Fig. 2: Different combinations of GPCR helix bundles and C-termini orchestrate distinct, GRK-dependent β-arrestin2 interactions.

a Schematic representation of the utilized receptor constructs and indication of the exchanged amino acids to create the chimeric constructs (b2V2 with the receptor transmembrane helix bundle of the b2AR and C-terminus of the V2R and vice versa for the V2b2) after Oakley et al.¹⁶. b (Partial) amino acid sequence of the b2AR and V2R C-terminus, including a schematic representation of the phospho-sites, targeted with specific antibodies. Antibody shape created in BioRender. Klement, L. (2025) https://BioRender.com/721vno0. c–j Measurements of proximal and distal C-terminal phosphorylation of b2AR (c, g), V2b2 (d, h), b2V2 (e, i) and V2R (f, j) in quadruple GRK2/3/5/6 knockout cells (ΔQ-GRK) or Control cells, utilizing a bead-based GPCR phosphorylation immunoassay³⁰. The stably expressed b2AR (c, g) and b2V2 (e, i) were stimulated with different concentrations of Iso and the V2b2 (d, h) and V2R (f, j) with AVP, as indicated. Data are shown as optical density (OD) at 405 nm ±SEM of n = 5 independent experiments, normalized to the maximum ligand concentration for each receptor in Control cells, respectively. k–n β-arrestin2 recruitment to the b2AR (k), V2b2 (l), b2V2 (m) and V2R (n) in ΔQ-GRK or Control cells stimulated with Iso (k, m) or AVP (l, n) as indicated. Data for b2AR, b2V2 and V2R (k, m, n) were published in Drube et al.²⁶ and are shown again to allow a direct comparison. For each receptor, Δ net BRET changes ±SEM of n = 3 independent experiments measured in ΔQ-GRK cells are shown in percent, normalized to the respective maximal change in Control cells. o–r β-arrestin2-F5 conformational changes in presence (Control) or absence of endogenous GRK2/3/5/6 (ΔQ-GRK) when coupling to the b2AR (o), V2b2 (p), b2V2 (q) or V2R (r). Data of n = 3−5 independent experiments are shown as Δ net BRET change (%) ± SEM (exact n numbers for each receptor-, GRK- and sensor-specific condition can be accessed in the source data). s–v Complete β-arrestin2 conformational change fingerprints are shown for each receptor in Control and ΔQ-GRK cells, stimulated with 10 µM Iso (s, u) or 3 µM AVP (t, v), as Δ net BRET change (%) in radar plots. Sensor conditions, which did not fulfill the pharmacological parameters (Hill slope and EC₅₀ analysis as described in methods) were classified as non-responding conditions and assigned zero. Source data are provided as a Source Data file.