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Increasing evidence supports a relationship between lipid metabolism and mental health. In particular, the biostatus of polyunsaturated fatty acids (PUFAs) correlates with some symptoms of psychiatric disorders, as well as the efficacy of pharmacological treatments. Recent findings highlight a direct association between brain PUFA levels and dopamine transmission, a major neuromodulatory system implicated in the etiology of psychiatric symptoms. However, the mechanisms underlying this relationship are still unknown. Here we demonstrate that membrane enrichment in the n-3 PUFA docosahexaenoic acid (DHA), potentiates ligand binding to the dopamine D2 receptor (D2R), suggesting that DHA acts as an allosteric modulator of this receptor. Molecular dynamics simulations confirm that DHA has a high preference for interaction with the D2R and show that membrane unsaturation selectively enhances the conformational dynamics of the receptor around its second intracellular loop. We find that membrane unsaturation spares G protein activity but potentiates the recruitment of β-arrestin in cells. Furthermore, in vivo n-3 PUFA deficiency blunts the behavioral effects of two D2R ligands, quinpirole and aripiprazole. These results highlight the importance of membrane unsaturation for D2R activity and provide a putative mechanism for the ability of PUFAs to enhance antipsychotic efficacy.

The four vertebrate arrestins play a key role in the desensitization and internalization of G protein-coupled receptors (GPCRs) and also mediate receptor-dependent signaling. Recent work has shown that bias for arrestin vs G protein signaling could offer certain therapeutic advantages (or disadvantages) in different systems, making assays that measure arrestin binding to receptors important for drug discovery efforts. Herein, we briefly review several commonly used techniques for measuring arrestin binding to receptors, as well as provide an in-depth and methodologically focused review of two methods that do not require receptor modification. The first approach measures direct binding between purified arrestin and rhodopsin, and the second measures the recruitment of arrestin to receptors in living cells.

Mutations in the GNB1 gene, encoding the G beta(1) subunit of heterotrimeric G proteins, cause GNB1 Encephalopathy. Patients experience seizures, pointing to abnormal activity of ion channels or neurotransmitter receptors. We studied three G beta(1) mutations (K78R, I80N and I80T) using computational and functional approaches. In heterologous expression models, these mutations did not alter the coupling between G protein-coupled receptors to G(i/o), or the G beta gamma regulation of the neuronal voltage-gated Ca2+ channel Ca(V)2.2. However, the mutations profoundly affected the G beta gamma regulation of the G protein-gated inwardly rectifying potassium channels (GIRK, or Kir3). Changes were observed in G beta(1) protein expression levels, G beta gamma binding to cytosolic segments of GIRK subunits, and in G beta gamma function, and included gain-of-function for K78R or loss-of-function for I80T/N, which were GIRK subunit-specific. Our findings offer new insights into subunit-dependent gating of GIRKs by G beta gamma, and indicate diverse etiology of GNB1 Encephalopathy cases, bearing a potential for personalized treatment.

G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, signaling mediated by β-arrestin has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either G protein or β-arrestinsignaling pathways. However, nearly all screening techniques for measuring β-arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure β-arrestin recruitment to the membrane or early endosomes by unmodified receptors. Our strategy uses NanoLuc, an engineered luciferase from Oplophorus gracilirostris (deep-sea shrimp) that is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. We have identified a unique split site within NanoLuc and fused the corresponding N-terminal fragment to either a plasma membrane or early endosome tether and the C-terminal fragment to β-arrestins, which form the basis for the MeNArC and EeNArC assays, respectively. Upon receptor activation, β-arrestin is recruited to the membrane and subsequently internalized in an agonist concentration-dependent manner. This recruitment promotes complementation of the two NanoLuc fragments, thereby reconstituting functional NanoLuc, allowing for quantification of β-arrestin recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification and has promise as a new generic assay for measuring β-arrestin recruitment to diverse GPCR types in heterologous or native cells.

Heterotrimeric G proteins are the main signalling effectors for G protein-coupled receptors. Understanding the distinct functions of different G proteins is key to understanding how their signalling modulates physiological responses. Pertussis toxin, a bacterial AB

The four vertebrate arrestins play a key role in the desensitization and internalization of G protein-coupled receptors (GPCRs) and also mediate receptor-dependent signaling. Recent work has shown that bias for arrestin vs G protein signaling could offer certain therapeutic advantages (or disadvantages) in different systems, making assays that measure arrestin binding to receptors important for drug discovery efforts. Herein, we briefly review several commonly used techniques for measuring arrestin binding to receptors, as well as provide an in-depth and methodologically focused review of two methods that do not require receptor modification. The first approach measures direct binding between purified arrestin and rhodopsin, and the second measures the recruitment of arrestin to receptors in living cells.

G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, G protein-independent signaling via the arrestin pathway has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either the G protein or arrestin-signaling pathways. Currently available screening techniques that measure arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure arrestin recruitment to any unmodified receptor.NanoLuc, an engineered luciferase from ophlorus gracilirostris (deep sea shrimp), is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. Here, we have identified a novel split site and have fused the N-terminal fragment to a membrane tether and the C-terminal fragment to the N-terminus of either β-arrestin 1 or 2. Upon receptor activation, arrestin is recruited to the plasma membrane in an agonist concentration-dependent manner and the two NanoLuc fragments complement to reconstitute functional luciferase, which allows quantification of recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification. The split NanoLuc arrestin recruitment assay has promise as a new generic assay for measuring arrestin recruitment to diverse GPCR types in heterologous or native cells.