Fig. 2: Apelin receptor variants show significant differences in expression and peptide ligand binding.

a Experimental pipeline schematic for in vitro characterisation of apelin receptor variants. CHO-K1 cells were transiently transfected with wild-type (WT) or variant apelin receptor constructs tagged C-terminally with an eGFP reporter. In high content screening studies (upper track), cells were plated and treated with fluorescently labelled apelin647 (300 nM) or ELA647 (1 µM) for 90 min before fixation and visualisation. In radioligand binding studies (lower track), cells were lysed and the membranes harvested before treatment with a concentration range (up to 1 nM) of [¹²⁵I]-apelin-13. Bound radioactivity was counted to determine receptor density and affinity. Created in BioRender. Davenport, A. (2024) https://BioRender.com/o44f254. b Representative confocal fluorescent images (n ≥ 3 independent experiments). Panels show a merge (eGFP reporter in green and 647 nm fluorescent ligand in red) or the 647 nm fluorescent peptide signal in isolation. Non-specific binding (NSB) was determined in WT cells treated with fluorescent peptide in the presence of 10 µM unlabelled [Pyr¹]apelin-13. Scale bars 50 µm. c Bar chart showing mean ± SD membrane eGFP fluorescence (expressed as % WT) from cells imaged over 12 regions, pooled from n = 4 independent experiments in triplicate. Untrans=cells untransfected with apelin receptor cDNA. d Bar chart showing mean ± SD count (n = 4 independent experiments in triplicate) of eGFP positive cells per region. e Grouped bar chart showing mean membrane apelin647 (solid bars) or ELA647 (slashed bars) fluorescence (expressed as %WT, n = 4 independent experiments in triplicate). For all data expressed in bar charts, statistical significance was determined using a one-way ANOVA, with Dunn’s correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. f Saturation binding curves showing specific binding of [¹²⁵I]-apelin-13 in transfected membrane preparations. Data are mean ± SD, n = 3 independent experiments. NSB was determined in the presence of 10 µM unlabelled [Pyr¹]apelin-13. g Competition radioligand binding curves showing ELA-11 peptide competing for binding (% specific) with a single 0.1 nM concentration of [¹²⁵I]-apelin-13 at WT or T/M89^2.64 variant apelin receptor in membrane preparations. Data expressed as mean ± SD, n = 3 independent experiments. Source data are provided as a Source Data file.