Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A_2A and dopamine D₂ receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A_2A and D₂ receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A_2A and D₂ receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson’s disease.

Parkinson’s disease (PD) is a dopaminergic-related pathology in which basal ganglia functioning are altered. It has been postulated that a direct receptor-receptor – i.e. dopamine D₂ receptor (D₂R) and adenosine A_2A receptor (A_2AR) – interaction may be finely regulating this brain area. Accordingly, elucidating whether the pathology prompts changes on these structures could grant valuable information for the design of new PD therapies. Here, we first resolved a long-standing question concerning D₂R-A_2AR assembly in native tissue. Thus, by means of different complementary experimental approaches (i.e. immunoelectron microscopy, proximity ligation assay and TR-FRET), we unambiguously identified native D₂R/A_2AR oligomers in rat striatum. Subsequently, we determined that under pathological conditions (i.e. in a rat PD model) D₂R-A_2AR interaction was impaired. Collectively, these results provide definitive evidence for a native D₂R/A_2AR oligomer alteration in experimental parkinsonism, thus conferring the rationale for appropriate oligomer-based PD treatments.

In the CNS, an antagonistic interaction has been shown between adenosine A_2A and dopamine D₂ receptors (A_2ARs and D₂Rs) that may be relevant both in normal and pathological conditions (i.e., Parkinson's disease). Thus, the molecular determinants mediating this receptor–receptor interaction have recently been explored, as the fine tuning of this target (namely the A_2AR/D₂R oligomer) could possibly improve the treatment of certain CNS diseases. Here, we used a fluorescence resonance energy transfer‐based approach to examine the allosteric modulation of the D₂R within the A_2AR/D₂R oligomer and the dependence of this receptor–receptor interaction on two regions rich in positive charges on intracellular loop 3 of the D₂R. Interestingly, we observed a negative allosteric effect of the D₂R agonist quinpirole on A_2AR ligand binding and activation. However, these allosteric effects were abolished upon mutation of specific arginine residues (217–222 and 267–269) on intracellular loop 3 of the D₂R, thus demonstrating a major role of these positively charged residues in mediating the observed receptor–receptor interaction. Overall, these results provide structural insights to better understand the functioning of the A_2AR/D₂R oligomer in living cells.

The EPH receptor A2 (EphA2) tyrosine kinase plays an important role in a plethora of biological and disease processes, ranging from angiogenesis and cancer to inflammation and parasitic infections. EphA2 is therefore considered an important drug target. Two short peptides previously identified by phage display, named YSA and SWL, are widely used as EphA2-targeting agents owing to their high specificity for this receptor. However, these peptides have only modest (micromolar) potency. Lack of structural information on the binding interactions of YSA and SWL with the extracellular EphA2 ligand-binding domain (LBD) has for many years precluded structure-guided improvements. We now report the high-resolution (1.53–2.20 Å) crystal structures of the YSA peptide and several of its improved derivatives in complex with the EphA2 LBD, disclosing that YSA targets the ephrin-binding pocket of EphA2 and mimics binding features of the ephrin-A ligands. The structural information obtained enabled iterative peptide modifications conferring low nanomolar potency. Furthermore, contacts observed in the crystal structures shed light on how C-terminal features can convert YSA derivatives from antagonists to agonists that likely bivalently interact with two EphA2 molecules to promote receptor oligomerization, autophosphorylation, and downstream signaling. Consistent with this model, quantitative FRET measurements in live cells revealed that the peptide agonists promote the formation of EphA2 oligomeric assemblies. Our findings now enable rational strategies to differentially modify EphA2 signaling toward desired outcomes by using appropriately engineered peptides. Such peptides could be used as research tools to interrogate EphA2 function and to develop pharmacological leads.