Figure 3: Ligand-dependent modulation of core interaction in Apoβ2V2Rphos–βarr1-Fab30 complex.
From: Functional competence of a partially engaged GPCR–β-arrestin complex
(a) A schematic representation showing that Apoβ2V2Rphos can potentially sample active like conformations and, therefore, might engage core interaction to some extent. Incubation with an inverse agonist is likely to ablate this basal level of core interaction yielding a ‘tail only’ complex while incubation with an agonist stabilizes the core interaction and results in a ‘fully engaged’ complex. (b) In-vitro assembly of Apoβ2V2Rphos complex with βarr1 in presence of Fab30 as assessed by ELISA approach. Incubation of this pre-formed complex with inverse agonist or agonist does not alter the physical assembly of the complex. (c) In-vitro assembly of Apoβ2V2Rphos complex with βarr1 in presence of Fab30 as measured by coimmunoprecipitation. Similar to ELISA approach, incubation of pre-formed complex with inverse agonist or agonist does not alter the complex assembly. This experiment was repeated three times with identical results and a representative image is shown. (d) Incubation of pre-formed Apoβ2V2Rphos complex with inverse agonist (carazolol) results in an increase in bimane fluorescence suggesting a loss of core binding, yet presumably stabilization of a ‘tail engaged’ complex. On the other hand, incubation of this complex with agonist (BI-167107) results in a further decrease in bimane fluorescence suggesting the engagement of receptor core and, therefore, stabilization of a ‘fully engaged’ complex. (e) Bimane fluorescence at emission λmax as measured in d is presented as a bar graph. (f) Incubation of pre-formed Apoβ2V2Rphos complex with a panel of ligands results in different extent of bimane fluorescence quenching, which directly correlates to the ligand efficacy. (g) Quantification of decrease in bimane fluorescence at emission λmax as measured in f is presented as a bar graph. Data in d and f represent mean of three independent experiments. Data presented in b, e and g represent mean±s.e.m. of three independent experiments and analysed using one-way ANOVA with Bonferroni post-test (*P<0.05; ***P<0.001).
