Extended Data Fig. 7: Biochemical and functional characterization of dimer interface mutations.
From: Structural insight into apelin receptor-G protein stoichiometry
a, Relative surface expression levels of mutants were monitored by FACS staining assay and normalized to the expression levels of WT-APJR. Data were presented as mean ± S.E.M of three biologically independent experiments. b, SEC profile of APJR mutants expressed in the absence of Gi protein. Single mutation F1013.24A disrupted the dimer formation (arrow showed F1013.24A shift towards the monomeric GPCR control (FLAG-BRIL-fused GPR52)). c, SEC profile of APJR mutants-Gi co-expression (arrow showed peak-shift of F1013.24A-Gi compared to WT-Gi). d, SEC profile and SDS-PAGE gel image of APJRWT-Gi or APJRF101A-Gi protein complex in the nanodisc system. Black arrow indicates peak shift from dimer to monomer species. e, Influence of dimer interface mutations on the downstream signaling measured by cAMP response in the presence of cmpd644. f, Influence of ‘dimer-switch’ F101A mutation on the downstream signaling measured by BRET2 bio-sensor assay for both ELA-32 and cmpd644 (normalized response relative to %WT Emax). Basal activity of F101A mutant relative to WT was denoted. For e and f, data were presented as mean ± S.E.M of three biologically independent experiments.
