Abstract
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
