Phosphorylation motif dictates GPCR C-terminal domain conformation and arrestin interaction

Arrestin dependent G protein-coupled receptor (GPCR) signaling pathway is regulated by the phosphorylation state of GPCR’s C-terminal domain, but the molecular bases of arrestin:receptor interaction are to be further illuminated. Here we investigated the impact of phosphorylation on the conformational features of the C-terminal region from three Rhodopsin-like GPCRs, the vasopressin V2 Receptor (V2R), the Growth Hormone Secretagogue or ghrelin Receptor type 1a (GHSR) and the β2-Adernergic Receptor (β2AR). Using phosphomimetic variants, we identified pre-formed secondary structure elements, or short linear motif (SLiMs), that undergo specific conformational transitions upon phosphorylation. Of importance, such conformational transition favors arrestin-2 binding. Hence, our results suggest a model in which the cellular signaling specificity of GPCRs is encoded in the phosphorylation-dependent structuration of the C-terminal regions, which will subsequently modulate arrestin conformation and therefore GPCR:arrestin signaling outcomes. ### Competing Interest Statement The authors have declared no competing interest. * ### Abbreviations IDP : intrinsically disordered protein IDR : intrinsically disordered region PTM : post-translational modifications GPCR : G protein-coupled receptor GRK : G protein receptor kinase V2R : vasopressin V2 receptor GHSR : ghrelin receptor 1a β2AR : β2-adreneric receptor wt-V2R-Cter : wild-type V2R C-terminus pm-V2R-Cter : phosphomimetic V2R C-terminus wt-GHSR-Cter : wild-type GHSR C-terminus pm-GHSR-Cter : phosphomimetic GHSR C-terminus wt-β2AR-Cter : wild-type β2AR C-terminus pm-β2AR-Cter : phosphomimetic β2AR C-terminus V2Rpp : synthetic fully phosphorylated peptide of V2R C-terminus C7pp : synthetic fully phosphorylated peptide of CXCR7 C-terminus pRho-Cter : phosphorylated C-terminus of rhodopsin receptor PPII : polyproline helix 2 CD : circular dichroism MS : mass spectrometry MST : microscale thermophoresis FRET : fluorescence resonance energy transfert SAXS : small angle X-ray scattering SDS-PAGE : sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEC-MALS : size exclusion chromatography – multi angle light scattering NMR : nuclear magnetic resonance SCS : secondary chemical shift CSP : chemical shift perturbation HSQC : heteronuclear single quantum coherence NOE : nuclear Overhauser effect PRE : paramagnetic relaxation enhancement RDC : residual dipolar coupling FM : flexible meccano EDTA : EthyleneDiamineTetraAcetic acid DTT : dithiothreitol Cou : L-(7-hydroxycoumarin-4-yl ethylglycine PMSF : phenylmethylsulfonyl fluoride TCEP : Tris(2-CarboxyEthyl)Phosphine