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Within the past decade, a large increase in structural knowledge from crystallographic studies has significantly fostered our understanding of the structural biology of G protein-coupled receptors (GPCRs). However, information on dynamic events upon receptor activation or deactivation is not yet readily accessed by these structural approaches. GPCR-based fluorescence resonance energy transfer or bioluminescence resonance energy transfer sensors or sensors for interacting proteins (e.g., G proteins or arrestins) can in part cover this gap. The principal design of such sensors was reported 15 years ago. Since then, sensors for almost 20 different GPCRs have been designed. If used with necessary controls and cautious interpretation, such sensors can contribute significantly to our understanding of the basic mechanisms of GPCR function and beyond. In this review, we will discuss the recent developments in this area of GPCR dynamics. [ABSTRACT FROM AUTHOR]

From 2004 to 2010, the Sino-German research group MAGIM (Matter fluxes of Grasslands in Inner Mongolia as affected by grazing) ran a grazing experiment in a typical steppe ecosystem in Inner Mongolia, North China. Multiple ecological effects of grazing, climate variability and topography on plant and animal productivity, plant species composition change, decomposition and mineralization, soil nitrogen and organic matter distributions and dynamics, soil physics and chemistry, and soil-atmosphere gas exchange were measured in fenced plots with defined stocking rates and under different grazing management systems. This paper reviews and synthesizes the most important outcomes, conclusions, and open questions from the different project groups, as published in 125 ISI articles. While greenhouse gas fluxes, plant properties, and livestock performance were particularly responsive to (inter-) annual climate variability, soil properties were more affected by grazing intensity. Various management options based on the project results for semi-arid grasslands under changing climatic conditions are discussed. [ABSTRACT FROM AUTHOR]

Two types of increased atmospheric dust concentration could be distinguished in the Xilingele grassland in the Chinese Province of Inner Mongolia, based on the dust origin i.e. local versus supra-regional type. While the local type is characterized by dust emission, dust events of the supra-regional type cause high dust passages and deposition rates. During dust events the temporal variability of the PM10- and PM1-concentrations, the particle size distribution and the friction velocity u * were measured in 5min intervals using a Laser Dust Monitor (by GRIMM Aerosol GmbH). The threshold friction velocity for local dust emission u *t, at which dust of local soils origin was measured, was above 0.6ms−1. The total suspended particles (TSP) was collected by MWAC catchers and measured by a Laser Particle Sizer (Analysette 22 by Fritsch GmbH). The average D[4/3] particle diameter of the TSP was 23.0μm and the greatest particles measured had sizes of up to 100μm. While fine dust of the PM10 category contained between 58% and 63% of the TSP-mass, coarser particles (>30μm) contributed to about a fourth of the TSP-mass. At the end of some strong dust storms, the dust concentrations remained at a high level even though wind speeds had already slowed down and u * was below 0.3ms−1. These phases were characterized by high deposition rates for dust particles greater than 60μm. [Copyright &y& Elsevier]

Interaction of G-protein-coupled receptors with β-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with β-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with β-arrestin-1-GFP or β-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y1 receptor stimulated with ADP strongly translocated β-arrestin-2-YFP, whereas only a slight translocation was observed for β-arrestin-1-GFP. The P2Y4 receptor exhibited equally strong translocation for β-arrestin-1-GFP and β-arrestin-2-YFP when stimulated with UTP. The P2Y6, P2Y11, and P2Y12 receptor internalized only when GRK2 was additionally co-transfected, but β-arrestin translocation was only visible for the P2Y6 and P2Y11 receptor. The P2Y2 receptor showed a β-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated β-arrestin-1-GFP and β-arrestin-2-YFP equally well, whereas ATP translocated β-arrestin-1-GFP to a much lower extent than β-arrestin-2-YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y2 receptor and β-arrestins. Thus, the P2Y2 receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of β-arrestins by ATP and UTP stimulation of P2Y2 receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with β-arrestins. [ABSTRACT FROM AUTHOR]

Abstract: Wind erosion and dust storms are common phenomena in the semi-arid Xilingele grassland (Inner Mongolia) and contribute considerably to matter balances. Dust emission and dust deposition affect particles of the silt and clay fraction and the organic matter predominantly, the carriers for nutrients in soils. Based on wind erosion measurements in spring 2005 and 2006 the organic carbon and total nitrogen contents of dust were analyzed to determine the contribution of wind erosion processes on C- and N-ratios to the grasslands. Dust deposition is important for the C and N balance in semi-arid grasslands. Despite large differences in dust storm intensities, the Corg and Nt contents in the trapped sediments were in the same range for both years with 35.1–39.1 mg g−1 (Corg) and 3.23–3.60 mg g−1 (Nt). These contents were significantly higher than the average soil contents of Corg and Nt, which were 24.5 mg g−1 and 2.53 mg g−1, respectively. Grazing intensity was the strongest influencing factor on material gains or losses and was reflected strongly in the vegetation conditions (vegetation height and vegetation coverage). A moderate grazing with 1 to 2 ewes per hectare was the grazing practice, in which dust deposition and dust emission were in equilibrium. Heavy grazing (>2 ewes per hectare) resulted in average organic carbon and nitrogen losses of 4.8 kg ha−1 (Corg) and 0.4 kg ha−1 (Nt) in the spring 2005 and 89.8 kg ha−1 (Corg) and 8.3 kg ha−1 (Nt) in the spring 2006. Lightly grazed sites (<1 ewe per hectare) gained between 14.7 and 52.7 kg ha−1 Corg and 1.4 to 4.8 kg ha−1 Nt in 2005 and 129 to 243 kg ha−1 Corg and 11.9 to 22.3 kg ha−1 Nt in 2006. The largest matter gain was measured at ungrazed sites with on average 238.9 kg ha−1 Corg and 20.0 kg ha−1 Nt in 2006. [Copyright &y& Elsevier]

Activation of G protein-coupled receptors like the β1-adrenergic receptor results in conformational changes that ultimately lead to signal propagation through a G protein to an effector like adenylyl cyclase. In this study we identified amino acids that seem to be critical for activation of the human β1-adrenergic receptor. Activation patterns of mutant receptors were analyzed using two structurally different ligands for β1-adrenergic receptors that both are mixed agonist/antagonists. Broxaterol and terbutaline are agonists at β2 and β3-receptors; however, they act as antagonists at the β1-subtype. We reasoned that this functional selectivity may be reflected by a corresponding sequence pattern in the receptor subtypes. Therefore, we exchanged single amino acids of the β1-adrenergic receptor for residues that were identical in the β2- and β3-subtypes but different in the β1-receptor. Pharmacological characterization of such receptor mutants revealed that binding of a panel of agonists and antagonists including broxaterol and terbutaline was unaltered. However, two of the mutants (1185V and D212N) were activated by broxaterol and terbutaline, which acted as antagonists at the wild-type receptor. Two additional mutants (V120L and K253R) could be activated by terbutaline alone, which is structurally more closely related to endogenous catecholamines like epinephrine than to broxaterol. A model of the human β1-adrenergic receptor showed that the four gain-of-function mutations are outside of the putative ligand-binding domain substantiating the lack of an effect of the mutations on binding characteristics. These results support the notion that Val-120, Ile-185, Asp-212, and Lys-253 are critically involved in conformational changes occurring during receptor activation. [ABSTRACT FROM AUTHOR]

Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a well-established method to monitor protein-protein interactions or conformational changes of individual proteins. But protein functions can be perturbed by fusion of large tags such as CFP and YFP. Here we use G protein–coupled receptor (GPCR) activation in living cells as a model system to compare YFP with the small, membrane-permeant fluorescein derivative with two arsen-(III) substituents (fluorescein arsenical hairpin binder; FlAsH) targeted to a short tetracysteine sequence. Insertion of CFP and YFP into human adenosine A2A receptors allowed us to use FRET to monitor receptor activation but eliminated coupling to adenylyl cyclase. The CFP/FlAsH-tetracysteine system gave fivefold greater agonist-induced FRET signals, similar kinetics (time constant of 66–88 ms) and perfectly normal downstream signaling. Similar results were obtained for the mouse α2A-adrenergic receptor. Thus, FRET from CFP to FlAsH reports GPCR activation in living cells without disturbing receptor function and shows that the small size of the tetracysteine-biarsenical tag can be decisively advantageous. [ABSTRACT FROM AUTHOR]

P2Y receptors are G protein-coupled receptors stimulated by extracellular nucleotides. Both the P2Y1 and the P2Y6 receptors are preferentially activated by nucleoside 5′-diphosphates, but favor different base moieties. In the case of the P2Y1 receptor the preferred base is adenine, while the P2Y6 receptor is activated by uracil nucleotides. To identify potential amino acid domains that interact with the base moiety, we used a chimeric receptor approach, employing the human P2Y1 receptor as core structure to investigate the role in receptor activation of extracellular loops (ELs) and transmembrane domains (TMs) of the rat P2Y6 receptor. The chimeric receptors were expressed in COS-7 cells and measured for stimulation of phospholipase C (PLC) induced by the potent P2Y1 receptor agonist 2-MeSADP or the potent P2Y6 receptor agonist UDP. Replacement of the N-terminus or EL2 resulted in low (∼50μM) potency of the agonist 2-MeSADP, thus confirming the importance of EL2 in ligand recognition. Upon replacement of several regions, the potency of the P2Y1 agonist 2-MeSADP was either 1–2μM (N-terminus and EL1, or EL1 and EL3) or 72μM (N-terminus and EL3). Concurrent replacement of three regions (N-terminus, EL1, and EL3) completely precluded activation by 2-MeSADP. Our study identified domains of the P2Y6 receptor that contribute to receptor activation by UDP and hence seem to be involved in uracil recognition. Upon replacement with extracellular domains of the P2Y6 receptor sequence we observed a trend toward gain of receptor-induced PLC activation by UDP, for example, in the chimera containing replacements of both the N-terminus and EL1. Exchange of three receptor domains led to a construct with an EC50 value for UDP of 19μM and a maximal inositol phosphate accumulation similar to the native P2Y6 receptor. Within receptor constructs of combined domain exchanges the additional substitution of Tyr110 by the corresponding Asn from the P2Y6 receptor showed a significant increase for activation by UDP, but only when combined with the N-terminal domain and TM1. The residue Tyr110 was identified to play an important role in the recognition of the nucleobase in the P2Y1 and P2Y6 receptors. [Copyright &y& Elsevier]

Constitutive activity and inverse agonism have been described for numerous G-protein-coupled receptors, including the β2-adrenergic receptor. We have investigated whether these properties also exist for the β1-adrenergic receptor. One line of experiments was done with cell lines transiently or stably expressing the human β1-adrenergic receptor measuring intracellular cAMP or adenylyl cyclase activity. In a second set of experiments, we used transgenic mice with cardiac overexpression of the human β1-adrenergic receptor and measured spontaneous beating frequency of isolated atria as a physiological read-out. Both sets of experiments revealed that the human β1-adrenergic receptor displays constitutive activity, but this constitutive activity is considerably lower than that of the β2-subtype. We then tested various β-adrenergic antagonists for their ability to modulate this constitutive activity. In the β1-receptor-transgenic atria, the β1-selective antagonists metoprolol and bisoprolol showed significant inverse agonistic activity, whereas carvedilol behaved as a neutral antagonist. In contrast, in cellular cAMP-assays, metoprolol, bisoprolol, propranolol and carvedilol all displayed similar inverse agonist activities. We conclude that also the human β1-adrenergic receptor displays constitutive activity and that—depending on the read-out—β-adrenergic “antagonists” currently in clinical use may differ in their inverse agonistic activity at this receptor. [Copyright &y& Elsevier]

Desensitisation of the mu-opioid receptor (MOR) is proposed to underlie the initiation of opioid analgesic tolerance and previous work has shown that agonist-induced phosphorylation of the MOR C-tail contributes to this desensitisation. Moreover, phosphorylation is important for β-arrestin recruitment to the receptor, and ligands of different efficacies induce distinct phosphorylation barcodes. The C-tail 370TREHPSTANT379 motif harbours Ser/Thr residues important for these regulatory functions. 375Ser is the primary phosphorylation site of a ligand-dependent, hierarchical, and sequential process, whereby flanking 370Thr, 376Thr and 379Thr get subsequently and rapidly phosphorylated. Here we used GRK KO cells, phosphosite specific antibodies and site-directed mutagenesis to evaluate the contribution of the different GRK subfamilies to ligand-induced phosphorylation barcodes and β-arrestin2 recruitment. We show that both GRK2/3 and GRK5/6 subfamilies promote phosphorylation of 370Thr and 375Ser. Importantly, only GRK2/3 induce phosphorylation of 376Thr and 379Thr, and we identify these residues as key sites to promote robust β-arrestin recruitment to the MOR. These data provide insight into the mechanisms of MOR regulation and suggest that the cellular complement of GRK subfamilies plays an important role in determining the tissue responses of opioid agonists. CRISPR/Cas9 knock-out of GRKs combined with mutagenesis and phospho-specific antibodies reveal the sites of the mu-opioid receptor that underlie the differences in regulation of this receptor by morphine and fentanyl. [ABSTRACT FROM AUTHOR]

Studies the role of extracellular loops of G protein-coupled receptors (GPCR) in ligand recognition. Stimulation of GPCR P2Y1 receptor by extracellular adenosine diphosphate and adenosine triphosphate; Site-directed mutagenesis of the extracellular loops; Calculation of energetically-sound conformational hypothesis for the receptor.

Soil landscape research is faced with wide-ranging questions of soil erosion, precision farming, and agricultural risk management. Digital Soil Morphometrics is a powerful tool to provide respective answers or recommendations but requires soil data from the pedon-to-field scale with high horizontal and vertical resolutions, including the subsoil. We present an efficient sampling and measurement method for easily obtainable soil driving cores with low-destructive preparation. Elemental contents and soil organic and mineral matter composition were measured rapidly and in large numbers using a multi-sensor approach, i.e., visible and near-infrared (Vis-NIR), diffuse reflectance infrared Fourier transform (DRIFT), and X-ray fluorescence (XRF) spectroscopy. The suitability of the approach with respect to three-dimensional soil landscape models was tested using soils along a slope representing different stages of erosion and deposition in a hummocky landscape under arable land use (Calcaric Regosols, Calcic Luvisols, Luvic Stagnosols, Gleyic-Colluvic Regosols). The combination of soil core sampling, pedological description, and three spectroscopic techniques enabled rapid determination and interpretation of horizontal and vertical spatial distributions of soil organic carbon (SOC), soil organic and mineral matter composition, as well as CaCO 3 , Fe, and Mn contents. Depth profiles for SOC, CaCO 3 , and Fe contents were suitable indicators for site-specific degrees of erosion and matter transport processes at the pedon-to-field scale. Fe and Mn profiles helped identifying zones of reductive and oxic domains in subsoils (gleyzation). Further methodical developments should implement plant-availability of nutrients, characterization of Fe-oxides, and calibration of the spectroscopic techniques to field-moist samples. • An efficient sampling and measurement method for undisturbed soil cores is presented. • Detection of element contents and SOM composition in high horizontal and vertical resolution • Combination of pedological description, Vis-NIR, DRIFT, XRF, and laboratory analyses • Stages of erosion and deposition assigned to soil properties, e.g. SOC, CaCO 3 , Fe contents • Spatial Mn and Fe distributions (vertical, horizontal) reflect different redox domains. [ABSTRACT FROM AUTHOR]

Sepsis is characterized as life-threatening organ dysfunction caused by a dysregulated host response to an infection. Despite numerous clinical trials that addressed this syndrome, there is still no causative treatment available to dampen its severity. Curtailing the infection at an early stage with anti-infectives is the only effective treatment regime besides intensive care. In search for additional treatment options, we recently discovered the inhibition of the sphingosine 1-phosphate (S1P) lyase and subsequent activation of the S1P receptor type 3 (S1PR3) in pre-conditioning experiments as promising targets for sepsis prevention. Here, we demonstrate that treatment of septic mice with the direct S1P lyase inhibitor C31 or the S1PR3 agonist CYM5541 in the advanced phase of sepsis resulted in a significantly increased survival rate. A single dose of each compound led to a rapid decline of sepsis severity in treated mice and coincided with decreased cytokine release and increased lung barrier function with unaltered bacterial load. The survival benefit of both compounds was completely lost in S1PR3 deficient mice. Treatment of the murine macrophage cell line J774.1 with either C31 or CYM5541 resulted in decreased protein kinase B (Akt) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) phosphorylation without alteration of the mitogen-activated protein kinase (MAPK) p38 and p44/42 phosphorylation. Thus, activation of S1PR3 in the acute phase of sepsis by direct agonism or S1P lyase inhibition dampened Akt and JNK phosphorylation, resulting in decreased cytokine release, improved lung barrier stability, rapid decline of sepsis severity and better survival in mice. [Display omitted] • Increased survival rate by S1PR3 stimulation in the advanced phase of sepsis. • Signaling via reduced Akt and JNK phosphorylation. • Decrease of cytokine release and increase of lung barrier stability. • Activity of S1PR3 agonist CYM5541 and S1P lyase inhibitor C31 in severe sepsis. • S1PR3 agonists as potential treatment option in critically ill sepsis patients. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent β-arrestin recruitment. The ubiquitously expressed GRKs are classified into cytosolic GRK2/3 and membrane-tethered GRK5/6 subfamilies. GRK2/3 interact with activated G protein βγ-subunits to translocate to the membrane. Yet, this need was not linked as a factor for bias, influencing the effectiveness of β-arrestin-biased agonist creation. Using multiple approaches such as GRK2/3 mutants unable to interact with Gβγ, membrane-tethered GRKs and G protein inhibitors in GRK2/3/5/6 knockout cells, we show that G protein activation will precede GRK2/3-mediated β-arrestin2 recruitment to activated receptors. This was independent of the source of free Gβγ and observable for Gs-, Gi- and Gq-coupled GPCRs. Thus, β-arrestin interaction for GRK2/3-regulated receptors is inseparably connected with G protein activation. We outline a theoretical framework of how GRK dependence on free Gβγ can determine a GPCR's potential for biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we anticipate generation of β-arrestin-biased ligands to be mechanistically challenging for the subgroup of GPCRs exclusively regulated by GRK2/3, but achievable for GRK5/6-regulated receptors, that do not demand liberated Gβγ. Accordingly, GRK specificity of any GPCR is foundational for developing arrestin-biased ligands. Using cytosolic or membrane-tethered GRK2/3 mutants and G protein inhibitors, authors show that G protein activation, releasing free Gβγ, and GRK2/3-mediated β-arrestin recruitment are inseparably intertwined. This adds to the concept of biased agonism. [ABSTRACT FROM AUTHOR]

In the frame of the joint research project BonaRes ("Soil as a sustainable resource for the bioeconomy", Germany) a data infrastructure has been set-up to upload, manage, store, and provide soil and agricultural research data, accompanying data (e.g. management, socioeconomic or meteorological data) and associated metadata. A consistent and standardized storage and provision of well-described research data within the BonaRes data infrastructure enhance the research visibility and data accessibility via the BonaRes Data Portal, enable the long-term availability and re-usability, and, not least, facilitate interoperability with international data infrastructures. The developed BonaRes Metadata Schema (Specka et al., submitted) is based on existing and accepted international schemes, combines all elements from DataCite and INSPIRE and uses controlled vocabularies (Agrovoc and GEMET) to indexing data sets. The BonaRes data infrastructure thus fulfils FAIR data principles (data should be "Findable, Accessible, Interoperable, and Re-usable") and requirements for Open Data.We present the current state of the BonaRes Data Portal, show typical data workflows from data owner to data user, discuss solutions to manage and standardize highly diverse research data e.g. from agricultural long-term experiments, give insights to metadata elements, demonstrate data accessibility, introduce into data policy elements, e.g. embargo times and licenses, and provide an outlook of future progress.Reference:Specka, X. Gärtner, P., Svoboda, N., Stecker, M., Einspanier, U., Senkler, K., Hoffmann, C., Zoarder, M.A.M., Kühnert, T., Heinrich, U. (submitted to Computer & Geoscience): The BonaRes Metadata Model for geospatial soil-agricultural research data – Merging INSPIRE and DataCite metadata schemes. [ABSTRACT FROM AUTHOR]

The interaction effects of different fertilisation regimes and weather variability on crop yield is a challenge that requires long-term investigation. Therefore, yield data for spring barley (SB) in an agricultural long-term field experiment, established in 1963 in Müncheberg, northeast Germany, were analysed to reveal the effects of 21 fertiliser regimes and different weather conditions on SB yields. SB yields were significantly affected by fertilisation regimes (11%), annual weather conditions (55%) and their interaction effect (8%). Mineral N fertilization decreased overall yield variability across seasons as compared to no fertilization and organic fertilization regimes showed higher yield variability. A suitable combined application of mineral nitrogen and organic fertiliser was found to be an effective way to produce higher SB yields than the application of either mineral nitrogen or organic fertiliser alone. A Bayesian linear regression model showed total precipitation during the growing season (April–July) positively affected on SB yields when high mineral N was supplied. At the early growth stage, a precipitation rate (March) and temperature (April or sowing day) negatively affected on SB yield. [ABSTRACT FROM AUTHOR]

A spatial data infrastructure (SDI) for the upload and provision of soil-agricultural research data in Germany was developed and launched in 2017. The precondition for the new SDI were to be compliant with the European initiative for spatial information (INSPIRE), to consider FAIR data principles, to be interoperable with other disciplinary national and international SDIs and to support dataset registrations with digital object identifiers (DOI). To meet these requirements, the new SDI had to support both the INSPIRE and DataCite metadata standards. As there was no metadata standard available that supported both metadata schemas from different disciplines, it became necessary to merge these two existing standards. The objective of this paper is to describe the developmental steps of the newly created BonaRes metadata schema for geospatial soil-agricultural research data. First, we analyzed the INSPIRE and DataCite metadata schemas, identified and compared semantically equivalent metadata elements, including properties and allowed content values, for potential mapping. In the next step, we specified the new metadata model, whose elements were derived from INSPIRE or DataCite under consideration of the previously developed mapping. Third, additional metadata elements were defined to directly describe the underlying data model. The advantages and disadvantages of the method that was applied to define the BonaRes metadata schema elements originating from INSPIRE and DataCite schemas are presented and discussed. By implementing the new metadata model, the SDIs can achieve cross-portal metadata interoperability with other INSPIRE-compliant SDIs. Furthermore, researchers who store their geospatial research data in SDIs to implement the BonaRes metadata schema can increase the visibility and findability of their research, as the data can be assigned a DOI, which is a prerequisite for data citations and data publications. The new metadata elements of the BonaRes metadata schema allows targeted dataset queries, e.g., soil pH values, yields or parameters, which were measured by a specific method, using the underlying data model, and improves the discovery and reusability of research data. Image 1 • The BonaRes metadata schema supporting INSPIRE and DataCite is described. • Semantic mapping between INSPIRE and DataCite elements. • Specification of the elements using the steps of a metadata crosswalk. • Extended metadata elements for information on the data model. [ABSTRACT FROM AUTHOR]

β-arrestins play a key role in G protein–coupled receptor (GPCR) internalization, trafficking, and signaling. Whether βarrestins act independently of G protein–mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., βarrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2- adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR–Cas9 gene edited for Gαs, β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and β-arrestins in controlling gene expression. We found that Gαs is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity. [ABSTRACT FROM AUTHOR]

Advances in resolving crystal structures of GPCRs and their binding partners as well as improvements in live-cell microscopy and the fluorescent proteins pallet has greatly driven new ideas for designing optical sensors for the same. Sensors have been developed to monitor ligand binding as well as the ensuing ligand-induced conformational changes in GPCRs, G-proteins and arrestins. In this review we will highlight the functionality of such sensor designs starting from monitoring ligand binding to receptor activation and interaction with arrestins. Furthermore, we will highlight the importance of sensor designs to monitor receptor-dependent arrestin conformations and give an idea about the various detected arrestin conformations and their possible implications. [ABSTRACT FROM AUTHOR]

Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phosphoacceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the a5 helix of Ga13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis. [ABSTRACT FROM AUTHOR]

G protein‐coupled receptors (GPCRs) constitute the largest family of transmembrane proteins and play a crucial role in regulating diverse cellular functions. They transmit their signaling via binding to intracellular signal transducers and effectors, such as G proteins, GPCR kinases, and β‐arrestins. To influence specific GPCR signaling behaviors, β‐arrestins recruit effectors to form larger signaling complexes. Intriguingly, they facilitate divergent functions for the binding to different receptors. Recent studies relying on advanced structural approaches, novel biosensors and interactome analyses bring us closer to understanding how this specificity is achieved. In this article, we share our hypothesis of how active GPCRs induce specific conformational rearrangements within β‐arrestins to reveal distinct binding interfaces, enabling the recruitment of a subset of effectors to foster specialized signaling complexes. Furthermore, we discuss methods of how to comprehensively assess β‐arrestin conformational states and present the current state of research regarding the functionality of these multifaceted scaffolding proteins. [ABSTRACT FROM AUTHOR]

In the last few years, fluorescence resonance energy transfer (FRET) receptor sensors have contributed to the understanding of GPCR ligand binding and functional activation. FRET sensors based on muscarinic acetylcholine receptors (mAChRs) have been employed to study dual-steric ligands, allowing for the detection of different kinetics and distinguishing between partial, full, and super agonism. Herein, we report the synthesis of the two series of bitopic ligands, 12-Cn and 13-Cn, and their pharmacological investigation at the M1, M2, M4, and M5 FRET-based receptor sensors. The hybrids were prepared by merging the pharmacophoric moieties of the M1/M4-preferring orthosteric agonist Xanomeline 10 and the M1-selective positive allosteric modulator 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) 11. The two pharmacophores were connected through alkylene chains of different lengths (C3, C5, C7, and C9). Analyzing the FRET responses, the tertiary amine compounds 12-C5, 12-C7, and 12-C9 evidenced a selective activation of M1 mAChRs, while the methyl tetrahydropyridinium salts 13-C5, 13-C7, and 13-C9 showed a degree of selectivity for M1 and M4 mAChRs. Moreover, whereas hybrids 12-Cn showed an almost linear response at the M1 subtype, hybrids 13-Cn evidenced a bell-shaped activation response. This different activation pattern suggests that the positive charge anchoring the compound 13-Cn to the orthosteric site ensues a degree of receptor activation depending on the linker length, which induces a graded conformational interference with the binding pocket closure. These bitopic derivatives represent novel pharmacological tools for a better understanding of ligand-receptor interactions at a molecular level. [ABSTRACT FROM AUTHOR]

Traditional methods to analyze G protein-mediated signaling mostly are done using biochemical methods requiring cell homogenization. To analyze signal transduction processes in living cells, we established a fluorescence-resonance-energytransfer (FRET-) based assay to monitor the interaction of P2Y1 receptors with Gq proteins. To do so, YFP was fused to the C terminus of the P2Y1 receptor, and coexpressed with Gαq β1 CFP-γ2 in HEK293T cells. We recorded single-cell fluorescence while perfusing the cell with the agonist ADP. Upon agonist stimulation, an increase in YFP and a decrease in CFP fluorescence was observed, leading to an increase in ratiometric FRET (Fig. 1) which, in amplitude and onset kinetics, depended on agonist concentration. This FRET-based assay will allow to characterize early steps in P2Y1 receptor signaling in living ceils. Fig. 1: P2Y1-YFP and Gαq β1 CFP-γ2 expressing cells were stimulated with the agonist ADP, leading to an increase in ratiometric FRET. [ABSTRACT FROM AUTHOR]

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening. A G protein-coupled receptors (GPCRs) phosphorylation assay for cell culture plates can be used for ligand profiling and inhibitor screening, as evidenced by the identification of two GRK inhibitor compounds. [ABSTRACT FROM AUTHOR]

In two swordtail species of the genus Xiphophorus, the onset of puberty has been shown to be modulated at the P locus by sequence polymorphism and gene copy-number variation affecting the type 4 melanocortin hormone receptor Mc4r. The system works through the interaction of two allelic types, one encoding wild type and the other dominant-negative receptors. We have analyzed the structure and evolution of the P locus in the platyfish Xiphophorus maculatus, where as many as nine alleles of P determining the onset of sexual maturity in males and females, fecundity in females, and adult size in males are located on both the X and Y chromosomes in a region linked to the master sex-determining locus. In this species, mc4r has been amplified to up to 10 copies on both the X and Y chromosomes through recent large serial duplications. Subsequently, mc4r paralogues have diverged considerably into many different subtypes. Certain copies have acquired new untranslated regions through genomic rearrangements, and transposable element insertions and other mutations have accumulated in promoter regions, possibly explaining observed deviations from the classical mc4r transcriptional pattern. In the mc4r-coding sequence, in-frame insertions and deletions as well as nonsense and missense mutations have generated a high diversity of Mc4r-predicted proteins. Most of these variants are expressed in embryos, adults, and/or tumors. Functional receptor characterization demonstrated major divergence in pharmacological behavior for Mc4r receptors encoded by different copies of platyfish mc4r, with differences in constitutive activity as well as binding and stimulation by hormones. The high degree of allelic and copy-number variation observed between individuals can explain the high level of polymorphism for sexual maturation, fecundity, and body size in the platyfish: multiple combinations of Mc4r variants with different biochemical properties might interact to modulate the melanocortin signaling that regulates the hypothalamus--pituitary--gonadal axis. [ABSTRACT FROM AUTHOR]

Agonist-induced phosphorylation is a crucial step in the activation/deactivation cycle of G protein-coupled receptors (GPCRs), but direct determination of individual phosphorylation events has remained a major challenge. We have recently developed a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in 96-well plates, thus eliminating the need for western blot analysis. In the present study, we adapted this assay to three novel phosphosite-specific antibodies directed against the neurokinin 1 (NK1) receptor, namely pS338/pT339-NK1, pT344/pS347-NK1, and pT356/pT357-NK1. We found that substance P (SP) stimulated concentration-dependent phosphorylation of all three sites, which could be completely blocked in the presence of the NK1 receptor antagonist aprepitant. The other two endogenous ligands of the tachykinin family, neurokinin A (NKA) and neurokinin B (NKB), were also able to induce NK1 receptor phosphorylation, but to a much lesser extent than substance P. Interestingly, substance P promoted phosphorylation of the two distal sites more efficiently than that of the proximal site. The proximal site was identified as a substrate for phosphorylation by protein kinase C. Analysis of GPCR kinase (GRK)-knockout cells revealed that phosphorylation was mediated by all four GRK isoforms to similar extents at the T344/S347 and the T356/T357 cluster. Knockout of all GRKs resulted in abolition of all phosphorylation signals highlighting the importance of these kinases in agonist-mediated receptor phosphorylation. Thus, the 7TM phosphorylation assay technology allows for rapid and detailed analyses of GPCR phosphorylation. [ABSTRACT FROM AUTHOR]

Aims: The aims of this study were to identify areas of wind erosion and dust deposition and to quantify the effects of different grazing intensities on soil redistribution rates in grasslands based on the Cs technique. Because the method uses a reference inventory as threshold for erosion or deposition, the classification of any other site as source or sink for dust depends on the accurate selection of this reference site. Methods: Measurements of Cs inventories and depth distributions were carried out at pasture sites with predominant species of Stipa grandis and Leymus chinensis which are grazed with different intensities. Additional measurements were made at arable land, plant-covered sand dunes and alluvial plains. Wind-induced soil erosion and dust deposition rates were calculated from Cs inventories by means of the 'Profile-Distribution' and the 'Mass Balance II' models. Results: The selection of the reference site was based on fluid dynamical and process-determining parameters. The chosen site should meet the following four conditions: (i) located at a summit position with obviously low deposition rates, (ii) sufficient vegetation cover to prevent wind erosion, (iii) plane to exclude water erosion and (iv) in the wind/dust shadow of a higher elevation. The measured reference inventory of Cs was 1967(±102) Bqm located at a summit position of moderately grazed Leymus chinensis steppe. The Cs inventories at other sites ranged from 1330 Bqm at heavily grazed sites to 5119 Bqm at river deposits, representing annual average soil losses of up to 130 tkm and deposits of up to 540 tkm, respectively. The calculated annual averages of dust depositions at ungrazed Leymus chinensis sites were related to the dust storm frequencies of the last 50 years resulting in a description of the temporal variability of annual dust depositions from about 154 tkm in the 1960s to 26 tkm at recent times. Based on this quantification already 80% of the total dust depositions can be related to the 20 years between the 1960s and the end of the 1970s and only 20% to the time between 1980 and 2001. Conclusions: Cs technique is a promising method to assess the effect of grazing intensity and land use types on the spatial variability of wind-induced soil and dust redistribution processes in semi-arid grasslands. However, considerable efforts are needed to identify a reliable reference site, because erosion and deposition induced by wind may occur at the same places. The combination of the dust deposition rates derived from Cs profile data with the dust storm frequencies is helpful for a better reconstruction of the temporal variability of dust deposition and wind erosion in this region. The calculated recent deposition rates of about 20 tkm are in good agreement with data of other authors. [ABSTRACT FROM AUTHOR]

The concept of "functional selectivity" or "biased signaling" suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the β2-adrenergic receptor (β2AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged β2AR FRET sensor. In this β2AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard "full" agonist isoproterenol. Furthermore, norepinephrine-induced changes in the β2AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial β2AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of Gs and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering β-arrestin2 recruitment to the cell surface and its interaction with β2AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the β2AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to Gs and less efficient in signaling to β-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the β2AR and link it to distinct conformational changes in the receptor. [ABSTRACT FROM AUTHOR]

Abstract: The melanocortin type 1 receptor (Mc1r) belongs to a family of G-protein-coupled receptors involved in various physiological processes in vertebrates. Melanocortins, the Mcr natural agonists, are pituitary peptide hormones including adrenocorticotropin and melanocyte-stimulating hormones. In mammals and birds, Mc1r is involved in pigmentation and expressed in melanocytes and melanoma. Activation of Mc1r leads to eumelanin production as well as to proliferation and survival of melanocytes in the epidermis. Here we report the molecular and evolutionary analysis of mc1r from three major fish models, the zebrafish Danio rerio, the medaka Oryzias latipes and the platyfish Xiphophorus maculatus. In contrast to some other melanocortin receptor genes, mc1r has been conserved as a single copy gene in divergent fish species. Its expression was detected in all organs tested in platyfish and medaka but was restricted to eyes, skin, brain and testis in zebrafish, this possibly reflecting differences in the distribution of extracutaneous melanophores. The mc1r gene was found to be expressed during embryogenesis, as well as in Xiphophorus hybrid melanoma, similar to human tumours. Protein sequence comparison between fish and mammalian Mc1r revealed a remarkable concordance between evolutionary and functional analyses for the identification of residues and regions critical for receptor function. [Copyright &y& Elsevier]

A large variety of techniques has been used to monitor activation of G protein-coupled receptors (GPCRs) both in isolated membranes and in intact cells. However, most of these techniques cannot resolve receptor activation and signaling in space and in time. Here, we describe techniques that allow the temporally and spatially resolved monitoring of these processes by optical recording with energy transfer techniques. Fluorescence and bioluminescence resonance energy transfer, FRET and BRET, are based on energy transfer between two closely spaced probes. The exquisite sensitivity of FRET and BRET to the distance of the two probes makes these techniques ideal tools to study either protein–protein interactions (when the two probes are localized on two different proteins) or conformational changes within a given protein (when the two probes are localized on a single protein). Here, we review the latter approach as a tool to study receptor activation and the levels of the second messengers cAMP and cGMP in intact cells. [Copyright &y& Elsevier]

To analyze individual steps of GS-linked signaling in intact cells, we used fluorescence resonance energy transfer (FRET)-based assays for receptor-G protein interaction, G protein activation, and cAMP effector activation. To do so, we developed a FRET-based sensor to directly monitor GS activation in living cells. This was done by coexpressing a Gαs mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged Gβγ subunits and appropriate receptors in HEK293 cells. Together with assays for receptor activation and receptor-G protein interaction, it is possible to characterize large parts of the GS signaling cascade. When A2A-adenosine or β1-adrenergic receptors are coexpressed with GS in HEK293T cells, the receptor-GS interaction was on the same time scale as A2A receptor activation with a time constant of <50 ms. GS activation was markedly slower and around 450 ms with similar kinetics following activation of A2A- or β1-receptors. Taken together, our kinetic measurements demonstrate that the rate of GS activation limits initiation of GS-coupled receptor signaling. [ABSTRACT FROM AUTHOR]

The interaction of activated G protein-coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)-based assay, we measure directly and in living cells the interaction of YFP-labeled α2A-adrenergic receptors with CFP-labeled G proteins. Upon agonist stimulation, a small, concentration-dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein interaction were <100 ms and depended on expression levels of Gα. Simultaneously recorded G protein-regulated inwardly rectifying K+ channel currents revealed a maximal current response already at agonist concentrations producing submaximal FRET amplitudes. By analyzing FRET signals in the presence of a Gα mutant, which dissociates more slowly from activated receptors, it was demonstrated that only a fraction of wild-type G proteins interacts with the activated receptor at any time. Our data suggest that α2A-adrenergic receptors and G proteins interact by rapid collision coupling and indicate that there is no significant precoupling between these receptors and G proteins. [ABSTRACT FROM AUTHOR]

A3 adenosine receptor (A3AR) agonists have been reported to influence cell death and survival. The effects of an A3AR agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-β-d-ribofuranonamide (Cl-IB-MECA), on apoptosis in two human leukemia cell lines, HL-60 and MOLT-4, were investigated. Cl-IB-MECA (≥30 μM) increased the apoptotic fractions, as determined using fluorescence-activated cell sorting (FACS) analysis, and activated caspase 3 and poly-ADP-ribose-polymerase. Known messengers coupled to A3AR (phospholipase C and intracellular calcium) did not seem to play a role in the induction of apoptosis. Neither dantrolene nor BAPTA-AM affected the Cl-IB-MECA-induced apoptosis. Cl-IB-MECA failed to activate phospholipase C in HL-60 cells, while UTP activated it through endogenous P2Y2 receptors. Induction of apoptosis during a 48 hr exposure to Cl-IB-MECA was not prevented by the A3AR antagonists [5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate] (MRS 1220) or N-[9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-yl]benzeneacetamide (MRS 1523). Furthermore, higher concentrations of MRS 1220, which would also antagonize A1 and A2A receptors, were ineffective in preventing the apoptosis. Although Cl-IB-MECA has been shown in other systems to cause apoptosis through an A3AR-mediated mechanism, in these cells it appeared to be an adenosine receptor-independent effect, which required prolonged incubation. In both HL-60 and MOLT-4 cells, Cl-IB-MECA induced the expression of Fas, a death receptor. This induction of Fas was not dependent upon p53, because p53 is not expressed in an active form in either HL-60 or MOLT-4 cells. Cl-IB-MECA-induced apoptosis in HL-60 cells was augmented by an agonistic Fas antibody, CH-11, and this increase was suppressed by the antagonistic anti-Fas antibody ZB-4. Therefore, Cl-IB-MECA induced apoptosis via a novel, p53-independent up-regulation of Fas. [Copyright &y& Elsevier]

Long-term field experiments (LTE) are highly valuable infrastructures in agricultural- and soil sciences for understanding the long-term impacts of climate and management practices. While they are designed to run under constant conditions, climate change is expected to affect site conditions considerably. This needs to be quantified when interpreting experimental results and when redesigning the experimental setup. One way to achieve this is by utilizing vegetation growth and carbon dynamics, specifically the Net Primary Productivity (NPP), as a spatially explicit indicator. NPP facilitates the assessment and interpretation of yield performance in LTEs under future climatic conditions. Our study estimated the changes in NPP for 271 LTE sites in Germany, comparing a baseline (2000–2020) with two scenarios (2081–2100) that were based on the Shared Socioeconomic Pathways (SSPs) (SSP245) and SSP585) by the Intergovernmental Panel on Climate Change (IPCC). We used the NASA-CASA biogeochemical model to calculate NPP in baseline and IPCC scenarios using Germany as a test case. LTEs were grouped by land use (crop types) and soil information (soil type, texture), drawing on the geodata infrastructure "BonaRes Repository". The total annual terrestrial NPP for the baseline was calculated as 202.4 Mt C (sum of forests, grasslands, and arable lands) in Germany, while total NPP was up to 56.0 Mt C for different land use types. For both scenarios, NPP was projected to increase in LTEs located in southern Germany, indicating increased crop productivity, while a decrease was projected for the central Germany. The decrease in NPP of numerous LTEs in central Germany was estimated to extend to the LTEs in the eastern part corresponding to the worst-case scenario SSP585. Explicitly, the use of the multi-model ensemble mean as the climate driver in modelling may overestimate projected NPP by reducing inter-annual variability, highlighting the importance of methodological choices for accurate future projections. Besides, the results indicated that poor soils are projected to experience a further decline in productivity, primarily attributed to escalating water scarcity. Conversely, soils with high quality are likely to witness enhanced productivity, largely driven by the extension of the growing seasons. The outcomes of this study provide a basis for considering the future conditions of German LTEs and facilitate distinguishing between the effects of climate change and the impact of agricultural management on productivity at the regional level. These outputs enable planning and developing research strategies for selecting future LTE sites and redesigning existing or newly planned experiments. Moreover, the integrated modelling framework presented here highlights the potential of LTE data for large-scale modelling studies of ecosystem functions. [Display omitted] • The effects of climate change on the Net Primary Productivity (NPP) of Long-Term Field Experiments were simulated. • Metadata of multiple LTEs were incorporated with NPP modelling using Germany as a test case. • A remarkable NPP decrease was found in the LTEs in central Germany, shifting to the east in future. • Poor soils experienced a disproportionate reduction in NPP compared to good soils primarily related to water scarcity. • The importance of soil quality for crop productivity is likely to increase in the future.. [ABSTRACT FROM AUTHOR]

Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4. [ABSTRACT FROM AUTHOR]

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth. [ABSTRACT FROM AUTHOR]

The earlier an activation of a G protein-dependent signalling cascade at a G protein-coupled receptor (GPCR) is probed, the less amplificatory effects contribute to the measured signal. This is especially useful in case of a precise quantification of agonist efficacies, and is of paramount importance, when determining agonist bias in relation to the β-arrestin pathway. As most canonical assays with medium to high throughput rely on the quantification of second messengers, and assays affording more proximal readouts are often limited in throughput, we developed a technique with a proximal readout and sufficiently high throughput that can be used in live cells. Split luciferase complementation (SLC) was applied to assess the interaction of Gαq with its effector phospholipase C-β3. The resulting probe yielded an excellent Z’ value of 0.7 and offers a broad and easy applicability to various Gαq-coupling GPCRs (hH1R, hM1,3,5R, hNTS1R), expressed in HEK293T cells, allowing the functional characterisation of agonists and antagonists. Furthermore, the developed sensor enabled imaging of live cells by luminescence microscopy, as demonstrated for the hM3R. The versatile SLC-based probe is broadly applicable e.g. to the screening and the pharmacological characterisation of GPCR ligands as well as to molecular imaging. [ABSTRACT FROM AUTHOR]

β-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-β-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in β-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that β-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, they indicate that, following receptor interaction, the plasma membrane stabilizes β-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of β-arrestin function at the plasma membrane, revealing a critical role for β-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation. [Display omitted] • β-arrestin spontaneously preassociates with the plasma membrane via its C-edge • Preassociated β-arrestin interacts with receptors via lateral diffusion • Receptor-β-arrestin interactions are short lived and trigger β-arrestin activation • Active β-arrestin and receptors reach clathrin-coated pits separately via diffusion Spontaneous preassociation of β-arrestin with the plasma membrane facilitates its interactions with receptors and subsequent activation. [ABSTRACT FROM AUTHOR]

In agricultural landscapes, the spatio-temporal distribution of organic matter (OM) varies greatly across landscape structures and soil types. We investigated patterns of organic carbon (OC) content, polyvalent cations, and isotopic values for specific OM fractions along transects spanning topographic positions from erosional to depositional areas, including aquatic sediments within a single kettle hole. We hypothesized different drivers exist at different scales. At the transect scale, we hypothesized (1) landscape form and land management to explain patterns of isotopic and OC content from different OM fractions. At the aggregate scale, (2) we expected different OM-mineral associations to explain stabilized OM. We also hypothesized, (3) that shallow sediment δC and δN of the kettle hole reflected different terrestrial sources. We found that distinct differences in the OM turnover rates existed between the fractions suggesting that different processes are affecting the transformation rates that are recorded in the isotopic composition patterns. Erosion along with plant productivity drive mineral-associated fractions over the transect, while microbial decomposition and slurry influence freely available and aggregated OM fractions. The type and magnitude of OM-mineral associations changed along the transect while binding OM of different decomposition status. OM in mineral-associated fractions in kettle hole sediments were derived from clay- and silt-sized particles from the field, whereas OM in freely available and aggregated fractions potentially originated from macrophytes. We conclude that kettle holes constitute important sinks for terrestrial OM across the landscape. [ABSTRACT FROM AUTHOR]

Über die Art und den zeitlichen Verlauf der Aktivierung von G ‐ Protein ‐ gekoppelten Rezeptoren (GPCRs), insbesondere der muskarinischen Acetylcholinrezeptoren (mACh ‐ oder M ‐ Rezeptoren), ist trotz der enormen therapeutischen Relevanz von M ‐ Rezeptoren und GPCRs wenig bekannt. In der vorliegenden Arbeit haben wir deshalb einen dualsteren Liganden eingesetzt, der sowohl mit der orthosteren Bindungsstelle des Rezeptors, d. h. derjenigen des Neurotransmitters, als auch mit einer allosteren Bindungsstelle interagiert, wobei synthetisch eine photoschaltbare (photochrome) Azobenzoleinheit eingebaut wurde. Wir haben die photophysikalischen Eigenschaften des BQCAAI benannten Liganden charakterisiert und seine Einsetzbarkeit als pharmakologisches Werkzeug mit einem Satz von FRET ‐ Techniken am M1 ‐ Rezeptor untersucht. BQCAAI stellt den ersten photoschaltbaren dualsteren Liganden dar, und seine Aktivität kann durch Licht reguliert werden. Wir haben BQCAAI eingesetzt, um den zeitlichen Ablauf verschiedener Prozesse der Rezeptoraktivierung zu untersuchen. Ein dualsterer Acetylcholinrezeptorligand, der eine photoschaltbare Azobenzoleinheit enthält und mit zwei Bindestellen des Rezeptors interagiert, wurde synthetisiert. FRET ‐ Studien zeigen, dass die Aktivität des Liganden durch Licht „gedimmt” werden kann und er ein geeignetes Werkzeug für die zeitliche Verfolgung von Rezeptoraktivierungsprozessen ist. [ABSTRACT FROM AUTHOR]

The investigation of the mode and time course of the activation of G-protein-coupled receptors (GPCRs), in particular muscarinic acetylcholine (mACh or M) receptors, is still in its infancy despite the tremendous therapeutic relevance of M receptors and GPCRs in general. We herein made use of a dualsteric ligand that can concomitantly interact with the orthosteric, that is, the neurotransmitter, binding site and an allosteric one. We synthetically incorporated a photoswitchable (photochromic) azobenzene moiety. We characterized the photophysical properties of this ligand called BQCAAI and investigated its applicability as a pharmacological tool compound with a set of FRET techniques at the M1 receptor. BQCAAI proved to be an unprecedented molecular tool; it is the first photoswitchable dualsteric ligand, and its activity can be regulated by light. We also applied BQCCAI to investigate the time course of several receptor activation processes. [ABSTRACT FROM AUTHOR]

G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation. [ABSTRACT FROM AUTHOR]

Background: Opioids are the gold standard for the treatment of acute pain despite serious side effects in the central and enteric nervous system. μ-opioid receptors (MOPs) are expressed and functional at the terminals of sensory axons, when activated by exogenous or endogenous ligands. However, the presence and function of MOP along nociceptive axons remains controversial particularly in naïve animals. Here, we characterized axonal MOPs by immunofluorescence, ultrastructural, and functional analyses. Furthermore, we evaluated hypertonic saline as a possible enhancer of opioid receptor function. Results: Comparative immunolabeling showed that, among several tested antibodies, which all provided specific MOP detection in the rat central nervous system (CNS), only one monoclonal MOP-antibody yielded specificity and reproducibility for MOP detection in the rat peripheral nervous system including the sciatic nerve. Double immunolabeling documented that MOP immunoreactivity was confined to calcitonin gene-related peptide (CGRP) positive fibers and fiber bundles. Almost identical labeling and double labeling patterns were found using mcherry-immunolabeling on sciatic nerves of mice producing a MOPmcherry fusion protein (MOP-mcherry knock-in mice). Preembedding immunogold electron microscopy on MOP-mcherry knock-in sciatic nerves indicated presence of MOP in cytoplasm and at membranes of unmyelinated axons. Application of [DAla ², N-MePhe4, Gly-ol]-enkephalin (DAMGO) or fentanyl dose-dependently inhibited depolarization-induced CGRP release from rat sciatic nerve axons ex vivo, which was blocked by naloxone. When the lipophilic opioid fentanyl was applied perisciatically in naïveWistar rats, mechanical nociceptive thresholds increased. Subthreshold doses of fentanyl or the hydrophilic opioid DAMGO were only effective if injected together with hypertonic saline. In vitro, using β-arrestin-2/MOP double-transfected human embryonic kidney cells, DAMGO as well as fentanyl lead to a recruitment of β-arrestin-2 to the membrane followed by a β-arrestin-2 reappearance in the cytosol and MOP internalization. Pretreatment with hypertonic saline prevented MOP internalization. Conclusion: MOPs are present and functional in the axonal membrane from naïve animals. Hypertonic saline acutely decreases ligand-induced internalization of MOP and thereby might improve MOP function. Further studies should explore potential clinical applications of opioids together with enhancers for regional analgesia. [ABSTRACT FROM AUTHOR]

R3465 --> 837.13 --> The D2 dopamine receptor (D2R) regulates numerous CNS functions including movement, cognition, emotion, and reward circuitry. Agonist binding to the D2R promotes the activation of G protein‐mediated signaling and the recruitment of β‐arrestin to the receptor. Activated D2Rs are also phosphorylated by members of the G protein‐coupled receptor kinase (GRK) family. GRK‐mediated receptor phosphorylation is canonically thought to promote β‐arrestin recruitment to GPCRs, however, previous research has shown that receptor phosphorylation is not required for β‐arrestin recruitment to the D2R. Thus, the role of GRKs in D2R‐β‐arrestin interactions remains unclear. In the current study, we examined the relationship between GRKs and β‐arrestin recruitment using GRK overexpression and GRK knockout cells engineered using CRISPR‐Cas9 technology. Overexpression of individual GRK isoforms in parental HEK293 cells potentiated D2R‐β‐arrestin interactions to varying degrees depending on the isoform. Complete knockout of all GRK isoforms in HEK293 cells (GRK‐KO) significantly abrogated, but did not eliminate, β‐arrestin recruitment to the D2R. Further, knockout of individual GRK isoforms had variable effects on D2R‐β‐arrestin interactions ranging from no effect to significant inhibition. Expression of individual GRK isoforms in the GRK‐KO cells rescued D2R‐mediated β‐arrestin recruitment to levels seen in the parental HEK293 cells. Overall, in these experiments, GRK2 seemed to exhibit the greatest modulatory effects on D2R‐β‐arrestin interactions. We next evaluated the role of receptor phosphorylation in GRK‐mediated effects using a phosphorylation‐deficient (PO4‐null) D2R mutant (Namkung et. al., JBC 284:34103, 2009). The WT‐D2R and PO4‐null‐D2R exhibited identical levels of agonist‐stimulated β‐arrestin interactions as well as receptor internalization. Notably, inhibition of GRK catalytic activity attenuated agonist‐stimulated β‐arrestin recruitment to both the WT‐D2R and PO4‐null‐D2R, suggesting the existence of an unknown GRK substrate. Upon recruitment to GPCRs, β‐arrestins undergo conformational changes that correlate with their downstream signaling effects. Consequently, we sought to investigate if GRKs play a role in β‐arrestin conformational changes seen upon D2R activation. To probe these phenomena, we used intramolecular fluorescent arsenical hairpin (FlAsH) nanoBRET β‐arrestin biosensors. We found that GRK2 increased β‐arrestin conformational changes induced by the WT‐D2R and PO4‐null‐D2R. Further, activation of the PO4‐null‐D2R induced greater conformational changes in β‐arrestin than with the WT‐D2R, suggesting that receptor phosphorylation does not mediate β‐arrestin activation by this GPCR. Taken together, these data suggest that GRKs function noncanonically to modulate D2R‐β‐arrestin interactions and highlight the need to identify additional GRK substrates to better inform our understanding of GPCR signaling. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This results in the recruitment of arrestins, leading to desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffolding partners. GRKs' versatility is also reflected by their diverse roles in pathological conditions such as cancer, malaria, Parkinson's-, cardiovascular-, and metabolic disease. Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRKs (GRK2, GRK3, GRK5, and GRK6) in Western blot analysis. We identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross-reactivity. Hence, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained Western blot results. Utilizing the most-suitable antibodies, we established the Western blot-based, cost-effective simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in nine commonly used cell lines, revealing differential isoform expression. [ABSTRACT FROM AUTHOR]

In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NOsensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e.g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions. [ABSTRACT FROM AUTHOR]

Semiarid steppe ecosystems account for large terrestrial areas and are considered as large carbon (C) sinks. However, fundamental information on topsoil sensitivity to grazing is lacking across different spatial scales including the effects of topography. Our interdisciplinary approach considering soil chemical, physical, and vegetation properties included investigations on pit scale (square-metre scale), plot scale (hectare scale), and the scale of a landscape section (several hectares). Five different sites, representing a grazing intensity gradient, ranging from a long-term grazing exclosure to a heavily grazed site were used. On the pit scale, data about aggregate size distribution, quantity of different soil organic carbon (SOC) pools, SOC mineralisation, hydraulic conductivity and shear strength was available for topsoil samples from representative soil profiles. Spatial variability of topographical parameters, topsoil texture, bulk density, SOC, water repellency, and vegetation cover was analysed on the basis of regular, orthogonal grids in differently grazed treatments by using two different grid sizes on the plot scale and landscape section. On the pit scale, intensive grazing clearly decreased soil aggregation and the amount of fresh, litter-like particulate organic matter (POM). The weak aggregation in combination with animal trampling led to an enhanced mineralisation of SOC, higher topsoil bulk densities, lower infiltration rates, and subsequently to a higher risk of soil erosion. On the plot scale, the effects of soil structure disruption due to grazing are enhanced by the degradation of vegetation patches and resulted in a texture-controlled wettability of the soil surface. In contrast, topsoils of grazing exclosures were characterised by advantageous mechanical topsoil characteristics and SOC-controlled wettability due to higher POM contents. A combined geostatistical and General Linear Model approach identified topography as the fundamental factor creating the spatial distribution of texture fractions and related soil parameters on the scale of a landscape section. Grazing strongly interfered with the topography-controlled particle relocation processes in the landscape and showed strongest effects on the aboveground biomass production and biomass-related soil properties like SOC stocks. We conclude that interdisciplinary multi-scale analyses are essential (i) to differentiate between topography- and grazing-controlled spatial patterns of topsoil and vegetation properties, and (ii) to identify the main grazing-sensitive processes on small scales that are interacting with the spatial distribution and relocation processes on larger scales. [ABSTRACT FROM AUTHOR]

Grazing of grasslands changes soil physical and chemical properties as well as vegetation characteristics, such as vegetation cover, species composition and biomass production. In consequence, nutrient allocation and water storage in the top soil are affected. Land use and management changes alter these processes. Knowledge on the impacts of grazing management on nutrient and water fluxes is necessary because of the global importance of grasslands for carbon sequestration. Soil water in semi-arid areas is a limiting factor for matter fluxes and the intrinsic interaction between soil, vegetation and atmosphere. It is therefore desirable to understand the effects of grazing management and stocking rate on the spatial and temporal distribution of soil moisture. In the present study, we address the question how spatio-temporal soil moisture distribution on grazed and ungrazed grassland sites is affected by soil and vegetation properties. The study took place in the Xilin river catchment in Inner Mongolia (PR China). It is a semi-arid steppe environment, which is characterized by still moderate grazing compared to other regions in central Inner Mongolia. However, stocking rates have locally increased and resulted in a degradation of soils and vegetation also in the upper Xilin River basin. We used a multivariate geostatistical approach to reveal spatial dependencies between soil moisture distribution and soil or vegetation parameters. Overall, 7 soil and vegetation parameters (bulk density, sand, silt and clay content, mean weight diameter, mean carbon content of the soil, vegetation cover) and 57 soil moisture data sets were recorded on 100 gridded points on four sites subject to different grazing intensities. Increasing stocking rates accelerated the influence of soil and vegetation parameters on soil moisture. However, the correlation was rather weak, except for a site with high stocking rate where higher correlations were found. Low nugget ratios indicate spatial dependency between soil or plant parameters and soil moisture on a long-term ungrazed site. However, the effect was not found for a second ungrazed site that had been excluded from grazing for a shorter period. Furthermore the most important soil and vegetation parameters for predicting soil moisture distribution varied between different grazing intensities. Therefore, predicting soil moisture by using secondary variables requires a careful selection of the soil or vegetation parameters. [ABSTRACT FROM AUTHOR]

Abstract: Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M1-, M3-, and M5-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M1- and M5-acetylcholine receptors and the amplitude of these signals was larger at the M1-acetylcholine receptor. Concentration–response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M1-, M3- and M5-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation. [Copyright &y& Elsevier]