Extended Data Figure 9. Differences in the bioenergetics of catalytic versus scaffold mechanisms of regulated β-arrestin trafficking and β-arrestin-dependent activation of ERK1/2 promoted by catalytic activation.

(a) Schematic depicting the proposed co-existence of catalytic and scaffolding mechanisms of β-arrestin trafficking tuned according to tail binding affinity, emphasizing the difference in tail versus core interactions (shaded boxes). The tail interaction, requiring GPCR phosphorylation (Rp) drives the scaffold mechanism through its essential role in stable GPCR/β-arrestin complex formation. The core interaction mediates catalysis by providing a kinetically favorable path for β-arrestin to remain captured at the PM irrespective of GPCR dissociation. Such capture requires phosphoinositide binding to the β-arrestin C-domain, explaining why the phosphoinositide requirement is specific to the catalytic mechanism and can be overcome by formation of a sufficiently sufficient stable scaffold complex requiring the phosphorylated GPCR tail. Primary energy inputs maintaining each proposed trafficking cycle are indicated by red arrows. The present results identify a specific requirement of the catalytic mechanism for phosphoinositide binding to the C-domain but they do not exclude binding also in the scaffold complex (which we think is likely). We also cannot presently rule out the possible existence of additional interaction(s) in the catalytic mechanism, such as phosphoinositide binding also to the β-arrestin N-domain that has the potential to displace the β-arrestin C-terminus²⁴. (b) Representative images (from 3 independent experiments) before and after 10 μM isoproterenol treatment of cells expressing chimeric FLAG-tagged β1AR-V2Rs and imaged live with TIRF microscopy. Profiles of FLAG-β2AR and β-arrestin-2–GFP average enrichment into CCSs in COS-1 cells expressing either an empty vector construct (c) or GRK2 (d) and treated with 10 μM isoproterenol (n=15 or 12 cells, respectively, from 3 independent experiments). (e) Difference in enrichment values between β-arrestin-2–GFP and β2AR from panels c and d showing the effect of GRK2 overexpression. (f) Representative western blot showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared from parental or β-arrestin knockout CRISPR HEK 293 cells expressing FLAG–β1AR and exposed to 10 μM isoproterenol for the indicated time period. (g) Quantification of ERK1/2 activation from the western blots in panel a (n=5 independent experiments, p=0.004 using a one-way ANOVA). (h) Representative western blot showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared from parental or β-arrestin knockout CRISPR HEK 293 cells expressing FLAG–β2AR and exposed to 10 μM isoproterenol for the indicated time period. (i) Quantification of ERK1/2 activation from the western blots in panel c (n=5 independent experiments). (f) and (h) show representative Western blots from 5 independent experiments. Data shown as mean ± s.e.m. For gel source data, see Supplementary Figure 1. Error bars represent s.e.m. ** p < 0.01