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

Guillien, Myriam; Mouhand, Assia; Sagar, Amin; Fournet, Aurélie; Allemand, Frédéric; Pereira, Glaécia A. N.; Thureau, Aurélien; Bernadó, Pau; Banères, Jean-Louis; Sibille, Nathalie

doi:10.1101/2023.02.23.529712

Cited by 3 publications

(2 citation statements)

References 74 publications

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“…For β-arrestin 1, we selected position 167 and 191, as they should report on the position of the receptor with respect to the arrestin N-and C-lobe. Moreover, these positions had already been modified for β-arrestin 1 intramolecular FRET experiments, and this has been shown not to affect the functional properties of this protein with regard to its interaction with GHSR in nanodiscs 30 . Although the 71 1.60 :167 distance poorly discriminates the different orientations of β-arrestin in the receptor:arrestin complex (Figure 5), it nevertheless reports on the core engagement of β-arrestin with GHSR, and as such allows to rule out any possible tail-only engaged complex.…”

Section: Pi(45)p2 Favors the Interaction Of Arrestin With Ghsrmentioning

confidence: 99%

“…mM HEPES, 150 mM NaCl, 0.5 mM EDTA, pH 7.4 buffer, active receptor fractions were purified using affinity chromatography with the biotinylated JMV2959 and homogeneous fractions of GHSR-containing discs finally obtained through a size-exclusion chromatography step on a S200 increase column (10/300 GL) using a 25 mM HEPES, 150 mM NaCl, 0.5 mM EDTA, pH 7.4 buffer as the eluent. The full-length cysteine-free β-arrestin 1 mutant and its pre-activated version (truncation at residue 382) with reactive cysteines at positions 68, 167 or 191 were produced as recently described 30 . For LRET measurement, a truncated receptor where a sortase ligation consensus sequence (LPERGGH) replaced the 346-366 region was produced using the protocol described above.…”

Section: Optimizing En For Isolated β-Arrestinmentioning

confidence: 99%

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LIPIDS MODULATE THE DYNAMICS OF GPCR:β-ARRESTIN INTERACTION

Gomes,

Di Michele,

Roessner

et al. 2024

Preprint

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β-arrestins are key privileged molecular partners of G-Protein Coupled Receptors (GPCRs), triggering not only their desensitization but also intracellular signaling. Existing structures point to a high conformational plasticity of β-arrestin:GPCRs interaction, with two completely different orientations between receptor and β-arrestin. The same set of structures also indicates that the C-edge loop of β-arrestin could contribute to its anchoring to the membrane, through an interaction with specific lipids, namely PI(4,5)P2. Combining molecular dynamics simulations and fluorescence spectroscopy, we show that β-arrestin 1 interacts with membranes even in the absence of a receptor, an interaction that is enhanced by PI(4,5)P2 presumably holding the β-arrestin 1 C-edge loop into the lipid bilayer. This key interaction helps β-arrestin 1 to adopt a receptor ready orientation. As a consequence, PI(4,5)P2 also favors the coupling of β-arrestin 1 to the ghrelin receptor (GHSR). In addition, we show that β-arrestin can adopt the two known extreme orientations when complexed with GHSR. Of importance, PI(4,5)P2 shifts the equilibrium between the two different arrangements, favoring one of them. Simulations performed on the GHSR:β-arrestin complex suggest that release of the C-edge loop is required for these transitions to occur and point to a different distribution of PI(4,5)P2 around the complex depending on the orientation of receptor-bound arrestin. Taken together, our results highlight how PI(4,5)P2 plays a true third player role in the β-arrestin:GPCRs interaction, not only by preparing β-arrestin for its further interaction with receptors but also by modulating its orientation once the protein:protein complex is formed.

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Section: Pi(45)p2 Favors the Interaction Of Arrestin With Ghsrmentioning

confidence: 99%

Section: Optimizing En For Isolated β-Arrestinmentioning

confidence: 99%

LIPIDS MODULATE THE DYNAMICS OF GPCR:β-ARRESTIN INTERACTION

Gomes,

Di Michele,

Roessner

et al. 2024

Preprint

Self Cite

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Control of G protein–coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains

Mancinelli,

Marx,

Gonzalez-Hernandez

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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G protein–coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD–membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here, we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction can regulate receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we then identify key conserved residues and cancer-associated mutations that modulate CTD–membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD–membrane interactions in live cells, which may be subject to regulation by CTD phosphorylation and changes in membrane composition. This work reveals an additional mechanism of GPCR modulation, suggesting that CTD–membrane binding may be a general regulatory mode throughout the broad GPCR superfamily.

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Control of G protein-coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains

Mancinelli,

Marx,

Gonzalez-Hernandez

et al. 2023

Preprint

View full text Add to dashboard Cite

G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction controls receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we identify key conserved residues and cancer-associated mutations that control CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells which can be modulated by disease-associated mutations or CTD phosphorylation. This work reveals a novel mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily.

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

Cited by 3 publications

References 74 publications

LIPIDS MODULATE THE DYNAMICS OF GPCR:β-ARRESTIN INTERACTION

LIPIDS MODULATE THE DYNAMICS OF GPCR:β-ARRESTIN INTERACTION

Control of G protein–coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains

Control of G protein-coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains

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