Figure 1: Assembly of partially and fully engaged β₂V₂R–βarr1-Fab30 complex.

(a) Schematic representation of biphasic GPCR–βarr interaction. βarr interacts with activated and phosphorylated GPCRs in a biphasic fashion where the first step is binding of βarr through the phosphorylated carboxyl terminus and the second step is the engagement of βarr with the 7TM core of the receptor. The receptor component is shown in grey, phosphorylated carboxyl terminus in yellow and βarr 1 in blue/magenta. (b) Schematic representation of an ELISA-based approach for in-vitro assembly of β₂V₂R–βarr1 complex. Purified Fab30 is immobilized on solid support as an anchor to capture the complex followed by incubation with purified β₂V₂R and βarr1. Formation of β₂V₂R–βarr1 complex is visualized using HRP-coupled anti-FLAG M2 antibody through detection of FLAG tagged β₂V₂R. (c) Fab 30 assisted in-vitro assembly of β₂V₂R–βarr1 complex. Agonist bound and phosphorylated β₂V₂R (^Actβ₂V₂R^phos) forms a stable complex while inverse agonist bound and non-phosphorylated β₂V₂R (^Inactβ₂V₂R^non-phos) does not exhibit any detectable complex formation. (d) An experimental set-up to assemble ‘tail only’ engaged and ‘fully’ engaged β₂V₂R–βarr1 complex in-vitro. β₂V₂R is coexpressed with GRK2^CAAX in cultured Sf9 cells and 66 h post-infection, cells are stimulated with a low-affinity agonist (Isoproterenol) to trigger receptor phosphorylation. Subsequently, the receptor is purified by affinity chromatography and the ligand is washed off during purification to yield ligand free phosphorylated β₂V₂R (^Apoβ₂V₂R^phos). Incubation with inverse agonist (carazolol) or high-affinity full agonist (BI-167107) yields ^Inactβ₂V₂R^phos and ^Actβ₂V₂R^phos, respectively. (e) Both, the ^Inactβ₂V₂R^phos and ^Actβ₂V₂R^phos form a stable complex with βarr1 as assessed by ELISA approach and potentially represent ‘tail only’ and ‘fully’ engaged complexes, respectively. (f) Formation of ‘tail only’ engaged and ‘fully’ engaged complexes as assessed by coimmunoprecipitation experiment. This experiment was repeated three times with identical results and a representative image is shown. Signals in c and e are normalized with ^Actβ₂V₂R^phos+βarr1+Fab30 condition as 100%. Data presented in c and e represent mean±s.e.m. of three independent experiments each carried out in duplicate and analysed using one-way ANOVA with Bonferroni post-test (***P<0.001).