Arrestin2-CLTC NTD interactions detected by arrestin2 NMR spectra.

(A) AlphaFold2 model of Arrestin21-418 showing the N- and C-domain (gray) followed by the clathrin-binding loop (CBL, orange), C-terminal β20 strand (yellow) and the disordered C-terminal tail (blue). The C-terminal strand β20 forms a parallel β-sheet with the N-terminal strand β1 (pink) of inactive arrestin2 and is released from the arrestin2 upon activation by phosphopeptides. Arrestin2 residues interacting with CLTC NTD are shown as green (red for main interacting residue I377) spheres. The clathrin and AP2 binding motifs are indicated in the arrestin2 sequence on the top. (B) Part of 1H-15N TROSY spectrum of apo arrestin21-393 (black) and upon addition of an equimolar amount of CLTC NTD (green). Assigned CBL arrestin21-393 residues are indicated. (C) Intensity ratios of assigned complexed vs. apo arrestin21-393 CBL resonances. The region between T374 and T381 undergoes significant intensity attenuation. (D) 1H-13C HMQC spectra of apo Ile-δ1-13CH3, 2H-labeled arrestin21-393 (black) and upon addition of an equimolar amount of the CLTC NTD (green). (E) Intensity attenuation (top) and chemical shift perturbation (bottom) of arrestin21-393 isoleucine 1H3-13Cδ1 resonances upon CLTC NTD addition. Out of 15 isoleucine residues, significant changes are observed only for I377. (F) Left: part of 1H-13C HMQC spectrum showing resonance shifts of I377 upon CLTC NTD binding. Right: detected chemical shift changes as a function of CLTC NTD concentration. The solid line depicts a global non-linear least-square fit to the data points with respective dissociation constant.

Arrestin2-CLTC NTD interactions detected by CLTC NTD NMR spectra.

(Left): intensity attenuation I/I0 of CLTC NTD 1H-15N TROSY resonances upon addition of equimolar amounts of various arrestin2 constructs: (A) apo arrestin21-393, (B) CCR5pp6-activated arrestin21-393, (C) full-length arrestin21-418 and (D) arrestin2ΔCBL with removed clathrin-binding motif. Regions that undergo significant attenuation upon interaction with arrestin2 are indicated by arrows. Right: residues undergoing significant intensity attenuation (one standard deviation below the average signal attenuation) are marked on the structure of CLTC NTD (PDB: 3GD1) together with a schematic representation of the respective arrestin2 construct.

Arrestin2 interaction with the C-terminal domain of the AP2β2.

(A) Structure of AP2β2701- 937 (orange) in complex with arrestin C-terminal peptide (blue) (PDB: 2IV8). Important residues from both chains stabilizing the interaction are depicted in stick representation. (B-F) SEC profiles of arrestin21-418, AP2β2701-937, various phosphopeptides and their mixtures. The annotated color coding below the SEC profile indicates the individual sample composition. In panels (B, C, E) ‘mixture’ refers to a SEC sample containing all of the individual components indicated on the left. In panels (D, F) the primary sample composition is indicated above the SEC profile, and the annotated color coding below the SEC profile indicates the added component to the primary sample composition. (G) Apparent affinities of the complexes derived by integrating the SEC peaks marked in Figure 4f and scaling the integrals by the respective extinction coefficients. ‘nd’ indicates ‘not detected’. (H, I) (Left): intensity attenuation I/I0 of AP2β2701-937 1H-15N TROSY resonances upon addition of equimolar amounts of apo arrestin21-418 (H) or CCR5pp6-activated arrestin21-418 (I). Regions that undergo significant attenuation upon interaction with arrestin2 are indicated by arrows. Right: residues undergoing significant intensity attenuation are marked on the structure of AP2β2 (PDB: 2IV8) together with a schematic representation of the respective arrestin2 construct.

CCR5 internalization in the presence of the chemokine ligands.

CCR5 internalization monitored in HeLa cells co-transfected with plasmids containing arrestin2-YFP (green) and CCR5 genes (magenta) stimulated with (A) [5P12]CCL5 (antagonist), (B) CCL5 (natural agonist), (C) [6P4]CCL5 (super-agonist). Cells were stimulated for 0, 10, and 30 min with chemokine ligands before fixation and preparation for fluorescence microscopy. Each image is accompanied by a zoomed region of interest (white squares) showing deconvolved CCR5 and arrestin2-YFP signals. (D) Mander’s colocalization coefficients of the CCR5 and arrestin2-YFP signals in the absence and presence of chemokine ligands 30 min after stimulation. Individual values, as well as mean and standard deviation are shown for N=3 biological replicates and n=45 ROIs from 20 cells; ****: P < 0.0001; ns: not significant (P > 0.9999). (E) Number of CCR5-positive (CCR5+) and CCR5/arrestin2-positive (CCR5+/arrestin2+) puncta after 30 min [6P4]CCL5 ligand stimulation. Individual values, as well as mean and standard deviation are shown for N=3 biological replicates and n=90 ROIs from 30 cells; ns: not significant (P > 0.9999). (F) CCR5 and arrestin2 fluorescence signals along the trajectory from the plasma membrane to the nucleus (blue line in panel c) 30 min after [6P4]CCL5 ligand stimulation. (G) Lysosomal trafficking of the CCR5 (magenta) and arrestin2 (cyan) complex in presence of the [6P4]CCL5 monitored in the HeLa cells using LAMP1 antibodies (yellow). No recruitment to the lysosome is observed.

Dependence of CCR5 internalization on arrestin2 interactions with clathrin or AP2 monitored in HeLa-arr2/3 cells.

(A) CCR5 internalization induced by 60 min [6P4]CCL5 ligand stimulation in HeLa cells co-transfected with plasmids containing CCR5 and arrestin2-YFP, arrestin2-YFPΔLIELD, arrestin2-YFPR395A, arrestin2-YFPΔAP2 or empty pcDNA3.1. (B, C) Internalization was quantified by counting the number of puncta (B) positive for CCR5 and arrestin2 (CCR5+/arrestin2+) or (C) only CCR5+ 60 min after ligand stimulation in the HeLa-arr2/3 cells. No significant difference in CCR5 internalization is detected for the arrestin2-YFPΔLIELD and arrestin2-YFPR395A constructs (ns, not significant: P > 0.9999) whereas the absence of the AP2 binding motif in the arrestin2ΔAP2-YFP construct causes a significant (****: P < 0.0001) reduction of CCR5 internalization 60 min after ligand incubation. Mean and standard deviation are shown for N=3 biological replicates and n=90 ROIs from 30 cells.

Overall scheme of arr-class A and b GPCR internalization.

Schematic difference of arrestin2-mediated internalization of arr-class B (left) vs. arr-class A (right) GPCRs. Arr-class B GPCRs bind stably to arrestin due to their high levels of phosphorylation. This results in a robust release of the arrestin C-terminus, a stable interaction with AP2 and formation of a long-lived GPCR•arrestin complex. Arr-class A GPCRs bind weakly to arrestin due to their poor phosphorylation. They require stabilization of the arrestin complex by membrane-bound PIP2 molecules. The arrestin C-terminus is not fully released and consequently the interaction with AP2 is unstable.