Structural Insights into the Intrinsically Disordered GPCR C-Terminal Region, Major Actor in Arrestin-GPCR Interaction

Abstract

Arrestin-dependent pathways are a central component of G protein-coupled receptor (GPCRs) signaling. However, the molecular processes regulating arrestin binding are to be further illuminated, in particular with regard to the structural impact of GPCR C-terminal disordered regions. Here, we used an integrated biophysical strategy to describe the basal conformations of the C-terminal domains of three class A GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR) and the β2-adernergic receptor (β2AR). By doing so, we revealed the presence of transient secondary structures in these regions that are potentially involved in the interaction with arrestin. These secondary structure elements differ from those described in the literature in interaction with arrestin. This suggests a mechanism where the secondary structure conformational preferences in the C-terminal regions of GPCRs could be a central feature for optimizing arrestins recognition.

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

“…2b), from residues 356 to 364 (called V2-1), suggesting an β-strand conformation in this region. Interestingly, we showed previously that V2-1 contained a helical conformation in the non-phosphorylated form (wt-V2R-Cter) (Guillien et al, 2022). This loss of helical content between the wt and the pm variant was also substantiated by the decrease of percentage of 3 JHNHA scalar coupling bellow 6 Hz (Fig.…”

“…A combined Size Exclusion Chromatography-Multi Angle Light Scattering (SEC-MALS), Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) analysis showed that these pm variants are monomeric and disordered domains with transient secondary structures (Fig. 1), as is the case of their non-phosphorylated (wt-GPCR-Cter) forms (Guillien et al, 2022). Indeed, SEC-MALS profiles of pm variants (Fig.…”

“…S2a-b) (BMRB accession codes: 51319 , 51328 and 51330 for pm-β2AR-Cter, pm-GHSR-Cter and pm-V2R-Cter respectively). We then used NMR to assess the structural features of the C-terminal domains in their phosphomimetic (pm) form and compared them to their non-phosphorylated (wt) form (Guillien et al, 2022). C-terminal domains were not affected by the protein concentration, as assessed by NMR (Table S1, Fig.…”

“…Intrinsically disordered proteins (IDPs) or IDRs are highly flexible with a low content of secondary structure, whose specific role in fundamental signaling and regulation processes started to be revealed two decades ago (Kriwacki et al, 1996; Olsen et al, 2017; Wright and Dyson, 2015). We have previously described the transient secondary structure contents of three truncated GPCR C-terminal domains by Nuclear Magnetic Resonance (NMR): the vasopressin V2 Receptor (V2R) (from residue 343 to 371), the Growth Hormone Secretagogue or ghrelin Receptor type 1a (GHSR) (from residue 339 to 366) and the β2-Adernergic Receptor (β2AR) (from residue 342 to 413) (Guillien et al, 2022). Only structures of class B receptors, which interact more strongly with arrestin, or chimeric class A receptors with the C-terminus of V2R (V2Rpp), have been reported in the literature until now (Bous et al, 2022; Huang et al, 2020; Lee et al, 2020; Staus et al, 2020; Yin et al, 2019).…”

Multi-omics approach to identifying isoform variants as therapeutic targets in cancer patients

“…For example, the unique isoforms and their IDRs (detected mainly in the 3 rd intracellular loop and the C-terminal tail) are typically enriched in tissue-specific isoforms, resulting in tissue-specific protein-protein interactions ( 90 ), including the GPCRs ( 97 ). Notably, the IDRs in the C-terminal of GPCRs are proposed to have a high potential for new target-binding partners ( 98 ). The importance of the N-terminal for the activation of two distinct GPCR isoforms was also highlighted ( 99 ).…”