberrant structural abnormalities, including disrupted cardiac fibers, deranged cellular structures, obscured intercellular border and increased cardiomyocyte transverse cross-sectional areas in diabetic model group Figure C-F. U0126 treatment significantly ameliorated the structural abnormalities in the hearts of diabetic mice. Consistent with the morphologic observations, the protein expression, markers of cardiac hypertrophy, were significantly increased in diabetic hearts, which were also reversed by U0126 treatment Figure G-H.In order to confirm whether U0126 could ameliorate diabetic cardiomyopathy, we conducted animal experiments in a well-established mouse model of streptozotocin (STZ)-induced type 1 diabetes  Figure I-K. We also detected the left ventricular hemodynamic alternation after administering. The result showed that diabetic mice has aggravated cardiac dysfunction, U0126 treatment decreased +dP/dt max and increased -dP/dtminFigure L-M. These data demonstrated U0126 administration exerted the beneficial effect on the left ventricle function in diabetic mice, even at a late stage.Before sacrifice, we evaluated the effects of U0126 on the cardiac function. Left ventricular performance was assessed by echocardiography. Compared to vehicle treated diabetic mice, mice treated with U0126 showed a significant reduction of fraction shorting and ejection fractionin vivo, we also determined the efficacy of U0126 on the phosphorylation of ERK1/2 in DCM mice. Consistent with the protective effects in H9C2 cells p-ERK1/2 was induced in STZ-induced in mice with DCM and U0126 markedly reversed this process Figure A.To determine whether U0126 ameliorates diabetic cardiomyopathy by inhibiting XBP1s SUMOylation and increasing XBP1s nuclear translocation in vitro, U0126 significantly attenuated XBP1s' SUMOylation induced by hyperglycemia in DCM mice Figure B. Likewise, U0126 treatment restored XBP1s' activity by promoting its nuclear translocation Figure C-D.Additionally, in congruent with the protective effect of U0126 in H9C2 cells Figure .Taken together, our results identified the crucial role of ERK1/2 pathway in DCM. Chronic hyperglycemia activates Ras/MEK/ERK cascades, leading to XBP1s' Ser 348 phosphorylation, subsequently resulting in XBP1s' K276 SUMOylation and finally restricting its nuclear translocation and activity. U0126 could ameliorates DCM by inhibiting Ras/MEK/ERK cascades to maintain XBP1s' nuclear translocation in vivo and complementary experiments in H9C2 cardiomyoblasts in vitro provide the following major findings: 1. XBP1s' nuclear translocation in cardiomyocyte correlates well with the DCM outcomes; 2. Chronic hyperglycemia induced activation of MAP kinase ERK1/2 can phosphorylate XBP1s on its serine residue 348, which requires for its SUMOylation on lysine residue 276 and results in its cytoplasmic retention; 3. ERK1/2 inhibitor U0126 can promote XBP1s' nuclear translocation in cardiomyocyte by restricting its phosphorylation and SUMOylation in vitro and in vivo, provides novel therapeutic avenue for DCM.Effective therapeutic approaches are urgently needed to improve clinical outcomes of patients with DCM. However, despite intense research, the pro-pathogenic factors that can be modulated to prevent DCM remain poorly understood. In particular, the involvement of dysregulated proteostasis as result of ER stress in this process remains obscure. Our studies using a STZ induced mouse model of T1DM Studies showed that HF and case-fatality remains higher in T1DM than in people without diabetes mellitus, moreover T1DM patients have an even higher incidence of HF than T2DMDiabetic cardiomyopathy is initially characterized by myocardial fibrosis, dysfunctional remodeling, and associated diastolic dysfunction, later by systolic dysfunction, and eventually by clinical heart failurein vivo and high glucose treated H9C2 cardiomyoblasts in vitro. Contrarily, Wang et al. reported recently that in cultured H9C2 cardiomyoblasts high glucose promoted the nuclear translocation of XBP1s and CHOP expression, resulting in cardiomyocytes apoptosis in vitroin vitro and in kidney of diabetic animals in vivo as wellAs mentioned above, we observed a positive correlation between HF indices and nuclear translocation of XBP1s in T1DM mouse hearts via its phosphorylationet al. discovered a phosphorylation-dependent SUMOylation motif, which is composed of a SUMOylation consensus site and an adjacent proline-directed phosphorylation site (ΨKxExxSP)in vivo. In general, the complexity of the posttranslational modification cross-talks contributes to the fine tuning of the physiological and pathological proteomic network regulation and it necessitates further elucidation.Cross-talks between various protein posttranslational modifications are critical for the regulation of coordinated networks for physiological functions etc., have been identified to regulate the phosphorylation status of XBP1sNumerous kinases, such as MAPKs, CKD5, AKT, IKKbeta,Taken together, we have confirmed the previously unknown function of MEK/ERK pathway in regulation of ER-stress in DCM, and that was related to XBP1s' posttranslational modification cross-talks in cardiomyocyte. Targeting MEK/XBP1 pathway could be a therapeutic avenue for the treatment of DCM.Supplementary figures and table.Click here for additional data file.


GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55 and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.Cdk8 of the RNA polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the  et al. examine regulators of Cdk8-dependent Gal4 activity in a screen for defects in the LTA response of gal3 yeast. They identify TORC1 components tor1 and tco89 and the downstream PP2A/Cdc55 phosphatase as regulators of Gal4. Additionally, genetic analysis indicates that mutations in hom3, which encodes aspartate kinase, produce defects in TOR signaling. These results demonstrate that regulation of Gal4 by phosphorylation is modulated by the TOR nutritional signaling pathway through the activity of PP2A/Cdc55.Horvath, Hawe  These include suppressors of snf1 (ssn3) (srb10) (ume5) (gig2) (HO) URS2 (nut7) (swi6 (ssx7) (G-mediated repression (urr1) (YGP1 expression (rye5)  , negativ2 (nut7) , suppresn (urr1) , and regn (rye5) . Cdk8 acGAL genes in response to galactose  pathways, which are interconnected by cross-talk, and are conserved amongst eukaryotes . TOR-comGAL) in yeast have served as an important model for understanding mechanisms of transcriptional regulation in eukaryotes  to galactose . One class of this mutant collection revealed that phosphorylation of Gal4 by Cdk8 is opposed by PP2A/Cdc55 phosphatase downstream of TORC1. Here, we demonstrate that GAL induction is modulated by a mechanism involving TORC1 signaling and a balance of phosphorylation of the regulatory Cdk8-dependent site on Gal4.To identify mechanisms regulating Cdk8, we conducted a genetic screen that exploited the dependence of Cdk8 for growth of E. coli. Antibodies against the Gal4 DNA-binding domain were produced in rabbits against 6-His-Gal4 (1–147) purified from E. coli. The Hom3 D234A mutation in plasmid pNH01 was created by site directed mutagenesis using oligos NH01 (5′-GGCTATACCGcaTTATGTGCCG-3′) and NH02 (5′-ACGACCAACACCATTCAG-3′) with the NEB Q5 SDM system. Plates with nonfermentable carbon sources contained 5% glycerol, 2% lactic acid, and 2% ethanol. Ethidium bromide (EB) galactose plates  contained yeast extract-peptone supplemented with 2% galactose and 20 mg of EB per liter. YPD/AOA and EB gal/AOA plates contained 10 nM aminooxyacetic acid (AOA) (Sigma-Aldrich). Rapamycin was added to YPD plates at 5 ng/ml.All strains were derived from the W303-1A background and are detailed in GAL induction, gal four throttle (gft), was performed by UV mutagenesis of gal3 W303 strain YJR7::131; colonies of mutagenized cells, grown on minimal medium containing glycerol, ethanol, and lactic acid as the sole source of carbon, were replica plated onto EB gal plates. Mutants incapable of growth on EB gal were transformed with a plasmid expressing WT GAL3 and re-examined for growth on EB gal; mutants incapable of growth when GAL3+ likely represented gal mutants and were discarded. Diploids were produced with the remaining mutant strains by crossing with an MATα gal3 strain (YJR52); all of the mutants analyzed produced a recessive gft phenotype in that the resulting diploids grew on EB gal as well as did the parental WT gal3 strain . Transformants were selected on SC-trp plates and the resulting colonies replica plated onto EB gal. Colonies that grew on EB gal were re-streaked on SC-trp and re-examined for growth on EB gal. Library plasmids were recovered from EB gal+ clones by transformation into DH5α E. coli, and analyzed by sequencing with the T7 (5′-TAATACGACTCACTATAGGG-3′) and T3 (5′-ATTAACCCTCACTAAAG-3′) promoter sequencing primers. Of the five clones sequenced, four contained DNA fragments spanning a region containing HOM3 on chromosome V  and IS2898 (5′-cggCTCGAGACCCTGACATTACATTTAGGGAA-3′), digestion with XhoI/NotI and cloning into pBSKSII. Similarly, gft7-1 clones were produced by amplification with oligos IS2899 (5′-ccgGCGGCCGCTGGCACGATGATTAAGG-3′) and IS2900 (5′-ccgGGATCCCGTTTTACCTCGAAAGCATTAGCTA-3′), digestion with BamHI/NotI and cloning into pGEM11Z. Multiple clones from each ligation and cloning were sequenced with nested sets of forward and reverse primers specific for the HOM3 and TCO89 ORF and 1 kb flanking DNA, respectively.Clones of the GAL1-GFP reporters were grown at 30°C in SC media containing glycerol, lactic acid, and ethanol as the sole carbon source, to an OD A600 ∼ 1.0 and then induced with 2% galactose. Samples were taken at the indicated times and diluted in PBS to 400,000 cells/ml and analyzed for GFP expression and side scatter, indicating cellular granularity produced by internal cellular complexity and physiological state, using a Guava Easycyte Flow Cytometer (MilliporeSigma). Mean fluorescence intensity (MFI) was calculated using FlowJo software. Expression of the GAL1-LacZ reporter was determined by assay of β-galactosidase activity from permeabilized cells as previously described , expressing GAT1-GFP or pJP01, expressing CDK8-GFP. Cells were grown in SC medium with glycerol, ethanol, and lactic acid or 2% raffinose as carbon source to an OD A600 ∼ 1.0 and then induced with 2% galactose for 2 h. Cells were recovered by centrifugation, washed in PBS, suspended in 1/4 original volume PBS, and examined using a Leica laser scanning confocal microscope; GFP and mCherry images were captured in separate channels for analysis using Image J. Cells expressing nuclear Gat1-GFP was quantitated using the Image J cell counter plugin, and represents the proportion of cells that have concentrated GFP expression co-localized with Nab2-mCherry  , and resuspended in 1 ml KLB/100 ml culture. Cells were lysed by vortexing with glass beads, and the lysates clarified by centrifugation at 13,000×g for 10 min at 4°C. FLAG-tagged Srb10/Cdk8 was recovered by immunoprecipitation with anti-FLAG-conjugated M2 agarose beads (Sigma). Samples were washed two times in KLB, twice in kinase assay buffer (KAB) , and the beads suspended in 50 μl KAB. Reactions contained 5 μl of the bead suspension and 1 μg substrate protein in 10 μl KAB. Two picomoles of [γ-32P] ATP were added, and the reactions incubated at 30°C for 20 min. Reactions were stopped by addition of 10 μl 2× SDS-PAGE sample buffer, boiled for 2 min, and resolved on 10% SDS-PAGE. The gels were stained with Coomassie blue . EB inhibits mitochondrial function and forces utilization of sugars as carbon source by fermentation  mutants, a collection which comprised several groups with distinct phenotypes that will be detailed in a separate report. The identity of one mutant, gft1, was determined by complementation with a plasmid library from WT yeast , and we found that gft1-1 mutant strains do not produce Hom3 protein as determined by immunoblotting , a residue conserved within the catalytic domain of amino acid kinase enzymes  is an enzyme required for synthesis of homoserine, the precursor for threonine and methionine biosynthesis . We dete of HOM3 . Neither of HOM3 , in eithl plates . The D23 protein , which igft1-1 mutation, and hom3 disruption, were more sensitive to sub-lethal concentrations of rapamycin compared with wild-type W303 or a strain bearing a gal3 disruption . FKBP12 binds the immunosuppressive agent rapamycin, and this receptor ligand complex inhibits TORC1 kinase function by direct interaction . Consequently, we examined whether disruption of genes encoding TOR signaling components also produce the gft phenotype in combination with a gal3 mutation. The only nonessential genes encoding proteins of the TORC1 complex are TOR1, and TCO89  comprised the redundant Pph21/Pph22 catalytic subunits and the nonessential Tpd3 and Cdc55 regulatory subunits . To exam mutants . Similarn EB gal , indicatGAL gene expression requires phosphorylation of Gal4 at serine 699 , and recombinant Gal4 protein as substrate. In these assays wild-type Cdk8-flag phosphorylates GST-CTD and Gal4, but not GST, but none of these substrates are phosphorylated by Cdk8 bearing a D290A mutation which inactivates kinase function .The function . In thespe cells , which igft1/hom3 and tor1 mutations on Gal4 phosphorylation in vivo. Gal4 is phosphorylated on multiple sites which produce distinctive alterations in mobility in SDS-PAGE (in vivo sites of phosphorylation (683–881)  or cdc55 (lane 3). However, we note that strains bearing disruption of tor1 (lane 2), hom3 (lane 5), or the gft1-1 mutation (lane 4) produce a faster migrating species typical of loss of the Cdk8-dependent P-S699 phosphorylation . However, we note that addition of excess aspartate to the growth medium is unlikely to affect intracellular concentrations without additional overexpression of the corresponding transporter . In contrast, AOA had no effect on growth of tor1 gal3 yeast on EB gal. This observation indicates that inhibition of aspartate biosynthesis reverses the effect of hom3 mutation on growth of gal3 yeast, suggesting that accumulation of aspartate may inhibit TOR signaling.Mutations of apamycin  (Xie et apamycin  which raapamycin , which md by AOA  (Antti ad by AOA , YPD/AOAGAL genes represent an important model for understanding eukaryotic gene regulation in response to signal transduction. In this study, we exploited the LTA/delayed GAL gene induction phenotype of gal3 yeast, which is Cdk8-dependent (GAL genes is modulated by nutrient signals that control TORC1 activity. Our results indicate that defects in TORC1 signaling do not directly affect Cdk8 kinase activity, but rather cause enhanced dephosphorylation of the Cdk-dependent phosphorylation site on Gal4 at S699 through hyperactivation of PP2A/Cdc55. These observations are consistent with known effects caused by inhibition of TORC1 activity, typified by dephosphorylation of transcription factors involved in nutrient response, including Gln3 and Gat1 which enables their nuclear translocation and activation of genes required for nutrient response (GAL genes is dependent upon phosphorylation of Gal4 at S699 (GAL induction through dephosphorylation of Gal4 P-S699, which would implicate this specific phosphorylation as a regulatory substrate for PP2A (GAL expression to levels appropriate for the growth environment.The yeast for PP2A . Howeverhom3 disruptions were found amongst the most sensitive to sub-lethal concentrations of rapamycin within the nonessential yeast deletion set (gft phenotype, as does hom3 disruption, but this effect is not produced by mutation of downstream genes involved in homoserine biosynthesis (gft phenotype is only produced by hom3 mutation suggests that aspartate kinase has an additional, more elaborate regulatory role for TOR signaling. Interestingly, a previous relationship between homoserine biosynthesis and Cdk8 activity was noted in that accumulation of β-aspartate semialdehyde caused by disruption of hom6 (hom3 might cause inhibition of TOR signaling (hom3 deletion on GAL expression (Regulation of TOR signaling is complex and involves sensing of nutrient availability through intracellular abundance of amino acids, most particularly glutamine (tion set . Impairiynthesis . These o of hom6  promotesignaling  and 11. ignaling , and thaignaling  suppresspression . Hom3 prpression . Overallgft mutant screen was designed to address this issue, and characterization of one mutant revealed a role of TOR signaling for regulation of GAL gene expression. The LTA phenotype is among the earliest phenotypic differences described between various laboratory strains of Saccharomyces cerevisiae, and extensive analysis of this phenotype had indicated an important role for nutrient signaling regulating delayed GAL gene induction (GAL expression is consistent with these previous observations. Considering that these signaling molecules are conserved amongst eukaryotes, it will be interesting to determine whether mTORC1 activity may regulate Cdk8/19 target factors in human cells. Characterization of additional mutants of the gft collection indicate that nutrient signaling must also regulate Cdk8 activity directly (The function of Cdk8 and the partially redundant Cdk19 protein kinase in humans are of considerable interest because a significant proportion of cancers may have alterations in their activity, particularly of melanoma and colorectal origin . Currentdirectly , and conAll data relating to this article are available in the article and in the GENETICS online.iyab168_Supplementary_DataClick here for additional data file.


Clitoria racemosa (Cli) mon-oculture, Acacia mangium (Aca) monoculture, Clitoria racemosa and Acacia mangium (Mix) bi-culture. We conducted mesocosm experiments where soil samples were incubated in anaerobic conditions for 35 days. To evaluate the effect of anaerobic conditions on biogeochemical cycles, we measured the following parameters related to iron-reducing bacteria and sulfate-reducing bacteria metabolism throughout the experiment: CO2 release, carbon dissolution, sulphide production and sulphate mobilization. We also monitored the solubilization of iron oxyhydroxides, manganese oxides, aluminum oxides and mercury in the culture medium. Iron-reducing bacteria (IRB) and sulfate-reducing bacteria (SRB) are described as the major players in the dynamics of iron, sulfur and metal elements including mercury in tropical environments. The results revealed two trends in these rehabilitated sites. In the Aca and Mix sites, bacterial iron-reducing activity coupled with manganese solubilization was detected with no mercury solubilization. In herbaceous sites, a low anaerobic activity coupled with sulphide production and mercury solubilization were detected. These results are the first that report the presence and activity of iron- and sulfate-reductive communities at rehabilitated mining sites and their interactions with the dynamics of metallic elements and mercury. These results report, however, the positive impact of ecological restoration of mining sites in French Guiana by reducing IRB and SRB activities, the potential mobility of mercury and its risk of transfer and methylation.Successive years of gold mining in French Guiana has resulted in soil degradation and deforestation leading to the pollution and erosion of mining plots. Due to erosion and topography, gold panning sites are submitted to hydromorphy during rainfall and groundwater increases. This original study focused on characterizing the impact of hydromorphic anaerobic periods on bio-geochemical cycles. We sampled soil from five rehabilitated sites in French Guiana, including sites with herbaceous vegetation and sites restored with fabaceous plants,  In the Amazon rainforest, several decades of gold mining and other anthropogenic activities has led to major environmental disruptions due to the cumulative effects of deforestation, acid mine drainage and soil and water pollution from arsenic, cyanide and mercury contamination . Mining Recent mining operations modify the topography of mining lands and expose the soils to sunlight and inclement weather conditions, and the absence of vegetation cover can facilitate erosion and the transport of particles containing trace elements into the water network . In addiAcidithiobacillus in aerobic environments can oxidize metal sulphides [Desulfovibrio can reduce iron-hydroxysulfate minerals to sulphides [Geobacter metallireducens, Clostridium sporogenes and Clostridium butyricum, solubilize iron oxides by reducing Fe(III) to Fe(II), using it either as a major final electron acceptor in anaerobic respiration or as a minor electron acceptor during fermentation metabolism [Soil microorganisms play an important role in the fate of metals in soils. Certain autotrophic bacteria of the genus ulphides ,8, thus ulphides ,10, whiculphides . Under aulphides  and affeulphides ,14. Undetabolism ,10,15. Ttabolism ,17. Recetabolism ,20,21,22tabolism ,18,23.In soils, mercury mobility modelling is complex, and its retention on clay minerals, on organic matter and on Fe-, Al- and Mn-(oxyhydr)oxides depends mainly on pH, redox conditions and the activity of anaerobic microbial communities , which iDue to the contributions of legislation in the management of gold mining sites, mining companies are now required to rehabilitate mining sites and, if possible, carry out an ecological restoration protocol . Legume Clitoria racemosa and Acacia mangium) and herbaceous species  were incubated under anaerobic conditions. The release of metals from soils into the culture medium and measurements of anaerobic microbial metabolism were monitored throughout the incubation period. This approach improves our knowledge of microbial communities at rehabilitated sites in Guyana and our ability to assess the effect of this rehabilitation on soil quality.The objective of this work is to evaluate the impact of different protocols of ecological restoration of mining soils on the mobility of the trace elements through the evaluation of two major bacterial activity in these soils, which are the iron-reductive activity and sulphate-reducing activity. Theses are recognized as the actors controlling not only the dynamics of iron in these soils but also that of toxic trace metal elements, including mercury, associated with iron, organic matter and other soil components. Soil samples from rehabilitated and restored plots with different fabaceous species , and no trees are present. This site was named Lyc to denote “Lycopodiella”. The third restoration site, in the region of Belizon, was restored by Clitoria racemosa in monoculture. The trees were planted with macro-cuttings in January 2013. This site was named Cli to denote “Clitoria”. The fourth restoration site, in the region of Yaoni, was restored by Acacia mangium in monoculture. The trees were planted with macro-cuttings in 1998. This site was named Aca to denote “Acacia”. The fifth restoration site, in the region of Yaoni, was restored by Acacia mangium and Clitoria racemosa (N 04°29.595′/W 052°26.984′). This site was named Mix to denote “Mixed fabaceous”. The trees were planted with macro-cuttings in 1998. Briefly, the first site, in the region of Yaoni, is only covered from 5% to 10% of the soil surface and is only dominated by herbaceous species, and this site has not been restored. This site was named NR to denote “Non-Restored”. The second selected site in the region of Belizon was rehabilitated, and more than 75% of the site is now mostly covered by n = 5) per site, with 3 sub-samples per plot that were pooled at the laboratory. The 5 sampling plots per site were selected based on the topography of the terrain, the accessibility of the plots and the feasibility of sampling. All plots were in similar topographic environments at similar altitudes. Soil samples were immediately sealed in sterile hermetic polyethylene bags for transportation to the Cayenne IRD laboratory. Samples were then air-dried at ambient temperature (25 °C) for approximately 3 weeks. These 75 collected samples (5 sites × 5 plots × 3 samples per plot) were then sieved at 2 mm to remove rocks, homogenized and hermetically sealed at 4 °C until use.The sampling campaign took place between April and June 2016 in French Guyana. This period usually corresponds to the main Guyanese rainy season, and the ambient humidity felt during the sampling was very high (not measured) with temperatures between 27 and 30 degrees during the sampling. At each site, soil samples were collected from depths between 0 and 10 cm with an auger. The sampling at the 5 sites consisted of sampling 5 plots  content, total trace element (Hg) content, total organic carbon content, granulometry and microbial biomass carbon (using the substrate-induced respiration (SIR) method) were determined to characterize the main physicochemical and biological properties; these measurements enabled us to assess the main differences between soils at initial conditions.3-HF (3:1 mL) using a hot block  following the ISO 11466 principles with some modifications advised by the device supplier. Samples (300 g) were warmed at 80 °C. Samples (300 mg) were then dissolved in 2 mL of concentrated HCl overnight at ambient temperature and then warmed for 4 h at 80 °C. Then, samples were diluted in 5% HNO3 before analysis by ICP-OES (Spectroblue).For all metal analyses, the samples were first ground to 63 µm. To obtain total metal  concentrations, soil samples were dissolved in cleaned Teflon digestion tubes (SCP Science) at 80 °C in a mixture of concentrated HNOMercury concentrations (HgT) in soil samples and in the culture medium were determined directly by thermal decomposition atomic absorption spectrometry after gold amalgamation using an automatic mercury analyzer  with a detection limit of 0.001 ng using the same method as described in a previous research article .Soil total carbon (Ctot) and total nitrogen (Ntot) were determined by the Dumas method (NF ISO 13878). Briefly, the Dumas method consists of flash combustion of the soil sample (at 1000 °C) using helium and oxygen. After purification of the combustion gases, determination of total carbon and nitrogen contents was performed by chromatography with a thermal conductivity detector (NA 1500 series 2 CARLO-ERBA).4 formed during acid digestion was measured after adding ammonium molybdate and ascorbic acid [® phosphate test (ref: 114848). The color produced by phosphomolybdenum (PMB) was measured by colorimetry at 885 nm .Total phosphorus (Ptot) was determined after the acid digestion of soil samples, as previously described. The HPObic acid  using thWater extractions were performed for total organic carbon (TOC), and concentrations were measured with a Shimadzu TOC-500 apparatus . Briefly, 1 g of soil sieved at 2 mm was shaken for 24 h in a polypropylene centrifuge tube with 10 mL of ultrapure water. The suspensions were centrifuged at 2000 rpm for 10 min, and supernatants were filtered at 0.2 µm  and directly analyzed on the TOC analyzer.Microcosms were prepared to study microbial activity under anaerobic conditions. Thus, 125 mL sterile hermetic glass plasma bottles were filled with 15 g of soil and 100 mL sterile ultra-pure water. Microcosms were performed with 3 replicates for each plot at each site for a total of 75 microcosms (5 sites × 5 plots per site × 3 microcosms per plot).2 for 10 min. The microcosms were incubated in the dark at 28 °C for 35 days without shaking. The measurements were taken on days 7, 14, 21, 28 and 35. Then, 8 mL of medium culture was collected with a sterile syringe through the butyl/PTFE shield cap, which was then analyzed for pH and the following concentrations in solution: sulfates, sulfides, ferrous iron, total iron, aluminum, manganese and mercury. The preparation of the experimental set-up is described in The microcosms obtained were mixed with sterile spatulas and sealed with a butyl/PTFE shield cap (VWR©). Anoxic conditions were then initiated by flushing microcosms with N2 evolution using micro gas chromatography .Immediately before sampling the culture medium, carbon mineralization was quantified in the microcosm atmosphere by measuring CODissolved organic carbon (DOC) concentrations in the culture medium were measured with a Shimadzu TOC-500 apparatus  after being filtered at 0.2 µm .42−) formation in the culture medium. This was carried out using a kit , similar to NF ISO 11048. This kit measures sulfate concentrations in the range of 5–250 mg SO42−/L. Samples were filtered  prior to analysis.Sulphur oxidation was followed by measuring sulfate (SO2−) formation in the culture medium. This was carried out using a kit , similar to NF ISO 10530. This kit measures sulphide concentrations in the range of 0.02–1.5 mg S2−/L. Samples were filtered  prior to analysis.Sulfate reduction was followed by measuring sulphide (SSoil pH was measured in the culture medium by using a pH meter (MetrOhm 744).4)2(SO4)2·6H2O).To avoid any oxidation reactions, all samples used for the determination of iron (II) were immediately acidified to pH 1 with nitric acid  and stored at 4 °C before analysis. Ferrous iron (Fe(II)) was measured in the medium using a colorimetric method with orthophenanthroline-chlorydrate . The absMetals were analyzed after filtration  and acidification at pH 1 with HCl (37%) by ICP-OES (Spectroblue).2, TOC, SO42−, S2−, Fe(II), Fe, Al, Mn and Hg), the results were also submitted to principal component analysis (PCA). The significance of each explanatory variable was tested using a Monte-Carlo permutation test. To calculate the correlations between the parameters measured during the experiment, Pearson correlations were performed. R software was used for all statistical analyses (R version 3.6.1 (5 July 2019)).The normality of data distributions and equal variance between treatments were tested using the Shapiro test and Bartlett’s test, respectively. To study the effect of vegetation mode, the effect of incubation and the interactions between soils and time, two-way ANOVA was performed. Tukey’s HSD multiple comparison method was used to test differences throughout the experiment at specific measurement points. To refine the analysis of the data, repeated-measures ANOVA was also carried out to determine the “sites” effect by taking into consideration the temporal evolution of the variables during the experiment. To analyze the effect of the different restoration processes and experimental treatments on biogeochemical data  and then rinsed several times with Milli-Q water before use. Concerning mercury assays in samples, the relative error was routinely 5% and was always under 10%. The detection limit (defined as three times the standard deviation (SD) of the blank) was 0.005 mg g−1. Concentrations obtained for repeated analyses of certified reference materials never exceeded the published range of concentrations . Total elements and trace elements were determined by inductively coupled plasma optical emission spectrometry . Calibration was conducted using standard solutions for spectroscopy . The analytical errors were estimated by carrying out 3 measurements for each sample and were <10%.To avoid contamination, all materials used in this work were acid-washed twice with HNOThe initial soil characteristics of the studied sites are summarized in Acacia mangium was more acidic than the other rehabilitated sites. Among the three sites restored with Fabaceae, the total carbon, nitrogen and phosphorus contents were always significantly the highest in Mix site. Macronutrient contents  for the Mix site were similar to the natural soil data in French Guiana [−1 at the Aca site and 7.01 g kg−1 at the Cli site. The total nitrogen content was 1.12 g kg−1 at the Aca site and 0.4 g kg−1 at the Cli site. The total phosphorus content was 0.37 g kg−1 at the Aca site and 0.17 g kg−1 at the Cli site. The total carbon, nitrogen and phosphorus contents at the spontaneous vegetation site NR were low. The five sites were rich in iron with a minimum of 44.56 g kg−1 at the Lyc site and a maximum of 113.3 g kg−1 at the Cli site. The aluminum contents at the sites were similar and were higher than the natural content in French Guiana. Manganese levels are low at the Lyc and Cli sites , and the Mix and NR sites had the highest concentration of total Mn. Mercury levels for NR, Lyc and Cli were lower than for other sites. The three sites restored with fabaceous species had significantly higher soil microbial biomass levels than the NR and Lyc sites. The maximum biomass was for the Mix site, while the minimum biomass was for the NR site, with values of 1103 mg kg−1 and 223 mg kg−1, respectively.The results for the granulometry demonstrated that the NR site was twice as clayey as other sites, with approximately 46.4% clay and 41.5% silt. In accordance with the soil triangle of texture, the Lyc site was silty with 65.8% silt on average. The Cli site was silt-clay-sand with 53.9% sand. The Aca and Mix sites had the same granulometry, with a dominance of sand over silt. Soil pH indicated that the site restored with h Guiana ,33,34. OSulfate A and sulHeterotrophic respiration C and orgThe ANOVA showed  that allConcentrations of both total iron A and iro−1 for the Cli and Mix sites, respectively.The evolution of soluble aluminum levels C in the −1 for Aca and Mix site samples, respectively.Measurements of soluble manganese in solution in culture media D showed −1 on day 28 before decreasing at the end of incubation.Measurements of soluble mercury in media E showed p < 0.05), but repeated-measures ANOVA showed that the pH change during incubation was similar between different soils, with no incubation time effect. The two sites without tree cover (NR and Lyc) had significantly more acidic pH levels compared to the restored sites . About trends, the Mix site had a similar pH variation of NR and Lyc sites in the first week of incubation, but after this time the pH behavior of the Mix site was similar to those in Cli and Aca sites.The ANOVA showed that medium pH F was afft-test comparisons enabled us to distinguish significant differences between the five sites with clusters (p = 0.001). The data clusters for the NR and Lyc sites were not significantly different from one another but were very different from the three clusters for the restored Cli, Aca and Mix sites. The cluster for the restored Cli site seemed to be relatively separate from the other restored sites, whereas the Aca and Mix sites were very close. In this study on the impact of the nature of the vegetation cover, the quality of ecological restoration was detected  or microbial biomass (p = 0.07) and no relationship to tree covering. The forms of iron in these soils could be different, which would impact solubilization kinetics. The distribution of manganese and aluminum also appeared to be heterogeneous among the various rehabilitated sites. Although the concentrations of oxides, clay, silt and sand in the soils were close to those for natural Oxisols or hydromorphic soil encountered in French Guyana ,38, the p = 0.0001) and microbial biomass (p = 0.0001), which is consistent with the literature, as described by Schuster et al. (1991) and Skyllberg et al. (2000), showing the high affinity of mercury for organic matter, particularly for sulphide groups [Mercury concentrations in these restored soils were still low when compared with other reported gold-mined soils in French Guiana, remaining within the normal range of Hg in this specific area . Neverthe groups ,41. In ae groups . VegetatFirstly, there were significant differences in soil microbial biomass, which were related with the density and quality of the vegetation cover on the restored sites. These results, already highlighted by various works on rehabilitated sites, confirm the contribution of successful ecological restoration in the recovery of soil microbial biomass ,42,43,44p < 0.001) results of the PCA , also observed by Gounou et al. (2010) [During the experiment, COsolution , which w Archaea , which s. 2010)  and Bous  and Bou. (2010) ,47. It i. (2010) .Iron-reducing activities were also supported by the rapid increase in sulfate levels in culture media, which may be associated with desorption of sulfates bound or adsorbed to Fe(III)-(hydr)oxides . AlthougThe low sulphide contents during the experiment in NR, Aca and Mix samples suggest that sulfate-reducing communities are not very active, and several factors could explain these low activities. In the Aca and Mix sites, the low SRB activities could be explained by high competition with iron-reducing bacteria, generally described as more competitive in an acidic environment ,51,52. F2S, indicating a priori the presence of iron sulphide, certainly linked to the presence of SRB.However, these chemical measurements may not reflect the observations of the culture media during the experiment. After 15 days of incubation, all the culture bottles had turned blackish, and the culture medium samples had a very marked smell of H2S degassing and potential metal sulphide precipitation [2S with iron under acidic conditions could explain the very low levels measured during the experiment [The very low sulphide contents measured could then be explained by both Hpitation . Indeed,periment ,57. Coleperiment , the preperiment . Thus, i2 = −0.621). These results confirmed that soil pH is a key factor affecting the adsorption–desorption behaviors and, hence, the bioavailability of heavy metals in soil, especially mercury [The dissolution of mercury in mesocosms under anaerobic conditions showed very contrasting results with other metallic elements, and the principal component analysis  clearly  mercury . In the  mercury , are at  mercury ,10,19,20 mercury . In this mercury , which s mercury . Althoug mercury  showed t mercury ,42. Roul mercury  work hig mercury , which i mercury  with lowAcacia mangium) and Mix (Acacia mangium and Clitoria racemosa) sites showed the positive effect of successful ecological restoration. Second, the simulation of flooding periods with the mesocosms showed interactions between iron- and sulfate-reducing activities depending on the rehabilitated sites. The two old restored sites (Aca and Mix) had predominant iron-reducing activities with the carbon and sulphur cycles coupled and a high potential for solubilization of iron and manganese. The restored Cli site and non-restored Lyc site had predominant sulfate-reducing activities and very low iron-reducing activities. Regarding the dynamics of mercury, its solubilization was twice as important at the two sites without tree cover restoration, which could lead to higher methylation potentials. Because these results provide information on the dynamics of microbial communities in association with the mobility of metallic elements, it seems necessary to further describe these results by characterizing the nature of metal oxides in the soils and estimating their environmental speciation according to the mode of ecological restoration.The ecological restoration of alluvial mining sites in French Guiana is recent and essential for the sustainable development of the gold industry in tropical regions. In this study, the comparison of the soil quality of five vegetation restoration modalities, as well as a simulation of the hydromorphic period with an anaerobic mesocosms experiment, revealed very contrasting results on reducing microbial activities and on the dynamics of metallic elements. First, the results showed a difference in terms of physicochemical properties between restored and non-restored sites. Aca (


The D2R is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, D2R arrestin-mediated signaling has been shown to have distinct physiological functions to those of G protein signalling. Relatively little is known regarding the patterns of D2R phosphorylation that might control these processes. We aimed to generate antibodies specific for intracellular D2R phosphorylation sites to facilitate the investigation of these mechanisms. We synthesised double phosphorylated peptides corresponding to regions within intracellular loop 3 of the hD2R and used them to raise phosphosite-specific antibodies to capture a broad screen of GRK-mediated phosphorylation. We identify an antibody specific to a GRK2/3 phosphorylation site in intracellular loop 3 of the D2R. We compared measurements of D2R phosphorylation with other measurements of D2R signalling to profile selected D2R agonists including previously described biased agonists. These studies demonstrate the utility of novel phosphosite-specific antibodies to investigate D2R regulation and signalling.The dopamine D DA effects are mediated by 5 members of the G protein-coupled receptor (GPCR) superfamily2. The dopamine D1 and D5 receptors (D1R & D5R) are coupled to stimulatory G proteins (Gαs or Gαolf) whereas the D2-like DRs  are coupled to inhibitory G proteins (Gαi/o/z). Dysregulation of dopamine signaling is associated with many CNS disorders. D2R agonists are used to treat the symptoms of Parkinson’s disease, whereas D2R antagonism is a necessary property of all clinically used antipsychotics3.The catecholamine neurotransmitter dopamine (DA) is involved in many physiological processes in the central nervous system (CNS) such as cognition, motor control and reward4. This inhibits G protein-signaling and leads to receptor internalization, dephosphorylation and recycling of receptors to the cell surface or trafficking to lysosomes for degradation. GRKs 2 and 3 primarily mediate agonist-stimulated D2R phosphorylation6 and overexpression of GRK2 has been shown to enhance D2R β-arrestin recruitment7. The D2R lacks the long C-terminal tail that is the site of GRK phosphorylation for many GPCRs. Mutagenesis studies from Namkung and colleagues identified eight serine/threonine (Ser/Thr) residues that are phosphorylated by GRK2/3 and a further five residues that are phosphorylated by protein kinase C (PKC) within intracellular loop (ICL) 38. A subsequent study by Cho and colleagues identified additional residues in ICL2 and 3 important for PKC-meditated desensitization of the D2R6.G protein signaling is rapidly desensitized by phosphorylation of the receptor by GPCR kinases (GRKs) followed by the recruitment of β-arrestins to the phosphorylated receptor9. Indeed, while Gαi/o/z G protein signaling appears to be responsible for many of the physiological consequences of D2R activation, a β-arrestin2-mediated signaling cascade involving protein phosphatase 2A, Akt (PKB) and glycogen synthase 3β has also been identified11. A global β-arrestin-2 knockout mouse displayed a reduction in DA-dependent locomotor activity11. Two studies that expressed mutant D2Rs, compromised either in the ability to recruit β-arrestin or to activate G protein-signaling relative to the other signaling process, in D2R-expressing medium spiny neurons (D2R-MSNs), provided evidence that D2R-β-arrestin signaling is sufficient for normal locomotor activity but not incentive motivation13. Elimination of β-arrestin2 specifically in D2R-MSNs reduced locomotor responses and blunted cocaine reward14. Together these data suggest that D2R-β-arrestin2 signaling may mediate physiological functions distinct from those controlled by D2R Gαi/o/z protein signaling15. Biased agonism describes a phenomenon whereby different ligands stabilize distinct conformations of a single receptor such that they differentially engage distinct signaling effectors16. If the therapeutic and deleterious side effects of a drug are mediated by distinct downstream signalling pathways through a single receptor, this concept may allow the development of biased pathways specific drugs that avoid such “on-target” side effects17. For the most part, however, the distinct GPCR signaling pathways in a specific tissue or cell that modulate particular physiological effects remains unclear18. Both arrestin and G protein biased ligands have been identified for the D2R19. Intriguingly, the action of one series of arrestin-biased ligands both to attenuate amphetamine-induced hyperlocomotion and avoid catalepsy in mice was diminished by global knockout of β-arrestin27.In addition to their role in receptor regulation, β-arrestins may act as scaffolding proteins to initiate signaling pathways2R phosphorylation by GRK2/3 is a key step in modulating downstream signaling to control distinct physiological responses to DA by terminating G protein signalling and engaging β-arrestins. We and others have shown that antibodies specific to phosphorylated residues of GPCRs are particularly useful in unravelling the complexities of such regulatory processes and in particular the hierarchy of phosphorylation patterns or barcodes22. In this study, we develop and characterize the first GRK phosphorylation site (phosphosite)-specific antibodies for the D2R and identify a site that is phosphorylated by GRK2 in response to D2R agonists. We compare the action of a number of agonists, including previously described biased agonists, to trigger receptor phosphorylation and correlate this to G protein activation, GRK2 recruitment and β-arrestin recruitment.Together these studies suggest that D2R was generated via artificial synthesis and cloned into pcDNA3.1 by Imagenes. The coding sequence for an amino-terminal HA-tag was added. Venus-1-155-Gγ2, Venus-156-239-Gβ1, masGRK3ct-NLuc were gifts from Nevin Lambert (Augusta University). hD2LR-Nluc was generated in house. GRK2-venus and YFP-βarrestin2 have been described previously23. The cDNA clones for human Gαi1-C351I or GαoA-C351I were obtained from the cDNA Resource Center (www.cdna.org).DNA for the long splice variant of the hD2R are shown in Table 2R third intracellular loop. After HPLC purification, the respective peptides were coupled to keyhole limpet haemocyanin. The conjugates were mixed 1:1 with Freund´s adjuvant and injected into groups of three rabbits (5095–5097) for anti-pThr287/Ser288 antibody production, (5098–5100) for anti-pThr293/Ser296 antibody production, (5101–5103) for anti-pSer317/Thr318 antibody production, and (5104–5106) for anti-D2R antibody production. The rabbits were injected at 4-week intervals. The serum was obtained 2 weeks after immunizations, beginning with the second injection. Specificity of the antisera was tested using dot blot analysis. Antibodies were affinity-purified against their immunizing peptide, immobilized using the SulfoLink kit (Thermo Scientific), for subsequent analysis. Anti-GRK2 , anti-GRK3 , anti-GRK5 (sc-518005) and anti-GRK6  antibodies were purchased from Santa Cruz Biotechnology. The anti-HA IgG C 488A antibody (SAB4600054) was obtained from Sigma-Aldrich, anti-HA IgG CF640R antibody (20240) was purchased from Biotium and the anti-rabbit IgG HRP-conjugated antibody (7074) was obtained from Cell Signaling Technologies.Peptide sequences used for generating phosphosite-specific antibodies against individual phosphorylated forms of the long splice variant of the DTerguride (ab144611) was obtained from Abcam. Aripiprazole (SML-0935), PMA (P8139) and pergolide mesylate (P8828) were purchased from Sigma-Aldrich. Apomorphine hydrochloride (2073), MLS1547 (6171), ropinirole (3680), quinpirole hydrochloride (1061), dopamine hydrochloride (3548), cabergoline (2664), bromocriptine mesylate (0427), forskolin (1099), SCH23390 (0925), PTX (3097), haloperidol hydrochloride (0931), L-741,626 (1003) and roxindole (1559) were obtained from Tocris. UNC9994 (A16087) was purchased from AdooQ Bioscience. Lambda-phosphatase (P0753S) was obtained from Santa Cruz. Compound 101 (HB2840) was obtained from Hello Bio. Terguride, PMA, forskolin, L-741,626, aripiprazole, pergolide, apomorphine, MLS1547, ropinirole, cabergoline, bromocriptine, haloperidol, roxindole, UNC9994 and compound 101 are DMSO-soluble and all the other mentioned compounds are water-soluble.2. HEK293 cells were stably transfected with TurboFect (ThermoFisher Scientific). Cells stably expressing HA-hD2R receptor were selected in medium supplemented with 400 µg/ml geneticin and cells stably transfected with HA-hD2R receptor and GIRK2-eGFP were selected in medium supplemented with 400 µg/ml geneticin and 300 µg/ml hygromycin. To increase the number of HEK293 cells stably expressing HA-hD2R receptor or HA-hD2R receptor in combination with GIRK-eGFP, fluorescence-activated cell sorting was used as described previously24.Human embryonic kidney 293 (HEK293) cells were obtained from the German Collection of Microorganisms and Cell Cultures GmbH . Cells were cultured in Dulbecco´s modified Eagle´s medium (DMEM), supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin at 37 °C and 5% COGRK2 (5′-AAGAAAUUCAUUGAGAGCGAU-3′), GRK3 (5′-AAGCAAGCUGUAGAACACGUA-3′), GRK5 (5′-AAGCAGTATCGAGTGCTAGGA-3′) and GRK6 (5′-AACACCUUCAGGCAAUACCGA-3′) and from GE Dharmacon a non-silencing RNA duplex (5′-GCUUAGGAGCAUUAGUAAA-3′ and 3′-UUUACUAAUGCUCCUAAGC-5′). HEK293 cells stably expressing HA-hD2 receptor were transfected with 150 nM siRNA for single transfection or with 100 nM of each siRNA for double transfection using HiPerFect. All experiments showed target protein abundance reduced by ≥ 80% 3 days after transfection.Chemically synthesized double-stranded siRNA duplexes (with 3′-dTdT overhangs) were purchased from Qiagen for the following targets: 21. HEK293 cells stably expressing the HA-hD2R were plated onto poly-l-lysine-coated 60-mm dishes and grown for 2 days to 80% confluency. Cells were treated with agonists or antagonists and subsequently lysed with detergent buffer  in the presence of protease and phosphatase inhibitors. Where indicated, cells were preincubated with GRK2/3 inhibitor compound 101 or D2 receptor antagonist for 30 min before agonist treatment. HA-tagged hD2R were enriched using anti-HA-agarose beads after 30 min centrifugation at 4 °C. Samples were inverted for 2 h at 4 °C. Where indicated, cell lysates were dephosphorylated with lambda protein phosphatase (Santa Cruz) for 1 h at 30 °C. Following sample washing, proteins were eluted using SDS sample buffer for 30 min at 50 °C. Protein separation was performed on 7.5% or 12% SDS–polyacrylamide gels. After electroblotting, membranes were incubated with 0.1 µg/ml antibodies to pThr287/Ser288 (5095), pThr293/Ser296 (5099) or pSer317/Thr318 (5102) overnight at 4 °C. Enhanced chemiluminescence detection (ECL) was used to detect bound antibodies (Thermo Fisher Scientific). Subsequently, blots were stripped and reprobed with the phosphorylation-independent antibody to the D2R (5106) to ensure equal loading of the gels.Western blot analysis was performed as described previously26. Initially, 2,500,000 Flp-In HEK 293 cells stably expressing the human D2LR were harvested into 10 cm dishes. 24 h after harvesting cells, the cells were transfected with cDNA constructs using linear polyethylenimine (PEI) in a ratio of 1 μg DNA: 6 μg PEI. Cells were transfected with pcDNA3.1 encoding the following constructs: 1 μg Venus-1–155-Gγ2, 1 μg Venus-156–239-Gβ1, 1 μg masGRK3ct-NLuc and 2 μg of either Gαi1-C351I or GαoA-C351I. 24 h after transfection the cells were harvested from dishes and plated into poly-d-lysine coated Greiner white 96-well TC treated plates. The cells were left to adhere for approximately 8 h and then treated with 100 ng/mL pertussis toxin (PTX) overnight. The following day, cells were washed once with Hank’s balanced salt solution (HBSS) pH 7.4 and left to equilibrate in HBSS 37 °C for 30 min before BRET detection. 10 min prior to addition of agonist, 10μL of Nano-Glo substrate (Promega) was added to each well . BRET was then measured using a PHERAstar FS microplate reader (BMG LABTECH). Luminescence was measured with the BRET1 plus filter for the emission signal of NLuc (445–505 nm) and Venus (505–565 nm) simultaneously. Measurements were taken 10 min after agonist addition. The counts from the Venus acceptor (505–555 nm) were then divided by the donor NLuc (465–505 nm) counts to give a BRET ratio. BRET ratios were then normalized to percent of the dopamine-induced maximal responses where indicated.The G protein activation assay was performed based on a previously reported bioluminescence resonance energy transfer (BRET) detection method27. HEK293 cells stably expressing the HA-hD2R and GIRK2-eGFP were plated into 96-well plates. After washing with Hank´s balanced salt solution (HBSS), buffered with 20 mM HEPES  cells were incubated with membrane potential dye  for 45 min at 37 °C. Final used injection volume of compounds and vehicle was 20 µl while the initial volume in the wells was 180 µL (90 µL buffer plus 90 µL dye); resulting in a final volume in the well of 200 µL and a 1:10 dilution of the compound. Therefore, the compounds were prepared at 10 × concentrations. Compounds or buffer were injected after a baseline reading for 60 s and measurements were recorded at 37 °C using a FlexStation 3 microplate reader (Molecular Devices). After data normalization to the baseline, the buffer-only trace for each corresponding data point was subtracted.Membrane potential change was measured as previously described28 and by others29 were improved by utilizing NanoBRET technology. Flp-In HEK 293 cells were initially harvested and transferred into plastic 10 cm2 dishes in DMEM with 10% FBS at a density of 2,000,000 cells. 24 h after transferring the cells to dishes, the cells were transfected using linear polyethylenimine (PEI) in a 1:6 ratio of DNA:PEI (μg). For GRK2 recruitment, 0.25 μg hD2LR-NLuc, 4 μg GRK2-Venus and 3.5 μg pcDNA3.1 were transfected. For β-arrestin2 recruitment, 0.25 μg hD2LR-NLuc, 2 μg GRK2 (untagged) and 5.5 μg YFP-β-arrestin2 were transfected. Approximately 30 h after transfection the cells were harvested from the dishes and plated into poly-D-lysine coated Greiner white 96-well TC treated plates in DMEM, 10% FBS. Approximately 20 h after cells were transferred to plates, the plate was washed with HBSS pH 7.4 and replaced with 80 μL HBSS. The cells were then left to equilibrate for 30 min at 37 °C before agonist addition. 10 min prior to agonist addition, 10 μL of Nano-Glo Luciferase Assay Substrate (Promega) diluted in HBSS was added to each well with a multi-step pipette . Changes in BRET were then detected 10 min after agonist addition in a PHERAstar FS microplate reader (BMG Labtech) at 37 °C. Individual wells were measured for the luminescence emission signal of NLuc (465–505 nm) and Venus/YFP (505–555 nm) simultaneously. Data was analysed by taking the counts from the acceptor Venus/YFP (505–555 nm) and dividing by the donor NLuc (465–505 nm) counts to give a BRET ratio. The BRET ratio was baseline-normalized to vehicle wells as well as 100% defined as the maximal BRET ratio obtained by stimulation with dopamine or quinpirole where indicated.GRK2 and β-arrestin2 recruitment assays were measured by means of BRET detection. The BRET assays previously reported by our group21. GraphPad Prism 5 software was used for data analysis and this was performed as described previously. Densitometry of every protein band was carried out with Image J. We used the same area size to perform densitometry for every protein band from the same experiment for every phosphorylation site as well as the total receptor. Accordingly, an equally sized empty area from the blot/film was measured to subtract this value as background signal from every measuring point. Finally, phosphorylation signals were normalized to the total receptor . SCR-controls were defined as 100% and phosphorylation of every target protein was calculated as percentage phosphorylation in comparison to the respective control. Statistical analysis was carried out with one-way ANOVA followed by Bonferroni correction. P values < 0.05 were considered statistically significant. Data sets of n less than 5 were not subjected to such statistical analyses.ImageJ 1.47v software was used for quantification of protein bands detected on Western blots as described previously50 is the molar concentration of agonist required to give a response half-way between maximal and minimal asymptote. Dose–response data were fitted to the following form of the operational model of agonism30 to allow the quantification of biased agonism:Em is the maximal possible response of the system, basal is the basal level of response, KA represents the equilibrium dissociation constant of the agonist (A) and τ is an index of the signalling efficacy of the agonist that is defined as RT/KE, where RT is the total number of receptors and KE is the coupling efficiency of each agonist-occupied receptor, and n is the slope of the transducer function that links occupancy to response. The analysis assumes that the transduction machinery utilized for a given cellular pathway are the same for all agonists, such that the Em and transducer slope (n) are shared between agonists. Data for all compounds for each pathway were fit globally to determine values of KA and τ. Note that in fitting the concentration response curves to Eq.  value of a reference agonist, in this case dopamine, is subtracted from the log(τ/KA) value of the agonists of interest to yield Δlog(τ/KA). The relative bias can then be calculated for each agonist at the two different signaling pathways by subtracting the Δlog(τ/KA) of one pathway from the other to give a ΔΔlog(τ/KA) value which is a measure of bias. A lack of biased agonism will result in values of ΔΔlog(τ/KA) not significantly different from 0 between pathways. Our previous studies have included an additional step of normalization in which the ∆log(τ/KA) of a particular agonist at a selected pathway is subtracted from the ∆log(τ/KA) determined at other pathways, termed the ∆∆log(τ/KA) or LogBias value (Supp. Table KA) values that are statistically compared we show these values. All affinity (pKi or pKA), potency (pEC50), and transduction ratio (log(τ/KA)) parameters were estimated as logarithms. When fold-changes in bias are described this was calculated by converting values of ΔΔlog(τ/KA) to the corresponding antilog value. However, we have previously demonstrated that such the distribution of these parameters does not conform to a normal (Gaussian) distribution whereas the logarithm of the measure is approximately Gaussian32. Thus, since the application of t tests and analyses of variance assume Gaussian distribution, estimating the parameters as logarithms allows valid statistical comparison. All results are expressed as the mean ± S.E.M. As such we performed a Brown-Forsythe test (Graphpad prism 8) to assure ourselves of equal variance when such parameters are compared.The results of concentration response experiments were analysed using Prism 8.0 (GraphPad Software Inc.). Concentration–response curves were fitted using the following three parameter equation:2R. Previous work identified putative GRK2 phosphorylation sites within the intracellular loops of the rat D2R (rD2R) using site-directed mutagenesis coupled with whole cell phosphorylation assays and auto-radiography including Thr287, Ser288, Thr293 and Ser8. Note that in the hD2R Ser317 is positioned next to another putative GRK site, Thr318, that is substituted for Asn in rD2LR  and short (D2SR) isoforms of the D2R.We set out to develop G protein-coupled receptor kinase (GRK) phosphosite-specific antibodies for the hD2LR, showing a diffuse band migrating between 60 and 80 kDa, consistent with preceding studies of N-terminally glycosylated D2Rs expressed in HEK293 cells8. While previous work suggested that Thr287, Ser288, and Thr293 are GRK2 phosphorylation sites8, we were unable to detect agonist-induced changes in phosphorylation with the pThr287/pSer288 antibody or the pThr293/pSer296 antibodies  or forskolin, that lead to activation of protein kinase C (PKC) and protein kinase A (PKA) family members, respectively  (vehicle control pEC50 = 7.29 (95% CI 7.25–7.33), cmpd101 pEC50 = 6.89 (95% CI 6.76–7.01) , cmpd101 Emax = 29.09 (95% CI 27.6–30.6)), (vehicle control pEC50 = 6.83 (95% CI 6.98–6.67), cmpd101 pEC50 = 6.82 (95% CI 6.72–6.93))  was rapid and sustained over time, with maximal signal obtained within 2.5 min  or arrestin (UNC9994) biased agonists19. We hypothesized that these biased ligands might display a differential ability to stimulate Ser317/Thr318 phosphorylation. For example, the arrestin biased agonist UNC9994 might be expected to stimulate Ser317/Thr318 phosphorylation even in the absence of G protein signalling. We have previously shown that the binding kinetics of D2R agonists can influence comparisons of agonist effects across measurements of different signaling endpoints28. This effect is driven, to an extent, by measurements of agonist action at different signaling endpoints at distinct timepoints. To negate this effect and allow comparison across assays, agonist-induced effects were measured 10 min after stimulation.Previous studies have shown that different ligands can induce different patterns of GPCR phosphorylation2LR , and a low affinity .There was a wide range in the maximal response of agonists to induce GRK2 recruitment to the D317/Thr318 phosphorylation induced by the twelve different agonists  concentration used for each agonist  that is a composite of the affinity of the agonist for the receptor-effector complex (KA) and the efficacy with which the agonist acts at that effector (τ) (Supp Table KA) obtained for DA from the values obtained for each agonist (∆log(τ/KA)), Fig. KA)) for the same ligand between different signaling pathways are, therefore, indicative of bias ) were not significantly different across the different signaling and regulatory endpoints indicating that these agonists do not display pathway bias  in agreement with our correlation of maximal effects  for regulatory events was significantly larger than for GαoA activation, Fig. oA activation, respectively  or G protein-biased (MLS1547) did not display these profiles in our hands. MLS1547 was able to stimulate robust G protein activation  and acted as a partial agonist in β-arrestin2 recruitment Tables . This paent Fig. K.35. In this study, we developed and characterized phosphosite-specific antibodies for the D2R against predicted phosphorylation sites within ICL3 and used them to provide an insight into the role GRK phosphorylation plays in D2R regulatory processes and how it is controlled by distinct agonists, including those thought to have pathway-biased actions. One of these antibodies enabled detection of Ser317 and/or Thr318 phosphorylation upon agonist activation mediated by GRK2/3. Comparison of these data with measurements of GRK2 and β-arrestin recruitment revealed that the relative efficacy of all tested agonists at the level pSer317/pThr318 is consistent with their relative efficacy at these other regulatory processes. Measurements of regulatory processes often entail the overexpression of one or more regulatory proteins that are often modified, for example through fusion to a fluorescent protein. The measurement of Ser317/Thr318 phosphorylation can be used in both heterologous expression systems and native tissue without the need for the overexpression of modified proteins. Relative levels of GRK2 expression have been shown to influence the action of agonists at cortical versus striatal D2Rs41. Phospho-site specific antibodies will allow us to understand the role of D2R phosphorylation in such observations.Phosphorylation is a key process to regulate GPCR signaling by promoting β-arrestin binding to inhibit G protein-mediated signaling. GPCR phospho-site specific antibodies have been useful tools with which to understand the hierarchical and sequential pattern of multisite phosphorylation upon agonist stimulation317/Thr318 phosphorylation. However, we also observed residual β-arrestin recruitment in the presence of this inhibitor, suggesting that β-arrestin can be recruited to activated D2Rs independently of Ser317/Thr318 phosphorylation, consistent with previous findings that a ‘phospho-null’ mutant rat D2R in which all putative phosphorylation sites were removed was still able to recruit arrestin8. Two other antibodies developed in this study recognized two additional sites, pThr287/pSer288 and pThr293/pSer296, that appear to be constitutively phosphorylated. The role of this constitutive phosphorylation is unclear, and we did not determine the kinases responsible for it. GRK2/3 phosphorylation has been shown to play a role in post-endocytic trafficking and re-sensitisation42. Thr287, Ser288, and Thr293 have been identified as GRK2 phosphorylation sites important for post-endocytic D2R trafficking8. Interestingly, while Ser317 is conserved in humans and rodents, Thr318 is absent in both mice and rats suggesting there may be species differences in the patterns of GRK phosphorylation. Other kinases are known to regulate D2Rs44, for example PKC can phosphorylate the D2R and regulate function through heterologous desensitization45. An antibody that recognizes a PKC phosphosite in ICL3 of the D2R has previously been described and reveals differences in phosphorylation between D2SR and D2LR46. In this study, we used double phosphorylated peptides to generate the antibodies in order to achieve a broad understanding of GRK mediated phosphorylation of the D2R ICL3. Future efforts to develop phospho-antibodies targeting other GRK and PKC sites, in combination with receptor mutants in which such phosphorylation sites are removed, will allow us to understand the roles of the individual phosphosites and the temporal pattern of this D2R phosphorylation in the modulation of D2R signaling. The antibodies described in the present study recognize phosphosites present in both D2SR and D2LR, although our study was limited to their action at the D2LR. Future work should confirm their activity at phosphosites in the D2SR. This is particularly relevant for the use of these antibodies in tissue or primary neuronal cultures as it will allow the detection of phosphorylation of both pre- and post-synaptic D2Rs.Overexpression of GRK2 enhanced arrestin recruitment and this enhancement was inhibited by a concentration of GRK2/3 inhibitor that prevented Ser47. There has been a surge of interest in D2R biased agonists over the last decade driven by their potential as safer treatments for schizophrenia and Parkinson’s disease53. In this study, we compared the relative ability of twelve agonists to stimulate Ser317/Thr318 phosphorylation to other measures of D2R activation including two ligands that have been described as biased agonists; MLS1547 (G protein-biased) agonist19 and UNC9994 (arrestin-biased)7. UNC9994 was shown to display differential activity at cortical and striatal D2Rs and that this was dependent on the level of GRK2 and βarrestin expression in these different neuronal populations41. We hypothesized that these two ligands may display differential activity at the level of Ser317/Thr318 phosphorylation.Recent interest in understanding GPCR regulatory processes such as β-arrestin recruitment has been driven, to some extent, by the appreciation that β-arrestin-mediated signaling may drive distinct physiological processes to those mediated by G protein signaling, and that one can selectively modulate these processes with biased agonists. GRK phosphorylation has been proposed to a key process that controls the balance between G protein- and β-arrestin-mediated signaling317/Thr318. In the presence of overexpressed GRK2, UNC9994 did not stimulate phosphorylation of Ser317/Thr318 and promoted submaximal recruitment of β-arrestin2. Together these data suggest that UNC9994 has low efficacy for GRK2 phosphorylation, GRK2 recruitment and β-arrestin2 recruitment. We found that UNC9994 also acted as a partial agonist in our measurements of G protein activation in agreement with previous experiments measuring GIRK channel opening in frog oocytes54. The atypical antipsychotic aripiprazole was initially described as a D2R partial agonist, but subsequent studies suggest it may act as a G protein biased agonist55. Analysis of our data using an operational model of agonism revealed that aripiprazole and UNC9994 do not display bias between G protein and β-arrestin recruitment relative to DA. Rather, they behaved in a manner consistent with that of low efficacy agonists. Stimulation with MLS1547 caused recruitment of β-arrestin2 as well as GRK2 recruitment and our analysis revealed no bias between G protein activation and β-arrestin2 recruitment. Previous studies have shown that MLS1547 can induce recruitment of β-arrestin256, and cause D2R internalization in striatal neurons suggesting that this ligand can activate these pathways57.In this present study we found that in the absence of GRK2 overexpression, UNC9994 did not stimulate β-arrestin2 recruitment or phosphorylation of Ser19, no agonism was detected in the unfavoured pathway and, thus, no quantitative measurement of bias could be made. In our studies, we observed sufficient effect in measurements of both G protein activation and β-arrestin recruitment to enable this quantification. The difference between these observations may stem from differences in assay sensitivity, receptor expression levels and/or signal amplification associated with the different studies. In the initial description of UNC9994 it was found to elicit no activity in a used as a measurement of Gi/o/z signalling. However, quinpirole is threefold more potent in the GαoA assay used in the present study, and aripiprazole displays an effect equivalent to that of quinpirole rather than the submaximal effect observed in the cAMP assay. This suggests that the GαoA assay is associated with a greater level of amplification and/or coupling efficiency than the cAMP assay. This can explain the observation that UNC9994 acts as an agonist in our GαoA assay but has no effect in the cAMP assay, as our data shows that UNC9994 is a lower efficacy agonist than aripiprazole. Gαo is proposed to be the primary G protein that the D2R is coupled to in the brain, suggesting that measuring the action of D2R agonists to activate this G protein in particular might be useful in order to understand their physiological effect58.In the original studies that identified MLS1547 and UNC9994 as biased agonists2R-mediated physiological effects60. The interpretation of these studies should be revisited considering our findings. Bromocriptine, cabergoline, terguride, pergolide and roxindole displayed an apparent preference for the regulatory pathways as compared to Gαo activation. These drugs are used to treat hyperprolactinaemia and/or Parkinson’s disease and, with the exception of roxindole, have an ergoline scaffold. It is not apparent how this bias might influence their therapeutic effect, but this observation warrants further investigation.Our results, then, are not contradictory with previous studies, but instead illustrate how experimental conditions and cellular context can greatly influence measurements of agonist action, particularly for low efficacy agonists. Measurement of the action of putative biased agonists at multiple differently amplified steps of a signaling cascade can provide further insight into their mechanism of action. Phosphorylation-site-specific antibodies may be useful tools as part of such characterizations. Biased agonists that display a preference for one pathway over another can be useful tools with which to interrogate the role of distinct downstream signals in a particular physiological process. MLS1547 and UNC9994 have been used to interrogate the contribution of β-arrestins and G proteins to DSupplementary Information 1.Supplementary Information 2.