Allosteric sodium binding cavity in GPR3: a novel player in modulation of Aβ production

Abstract

The orphan G-protein coupled receptor 3 (GPR3) belongs to class A G-protein coupled receptors (GPCRs) and is highly expressed in central nervous system neurons. Among other functions, it is likely associated with neuron differentiation and maturation. Recently, GPR3 has also been linked to the production of Aβ peptides in neurons. Unfortunately, the lack of experimental structural information for this receptor hampers a deep characterization of its function. Here, using an in-silico and in-vitro combined approach, we describe, for the first time, structural characteristics of GPR3 receptor underlying its function: the agonist binding site and the allosteric sodium binding cavity. We identified and validated by alanine-scanning mutagenesis the role of three functionally relevant residues: Cys2676.55, Phe1203.36 and Asp2.50. The latter, when mutated into alanine, completely abolished the constitutive and agonist-stimulated adenylate cyclase activity of GPR3 receptor by disrupting its sodium binding cavity. Interestingly, this is correlated with a decrease in Aβ production in a model cell line. Taken together, these results suggest an important role of the allosteric sodium binding site for GPR3 activity and open a possible avenue for the modulation of Aβ production in the Alzheimer’s Disease.

Harnessing Ion-Binding Sites for GPCR Pharmacology

“…In the case of the D 2.50 N(A) mutations (Massink et al, 2015;White et al, 2018) in adenosine A 2A AR, however, any cAMP activity (basal or induced) of the mutants was disrupted, suggesting that in addition to the Na 1 anchoring role, D 2.50 has other roles in the receptor activation, which may also be related to dynamic change in its protonation state (Vickery et al, 2018). A similar effect was recently observed for GPR3, where D 2.50 A mutation in a recently characterized sodium site completely abolished signaling (Capaldi et al, 2018).…”

Hybrid MM/CG Webserver: Automatic Set Up of Molecular Mechanics/Coarse-Grained Simulations for Human G Protein-Coupled Receptor/Ligand Complexes

“…The hybrid MM/CG protocol has been successfully used to predict ligand poses in a variety of hGPCRs ( Leguèbe et al, 2012 ; Marchiori et al, 2013 ; Sandal et al, 2015 ; Fierro et al, 2017 ; Capaldi et al, 2018 ; Fierro et al, 2019 ). Moreover, the Amber-based MM/CG poses improved significantly relative to those obtained by simple docking, especially for low resolution starting models ( Schneider et al, 2020 ).…”

“…For lower resolution models, however, the uncertainty in the structure (and particularly in the orientation of side chains) decreases the accuracy of the docking predictions and, thus, follow-up molecular dynamics (MD) simulations are recommended ( Kufareva et al, 2014 ; Cavasotto and Palomba, 2015 ; Esguerra et al, 2016 ; Heifetz et al, 2016 ; Fierro et al, 2017 ; Lupala et al, 2018 ; Rodríguez-Espigares et al, 2020 ). In an effort at addressing this issue, we have developed a Hybrid Molecular Mechanics/Coarse-Grained (MM/CG) simulation approach ( Neri et al, 2005 , 2008 ; Leguèbe et al, 2012 ; Marchiori et al, 2013 ; Sandal et al, 2015 ; Capaldi et al, 2018 ; Alfonso-Prieto et al, 2019 ; Fierro et al, 2019 ). The receptor/ligand interactions are described in atomistic detail, including explicit water molecules in the binding site (MM region), while the rest of the receptor is coarse-grained (CG region) ( Schneider et al, 2018 ).…”

Understanding Ligand Binding to G-Protein Coupled Receptors Using Multiscale Simulations

“…Although this approach has been used so far for a limited number of GPCR/ligand complexes (Leguèbe et al, 2012; Marchiori et al, 2013; Sandal et al, 2015), the excellent agreement of the computationally predicted binding poses with the experimental mutagenesis data [for the aforementioned three bitter taste receptor complexes (Marchiori et al, 2013; Sandal et al, 2015)] or the crystal structure [for the β2-adrenergic receptor (Leguèbe et al, 2012)] further supports the applicability of the MM/CG method to other GPCR/ligand complexes. Indeed, MM/CG simulations have been recently used to model the synthetic agonist diphenyleneiodonium chloride (DPI) bound to its target receptor GPR3 (Capaldi et al, 2018). Two of the predicted DPI binding residues were successfully validated a posteriori using mutagenesis and functional assays, as previously done for TAS2R38 (Marchiori et al, 2013) and TAS2R46 (Sandal et al, 2015).…”