Journal: bioRxiv

Article Title: Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

doi: 10.1101/2021.02.02.429298

Figure Lengend Snippet: A. Schematic representation of canonical GPCR and non-canonical 7TMR pairs activated by a common agonist. C5a, complement C5a; C5aR1, C5a receptor subtype 1; C5aR2, C5a receptor subtype 2; CCL7, chemokine CCL7; CCR2, C-C chemokine receptor subtype 2; D6R, decoy D6 receptor. B-C. Agonist-induced dissociation of heterotrimeric G-proteins for C5aR1-C5aR2, and CCR2-D6R pairs measured using NanoBiT complementation assay. HEK-293 cells expressing the indicated receptor and Sm/Lg-BiT constructs of G-protein α,β,γ sub-units were stimulated with corresponding ligands, and the change in luminescence signal upon NanoBiT dissociation was measured as a readout of G-protein coupling. Data represent three independent experiments, normalized with respect to baseline signal (i.e. vehicle treatment). D. Agonist-induced second messenger response measured using GloSensor assay (cAMP stimulation, and inhibition of forskolin-induced cAMP level), and Fluo-4 NW calcium mobilization assay. HEK-293 cells expressing the indicated receptor were used for these assays using standard protocols as described in the method section. For each of the second messenger assay, a well-established prototypical GPCR was included as a reference (V 2 R, vasopressin receptor subtype 2; B 2 R, bradykinin receptor subtype 2). Data are normalized with respect to maximum signal (treated as 100%) and represent mean±sem of four independent experiments.

Article Snippet: Wild-type (WT), C5aR1−/− and C5aR2−/− mice on a C57BL/6J genetic background (n=5-15) were administered with recombinant mouse C5a (Sino Biological, China) at a dose of 50 μg kg-1 via intravenous injection (tail vein).

Techniques: Expressing, Construct, Inhibition, Calcium Mobilization Assay

Journal: bioRxiv

Article Title: Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

doi: 10.1101/2021.02.02.429298

Figure Lengend Snippet: A. HEK-293 cells expressing the indicated receptor constructs were stimulated with agonist, lysed and incubated with purified βarr1/2. Subsequently, the receptor was immunoprecipitated using anti-FLAG M1 antibody agarose and co-purified βarrs were detected using Western blotting. A representative blot from three independent experiments is shown here. B-C. Agonist-induced trafficking of βarrs was measured in HEK-293 cells expressing the indicated receptor constructs and mYFP-tagged βarrs using live cell confocal microscopy. Cells were stimulated with saturating concentration of agonists (CCL7, 100 nM; C5a,100 nM) and trafficking of βarrs was monitored at indicated time-points. Representative images from three independent experiments are shown (scale bar is 10μm). D. Internalized D6R and C5aR2 co-localize with βarr2 as monitored by confocal microscopy. HEK-293 cells expressing the indicated receptor and βarr2-mYFP were stimulated with agonist, followed by fixation, permeabilization and staining of the receptor using DyLight-688 conjugated anti-FLAG M1 antibody. Localization of the receptor and βarr2 was visualized using confocal microscopy, and representative images from three independent experiments are shown (scale bar is 10μm). The Pearson’s Correlation Coefficient (PCC) were 0.68±0.05, 0.72±0.06, 0.94±0.01 and 0.96±0.01 for C5aR2, 0min (12 cells), C5aR2, 10min (13 cells), D6R, 0min (14 cells) and D6R, 10min (15 cells), respectively. E. Single particle analysis of C5aR2-V2-βarr1-Fab30 complex further corroborates the interaction of βarr1 with C5aR2. Sf 9 cells expressing a chimeric C5aR2 construct (C5aR2-V2), GRK2 CAAX and βarr1 were stimulated with C5a (100 nM), stabilized using Fab30, followed by affinity purification of the complex on anti-Flag M1 column. Subsequently, the fractions containing the complex were further isolated by size-exclusion chromatography and subjected to negative staining based single particle analysis. 2D-class averages based on approximately ten thousand particles are shown here, and a typical 2D class average is indicated together with a schematic representation of the complex.

Article Snippet: Wild-type (WT), C5aR1−/− and C5aR2−/− mice on a C57BL/6J genetic background (n=5-15) were administered with recombinant mouse C5a (Sino Biological, China) at a dose of 50 μg kg-1 via intravenous injection (tail vein).

Techniques: Expressing, Construct, Incubation, Purification, Immunoprecipitation, Western Blot, Confocal Microscopy, Concentration Assay, Staining, Single Particle, Affinity Purification, Isolation, Size-exclusion Chromatography, Negative Staining

Journal: bioRxiv

Article Title: Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

doi: 10.1101/2021.02.02.429298

Figure Lengend Snippet: A. CCL7 stimulation leads to robust ERK1/2 phosphorylation in HEK-293 cells expressing CCR2, however, it fails to elicit any detectable ERK1/2 phosphorylation for D6R as measured by Western blotting. B. C5aR1 stimulation exhibits a typical ERK1/2 phosphorylation pattern upon agonist-stimulation while C5aR2 cells display an elevated level of basal ERK1/2 phosphorylation, which decreases upon C5a-stimulation. C. PTX-treatment inhibits C5a-induced ERK1/2 phosphorylation downstream of C5aR1 but it fails to inhibit the elevated level of basal ERK1/2 phosphorylation for C5aR2. D. Treatment of cells with U0126, a MEK-inhibitor completely abolishes ERK1/2 phosphorylation for both, C5aR1 and C5aR2 suggesting the involvement of a canonical mechanism of ERK1/2 phosphorylation. The right panels show quantification based on densitometry data from 4-6 experiments analyzed using One- or Two-Way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ***p<0.0001).

Article Snippet: Wild-type (WT), C5aR1−/− and C5aR2−/− mice on a C57BL/6J genetic background (n=5-15) were administered with recombinant mouse C5a (Sino Biological, China) at a dose of 50 μg kg-1 via intravenous injection (tail vein).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

doi: 10.1101/2021.02.02.429298

Figure Lengend Snippet: A. Phospho-antibody array on HEK-293 cells expressing C5aR1 or C5aR2 reveals phosphorylation/dephosphorylation of a few common and several distinct cellular proteins. Of these, C5a-stiulation of C5aR2-expressing HEK-293 cells exhibits robust enhancement of p90RSK phosphorylation, which is also common to C5aR1. B. C5a-stimulation of C5aR2 expressing HEK-293 cells is validated using Western blotting, which validates the phospho-antibody array data. A representative blot from six experiments and densitometry-based quantification (average±sem) is presented (*p<0.05, ***p<0.001; One-Way ANOVA). C. C5a-stiulated p90RSK phosphorylation is βarr1 dependent as knock-down of βarr1 in HEK-293 cells expressing C5aR2 reduces p90RSK phosphorylation. A representative blot from five experiments and densitometry-based quantification (average±sem) is presented (**p<0.01; One-Way ANOVA). D. Stimulation of human macrophage derived monocytes (hMDMs) with either C5a or P32 (a C5aR2-selective agonist) results in significant p90RSK phosphorylation. Importantly, PMX53, a C5aR1-selective antagonist, does not block p90RSK phosphorylation suggesting that it is mediated by C5aR2. E. A significant component of C5a-induced polymorphonuclear leukocyte (PMN) mobilization depends on C5aR2. WT, C5aR1 −/− and C5aR2 −/− mice on a C57BL/6J genetic background (n = 5-15) were intravenously administered with recombinant mouse C5a (50 μg kg −1 ). Tail tip collected blood was smeared onto a slide followed by staining and counting of white blood cells, with the proportion of PMNs calculated. Data is presented as average±sem (*p<0.05, ***p<0.001; One- and Two-Way ANOVA).

Article Snippet: Wild-type (WT), C5aR1−/− and C5aR2−/− mice on a C57BL/6J genetic background (n=5-15) were administered with recombinant mouse C5a (Sino Biological, China) at a dose of 50 μg kg-1 via intravenous injection (tail vein).

Techniques: Ab Array, Expressing, De-Phosphorylation Assay, Western Blot, Derivative Assay, Blocking Assay, Recombinant, Staining

Journal: The ISME Journal

Article Title: The soil microbial food web revisited: Predatory myxobacteria as keystone taxa?

doi: 10.1038/s41396-021-00958-2

Figure Lengend Snippet: Myxobacteria and potential prey organisms used in predation assays.

Article Snippet: The myxobacteria Haliangium ochraceum , Stigmatella aurantiaca , Kofleria flava , Corallococcus coralloides, and Chondromyces robustus were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Table ).

Techniques: Bacteria