Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Biased agonists at the human Y 1 receptor lead to prolonged membrane residency and extended receptor G protein interaction

doi: 10.1007/s00018-019-03432-7

Figure Lengend Snippet: Novel Y1R agonists display impaired arr-3 recruitment and receptor internalization, leading to a net bias towards the G protein pathway. a, b CRE reporter gene assay to determine the activity of the peptides at the Y1R in the endogenous Gi/o pathway in transiently transfected HEK293 (a) and SK-N-MC cells endogenously expressing the Y1R (b). c BRET experiments with Y1R fused to eYFP and RLuc8-arr-3 in transiently transfected HEK293 cells. Cells were stimulated with peptide variants for 5 min. d Internalization and arr-3 recruitment was detected by fluorescence microscopy prior to (w/o) and after stimulation with 100 nM of Y1R ligands. Y1R is C-terminally fused to eYFP (yellow) and arr-3 is C-terminally tagged with mCherry (red). Nuclei were stained with Hoechst33342 (blue), n ≥ 2. (scale bar = 10 µm). e, f Ligand bias plot generated from arr-3 recruitment versus G protein activation downstream of the native Gi/o or chimeric Giq pathway in transfected HEK293 cells (cf. Table ​Table1).1). Shown are the mean between-pathway differences (ΔΔlogEC50 ± SEM), and the statistical significance was tested by one-way ANOVA and Dunnett’s post test compared to NPY, **p < 0.01, ***p < 0.001

Article Snippet: Fluorescence microscopic studies were performed with a Zeiss Axio Observer.Z1 inverted microscope (filters 46 for YFP, 31 for mCherry, and 02 for Hoechst33342 stain) equipped with an ApoTome Imaging System and a Heating Insert P Lab-Tek S1 unit.

Techniques: Reporter Gene Assay, Activity Assay, Transfection, Expressing, Fluorescence, Microscopy, Staining, Generated, Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Biased agonists at the human Y 1 receptor lead to prolonged membrane residency and extended receptor G protein interaction

doi: 10.1007/s00018-019-03432-7

Figure Lengend Snippet: An internalization deficient Y1R variant (Y1-NC) shows enhanced G protein signaling. a Y1-NC is expressed similar to the wild-type receptor under the conditions used for signal transduction studies (d, e). b This receptor variant does not recruit arr-3 and internalize following NPY stimulation. Arr3-mCherry is depicted in red, the receptor-eYFP fusion protein in yellow, cell nuclei stained with Hoechst33342 and depicted in blue; bar equals 10 μm. c Ligand affinities at the Y1 -NC were determined in competition binding experiments using 125I-PYY (75 pM) and membrane preparations of transiently transfected HEK293 cells. d, e Kinetic analysis of cellular inositol phosphates produced downstream of the Y1R variants. d displays the concentration response curves after 15, 30 and 60 min of stimulation, which is re-plotted on a time-axis in e. f Saturation BRET experiments of Y1-NC-RLuc8 with different concentrations of Gαo-Venus in transiently transfected HEK293 cells. As seen for the wild-type receptor, there is a saturable BRET signal in the basal state indicative of pre-assembly. g Kinetic BRET experiments of the same constructs at saturating F/L ratio (F/L > 0.02) to resolve ligand effects on Y1-NC-Gαo interactions. The BRET signal prior to stimulation was recorded for 2 min and the baseline was set to 0. Compared to the wild-type receptor, the BRET increase was substantially prolonged for both, the native NPY and the G protein-biased G34-NPY after addition of ligand (+). b, g display representative examples of three independent experiments; a, c, d, e, f display mean ± SEM of three independent experiments conducted in technical triplicate

Article Snippet: Fluorescence microscopic studies were performed with a Zeiss Axio Observer.Z1 inverted microscope (filters 46 for YFP, 31 for mCherry, and 02 for Hoechst33342 stain) equipped with an ApoTome Imaging System and a Heating Insert P Lab-Tek S1 unit.

Techniques: Variant Assay, Transduction, Staining, Binding Assay, Membrane, Transfection, Produced, Concentration Assay, Construct