Journal: Nature Communications

Article Title: A Vaccinia -based system for directed evolution of GPCRs in mammalian cells

doi: 10.1038/s41467-023-37191-8

Figure Lengend Snippet: a Comparison of the populations after selection with PTH’(1–34)-HL647 (left panel) or M-PTH(1–14)-HL647 (right panel) to wild-type PTH1R: sort 1 (red line), sort 2 (blue line), sort 3a (black line), sort 3b (green line, repetition of 3a), negative control (dark gray shaded). b Expression analysis of 43 evolved PTH1R variants assessed in live HEK293T cells by flow cytometry analysis with saturating concentrations of PTH’(1–34)-HL647. Expression levels are relative to wild-type receptor expression and are given as mean values ± s.e.m. of 2–3 independent experiments (Supplementary Table ). c cAMP accumulation of 43 evolved PTH1R variants after stimulation with 1 µM PTH(1–34). Data represent maximal cAMP concentrations relative to PTH1R. Bars represent mean values ± s.e.m. of 3–6 independent experiments performed in duplicates (Supplementary Table ). d Ligand affinities of of 43 evolved PTH1R variants in comparison to PTH1R. IC 50 values were derived from whole-cell ligand competition-binding experiments with M-PTH(1–14) or PTH(1–34). Bars represent the mean change ± s.e.m. in calculated affinity (∆pIC 50 ) for each mutant compared with wild-type receptor from 2–8 independent experiments performed in duplicates (Supplementary Table ). b – d Ligands used for selection are indicated below the bar plots. Source data are provided as a Source Data file.

Article Snippet: cDNA libraries for rat NTR1 and human PTH1R were custom-synthesized by MorphoSys AG as linear DNA fragments using the Slonomics® technology , , .

Techniques: Comparison, Selection, Negative Control, Expressing, Flow Cytometry, Derivative Assay, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: A Vaccinia -based system for directed evolution of GPCRs in mammalian cells

doi: 10.1038/s41467-023-37191-8

Figure Lengend Snippet: a Evolved PTH1R variants exhibit high-affinity agonist binding but equal or reduced affinity for partial or inverse agonists in cells. Competition ligand binding curves of full [M-PTH(1–14) and PTH(1–34)], partial [PTH(3–34)] and inverse agonist [IA-PTH(7–34)], measured in whole cells expressing wild-type PTH1R or 5 evolved variants. b High agonist affinity of evolved PTH1R variants is similar to that of wild-type PTH1R in the G protein-bound state. Ligand binding curves of M-PTH(1–14) to evolved PTH1Rs were measured in membrane fractions, obtained from cells expressing PTH1R wild-type or 5 evolved variants, in the absence (left panel) or presence (middle panel) of 12.5 µM mini-G s . Relative binding constants obtained by competition of M-PTH(1–14) binding with labeled M-PTH(1–14), measured in whole cells (from a ) vs. membrane fractions in the absence or presence of 12.5 µM mini-G s (right panel). Positive values thus reflect stronger binding on membrane fractions than on cells. c Increased agonist affinity is G protein-dependent. Binding of 40 nM M-PTH(1–14) was measured in membrane fractions supplemented with increasing concentrations of mini-G s . Data are relative to binding levels of PTH1R in the absence of mini-G s (gray area). Left panel: Variants showing an increased basal and G protein-dependent increase in ligand binding. EC 50 values are indicated as dotted vertical lines (PTH1R: 1.39 ± 0.2 µM, P34_05: 0.28 ± 0.06 µM, P34_13: 0.38 ± 0.1 µM). Right panel: Variants showing an increase in ligand binding independent of G protein. d G protein-independent increase in ligand binding is due to higher basal activity. cAMP levels were measured 30 min after addition of the phosphodiesterase inhibitor IBMX to cells expressing PTH1R variants and were normalized to receptor expression levels. Data represent mean values ± s.e.m. of 3 experiments performed in duplicates. ** p = 0.0044, **** p < 0.0001. Statistical significance was determined by one-way ANOVA and Bonferroni multi-comparison. Source data are provided as a Source Data file.

Article Snippet: cDNA libraries for rat NTR1 and human PTH1R were custom-synthesized by MorphoSys AG as linear DNA fragments using the Slonomics® technology , , .

Techniques: Binding Assay, Ligand Binding Assay, Expressing, Membrane, Labeling, Activity Assay, Comparison