Mini-G proteins: Novel tools for studying GPCRs in their active conformation
Fig 3
The β1AR−mini-Gs and A2AR−mini-Gs complexes.
(a) FSEC traces of GFP-mini-Gs with β1AR (retention volumes are given in parentheses): green, GFP-mini-Gs (15.1 ml); orange, GFP-mini-Gs with β1AR bound to the inverse agonist ICI118551 (15.1 ml); black, GFP-mini-Gs with β1AR bound to the agonist isoprenaline (8 ml, 12.1 ml and 15.1 ml). Representative chromatograms from at least two independent experiments are shown. (b) Measurement of GFP-mini-Gs affinity to DDM-solubilized β1AR using a fluorescent saturation binding assay (FSBA); blue circles, β1AR bound to the agonist isoprenaline (total binding); orange squares, β1AR bound to the inverse agonist ICI118551 (non-specific binding); green triangles, specific binding, with an apparent KD of 200 ± 1 nM (mean ± SEM, n = 2). Curves shown are from a representative experiment. (c) FSEC traces of GFP-mini-Gs with DDM-solubilised A2AR (retention volumes are given in parentheses): green, GFP-mini-Gs (15.1 ml); orange, GFP-mini-Gs with A2AR bound to the inverse agonist ZM241385 (15.1 ml); black, GFP-mini-Gs with A2AR bound to the agonist NECA (12.5 ml and 15.1 ml). Representative chromatograms from at least two independent experiments are shown. (d) Measurement of mini-Gs affinity to DDM-solubilized A2AR using FSBA: blue circles, A2AR bound to the agonist NECA (total binding); orange squares, A2AR bound to the inverse agonist ZM241385 (non-specific binding); green triangles, specific binding, with an apparent KD of 430 ± 24 nM (mean ± SEM, n = 2). (e) Analytical size exclusion chromatography (SEC) of mini-Gs bound to purified A2AR (retention volumes are given in parentheses): black, A2AR–mini-Gs complex, 153 kDa (13 ml); blue, A2AR, 133 kDa (13.3 ml); green, mini-Gs, 22 kDa (17.2 ml). Three panels to the right of the SEC traces are coomassie blue-stained SDS-PAGE gels of fractions from 3 separate SEC experiments: top panel, mini-Gs; middle panel, A2AR; bottom panel, mini-Gs mixed with NECA-bound A2AR (1.2:1 molar ratio).