Neutrophil Elastase and Proteinase-3 Trigger G Protein-biased Signaling through Proteinase-activated Receptor-1 (PAR1)

Agonist-induced phosphorylation of G protein–coupled receptors (GPCRs) is a key determinant for their interaction with β-arrestins (βarrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing βarr interaction and functional outcomes. Here, we find that several phosphorylation sites in the human vasopressin receptor (V2R), positioned either individually or in clusters, differentially contribute to βarr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V2R, suitably positioned to cross-talk with a key residue in βarrs, has a decisive contribution in βarr recruitment, and its mutation results in strong G-protein bias. Molecular dynamics simulation provides mechanistic insights into the pivotal role of this key phosphorylation site in governing the stability of βarr interaction and regulating the interdomain rotation in βarrs. Our findings uncover important structural aspects to better understand the framework of GPCR-βarr interaction and biased signaling.

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A structural basis for how ligand binding site changes can allosterically regulate GPCR signaling and engender functional selectivity

Science Signaling ◽

10.1126/scisignal.aaw5885 ◽

2020 ◽

Vol 13 (617) ◽

pp. eaaw5885 ◽

Cited By ~ 5

Author(s):

Marta Sanchez-Soto ◽

Ravi Kumar Verma ◽

Blair K. A. Willette ◽

Elizabeth C. Gonye ◽

Annah M. Moore ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

G Protein ◽

Structural Basis ◽

Protein Signaling ◽

Ligand Binding Site ◽

Intracellular Loop ◽

Gpcr Signaling ◽

Biased Signaling ◽

Dynamics Simulations

Signaling bias is the propensity for some agonists to preferentially stimulate G protein–coupled receptor (GPCR) signaling through one intracellular pathway versus another. We previously identified a G protein–biased agonist of the D2 dopamine receptor (D2R) that results in impaired β-arrestin recruitment. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with β-arrestin. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired β-arrestin recruitment while maintaining G protein signaling. To investigate the mechanism of this signaling bias, we used an active-state structure of the β2-adrenergic receptor (β2R) to build β2R-WT and β2R-Y1995.38A models in complex with the full β2R agonist BI-167107 for molecular dynamics simulations. These analyses identified conformational rearrangements in β2R-Y1995.38A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in β2R-Y1995.38A, which is predicted to affect its interactions with β-arrestin. Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling.

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