Fig. 7: Putative phosphorylation motifs in IL3 and C-terminus of GPCRs are not associated with GRK-specific β-arrestin recruitment.

Analysis of the abundance and position of identified putative serine and threonine (Ser/Thr) phosphorylation sites, clusters⁶² (PPP, PXPP) and patterns (PXXPXXP, PXPXXP, as identified by Zhou et al.⁶¹) in the intracellular loop 3 (IL3) and C-terminus (C-term; information from GPCRdb.org) of GPCRs listed in Supplementary Table 2 with the exception of the unphysiological b2V2 chimaera receptor. While X represents any amino acid, P may be a Ser, Thr or a negatively charged amino acid (glutamic acid, Glu; aspartic acid, Asp). The number and positions of potential phosphorylation sites, clusters and patterns were detected using Python 3.8.7. In order to compare positions of potential phosphorylation sites or motifs between GPCRs with varying lengths of IL3 and C-term, their relative position was calculated as the position index in relation to the full length of the respective peptide stretch. Consequently, the relative position of 0 corresponds to the beginning (N), whereas a relative position of 1 corresponds to the end (C) of the respective peptide stretch. All relative positions are displayed as dot plots with the corresponding median, indicated by bars. Positions between 0.25 and 0.75 were categorised as central (grey) while positions between 0.0 and 0.25 as well as 0.75 and 1.00 were considered peripheral (orange). Dashed lines mark 0.25 and 0.75 breakpoints. a Relative position of potential phosphorylation sites (Ser/Thr) in the IL3 and C-term of analysed GPCRs grouped according to their GRK-specific β-arrestin (βarr) recruitment. In addition to the three groups defined in Fig. 3i, j (GRK2/3-regulated, GRK2/3/5/6-regulated and not significant), GPCRs displaying βarr pre-coupling (AT1R and V2R) were compiled in a fourth group (pre-coupling). b The analysed receptors were grouped as class A or B according to Oakley et al.⁶² (Supplementary Table 2) and each class is represented as one pie chart. In each pie chart, all GPCR–βarr pairs are assigned to the different GRK-specific βarr recruitment groups. The absolute number of GPCR–βarr pairs presented in each group is indicated in brackets. Since GPCRs displaying βarr pre-coupling can be considered a subgroup of GRK2/3/5/6-regulated GPCRs, both slices are separated by a dashed line (right pie). c–f Abundance of PPP clusters (c), PXPP clusters (d), PXPXXP patterns (e) and PXXPXXP patterns (f) are displayed as dot plots grouped according to their GRK-specific βarr recruitment. g–n Relative positions of P-patterns as indicated. They were grouped according to their GRK-specific βarr recruitment (g–j), as well as the GPCR classification (k–n). Association between the position of PXPP and the GRK-specific βarr recruitment (h, p = 0.0005) or GPCR classification (l, p = 0.7328) was tested using Fisher’s exact test. Exact odds ratios and confidence intervals are provided in the Source Data files.