Genetic and biased agonist-mediated reductions in ß-arrestin recruitment prolong cAMP signaling at glucagon family receptors.

Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1R), glucose-dependent insulinotropic polypeptide (GIPR), and glucagon (GCGR) are important regulators of insulin secretion and energy metabolism. GLP-1R agonists have been successfully deployed for the treatment of type 2 diabetes, but it has been suggested that their efficacy is limited by target receptor desensitization and downregulation due to recruitment of ß-arrestins. Indeed, recently described GLP-1R agonists with reduced ß-arrestin-2 recruitment have delivered promising results in preclinical and clinical studies. We therefore aimed to determine if the same phenomenon could apply to the closely related GIPR and GCGR. In HEK293 cells depleted of both ß-arrestin isoforms the duration of G protein-dependent cAMP/PKA signaling was increased in response to the endogenous ligand for each receptor. Moreover, in wildtype cells, "biased" GLP-1, GCG, and GIP analogs with selective reductions in ß-arrestin-2 recruitment led to reduced receptor endocytosis and increased insulin secretion over a prolonged stimulation period, although the latter effect was only seen at high agonist concentrations. Biased GCG analogs increased the duration of cAMP signaling, but this did not lead to increased glucose output from hepatocytes. Our study provides a rationale for the development of GLP-1R, GIPR, and GCGR agonists with reduced ß-arrestin recruitment, but further work is needed to maximally exploit this strategy for therapeutic purposes.

Acylation of the Incretin Peptide Exendin-4 Directly Impacts Glucagon-Like Peptide-1 Receptor Signaling and Trafficking.

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitization and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cAMP. Here, we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analog, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and ß-arrestins, endocytic and postendocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency, but exendin-4-C16 showed ∼2.5-fold bias toward G protein recruitment and a ∼60% reduction in ß-arrestin-2 recruitment efficacy compared with exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting toward recycling pathways. These effects were associated with reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach and a ∼70% increase in insulin secretion in INS-1 832/3 cells. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. SIGNIFICANCE STATEMENT: Acylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. This work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking, and interactions with the plasma membrane, which may be therapeutically important.

Systematic protein-protein interaction mapping for clinically relevant human GPCRs.

G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.

Regulation of RhoA signaling by the cAMP-dependent phosphorylation of RhoGDIα.

RhoA plays a pivotal role in regulating cell shape and movement. Protein kinase A (PKA) inhibits RhoA signaling and thereby induces a characteristic morphological change, cell rounding. This has been considered to result from cAMP-induced phosphorylation of RhoA at Ser-188, which induces a stable RhoA-GTP-RhoGDIα complex and sequesters RhoA to the cytosol. However, few groups have shown RhoA phosphorylation in intact cells. Here we show that phosphorylation of RhoGDIα but not RhoA plays an essential role in the PKA-induced inhibition of RhoA signaling and in the morphological changes using cardiac fibroblasts. The knockdown of RhoGDIα by siRNA blocks cAMP-induced cell rounding, which is recovered by RhoGDIα-WT expression but not when a RhoGDIα-S174A mutant is expressed. PKA phosphorylates RhoGDIα at Ser-174 and the phosphorylation of RhoGDIα is likely to induce the formation of a active RhoA-RhoGDIα complex. Our present results thus reveal a principal molecular mechanism underlying G(s)/cAMP-induced cross-talk with G(q)/G(13)/RhoA signaling.

Novel repertoire of tau biosensors to monitor pathological tau transformation and seeding activity in living cells.

Aggregates of the tau protein are a well-known hallmark of several neurodegenerative diseases, collectively referred to as tauopathies, including frontal temporal dementia and Alzheimer's disease (AD). Monitoring the transformation process of tau from physiological monomers into pathological oligomers or aggregates in a high-throughput, quantitative manner and in a cellular context is still a major challenge in the field. Identifying molecules able to interfere with those processes is of high therapeutic interest. Here, we developed a series of inter- and intramolecular tau biosensors based on the highly sensitive Nanoluciferase (Nluc) binary technology (NanoBiT) able to monitor the pathological conformational change and self-interaction of tau in living cells. Our repertoire of tau biosensors reliably reports i. molecular proximity of physiological full-length tau at microtubules; ii. changes in tau conformation and self-interaction associated with tau phosphorylation, as well as iii. tau interaction induced by seeds of recombinant tau or from mouse brain lysates of a mouse model of tau pathology. By comparing biosensors comprising different tau forms (i.e. full-length or short fragments, wild-type, or the disease-associated tau(P301L) variant) further insights into the tau transformation process are obtained. Proof-of-concept data for the high-throughput suitability and identification of molecules interfering with the pathological tau transformation processes are presented. This novel repertoire of tau biosensors is aimed to boost the disclosure of molecular mechanisms underlying pathological tau transformation in living cells and to discover new drug candidates for tau-related neurodegenerative diseases.

Mitochondria-targeted melatonin photorelease supports the presence of melatonin MT1 receptors in mitochondria inhibiting respiration.

The presence of signaling-competent G protein-coupled receptors in intracellular compartments is increasingly recognized. Recently, the presence of Gi/o protein-coupled melatonin MT1 receptors in mitochondria has been revealed, in addition to the plasma membrane. Melatonin is highly cell permeant, activating plasma membrane and mitochondrial receptors equally. Here, we present MCS-1145, a melatonin derivative bearing a triphenylphosphonium cation for specific mitochondrial targeting and a photocleavable o-nitrobenzyl group releasing melatonin upon illumination. MCS-1145 displayed low affinity for MT1 and MT2 but spontaneously accumulated in mitochondria, where it was resistant to washout. Uncaged MCS-1145 and exogenous melatonin recruited ß-arrestin 2 to MT1 in mitochondria and inhibited oxygen consumption in mitochondria isolated from HEK293 cells only when expressing MT1 and from mouse cerebellum of WT mice but not from MT1-knockout mice. Overall, we developed the first mitochondria-targeted photoactivatable melatonin ligand and demonstrate that melatonin inhibits mitochondrial respiration through mitochondrial MT1 receptors.

A seeding-based neuronal model of tau aggregation for use in drug discovery.

Intracellular accumulation of tau protein is a hallmark of Alzheimer's Disease and Progressive Supranuclear Palsy, as well as other neurodegenerative disorders collectively known as tauopathies. Despite our increasing understanding of the mechanisms leading to the initiation and progression of tau pathology, the field still lacks appropriate disease models to facilitate drug discovery. Here, we established a novel and modulatable seeding-based neuronal model of full-length 4R tau accumulation using humanized mouse cortical neurons and seeds from P301S human tau transgenic animals. The model shows specific and consistent formation of intraneuronal insoluble full-length 4R tau inclusions, which are positive for known markers of tau pathology (AT8, PHF-1, MC-1), and creates seeding competent tau. The formation of new inclusions can be prevented by treatment with tau siRNA, providing a robust internal control for use in qualifying the assessment of potential therapeutic candidates aimed at reducing the intracellular pool of tau. In addition, the experimental set up and data analysis techniques used provide consistent results in larger-scale designs that required multiple rounds of independent experiments, making this is a versatile and valuable cellular model for fundamental and early pre-clinical research of tau-targeted therapies.

Measuring Protein-Protein Interactions of Melatonin Receptors by Bioluminescence Resonance Energy Transfer (BRET).

The melatonin receptor subfamily belongs to the G protein-coupled receptor superfamily and consists of three members in mammals, MT1, MT2, and GPR50. These receptors can interact with each other to form homo- and heterodimers that are part of larger molecular complexes composed of G proteins, ß-arrestins, and other membrane and cytosolic proteins. BRET (bioluminescence resonance energy transfer) is a versatile technique to follow protein-protein interactions on the nanometer scale, in real time, in living cells, which contributed largely to our understanding of the function of melatonin receptors. In this chapter, we describe our BRET protocols for melatonin receptors, which can also be applied to other GPCRs.

Melatonin receptors, brain functions, and therapies.

In mammals, including humans, the neurohormone melatonin is mainly secreted from the pineal gland at night and acts on two high-affinity G protein-coupled receptors, the melatonin MT1 and MT2 receptors. Major functions of melatonin receptors in the brain are the regulation of circadian rhythms and sleep. Correspondingly, the main indications of the currently available drugs for these receptors indicate this as targets. Yet these drugs may not only improve circadian rhythm- and sleep-related disorders but may also be beneficial for complex diseases like major depression, Alzheimer's disease, autism, and attention-deficit/hyperactivity disorders. Here, we will focus on the hypothalamic functions of melatonin receptors by updating our knowledge on their hypothalamic expression pattern at normal, aged, and disease states, by discussing their capacity to regulate circadian rhythms and sleep and by presenting the clinical applications of the melatonin receptor-targeting drugs ramelteon, tasimelteon, and agomelatine or of prolonged-release melatonin formulations. Finally, we speculate about future trends in the field of melatonin receptor drugs.

ß-Arrestin-2 BRET Biosensors Detect Different ß-Arrestin-2 Conformations in Interaction with GPCRs.

ß-Arrestins are critical regulators of G protein-coupled receptors (GPCRs) that desensitize G protein signaling, promote receptor internalization, and initiate signaling on their own. Recent structural findings indicate that ß-arrestins adopt different conformations upon interaction with agonist-activated GPCRs. Here, we established a ß-arrestin-2 conformational bioluminescence resonance energy transfer (BRET) sensor composed of the bright Nanoluc BRET donor and the red-shifted CyOFP1 BRET acceptor. The sensor monitors early intramolecular conformational changes of ß-arrestin-2 in complex with a wide panel of different class A and class B GPCRs upon agonist activation and with orphan GPCRs known to spontaneously recruit ß-arrestin-2. The introduction of the R170E mutant in the ß-arrestin-2 sensor allowed the detection of a partially active ß-arrestin-2 conformation, which is not dependent on receptor phosphorylation, while the deletion of the ß-arrestin-2 finger-loop region detected the "tail-conformation" corresponding to the interaction of ß-arrestin with the carboxyl-terminal domain of GPCRs. The new sensors combine the advantages of the BRET technique in terms of sensitivity, robustness, and suitability for real-time measurements with a high responsiveness toward early conformational changes to help to elucidate the different conformational states of ß-arrestins associated with GPCR activation in living cells.

Melatonin receptors: molecular pharmacology and signalling in the context of system bias.

Melatonin, N-acetyl-5-methoxytryptamine, an evolutionally old molecule, is produced by the pineal gland in vertebrates, and it binds with high affinity to melatonin receptors, which are members of the GPCR family. Among the multiple effects attributed to melatonin, we will focus here on those that are dependent on the activation of the two mammalian MT1 and MT2 melatonin receptors. We briefly summarize the latest developments on synthetic melatonin receptor ligands, including multi-target-directed ligands, and the characterization of signalling-biased ligands. We discuss signalling pathways activated by melatonin receptors that appear to be highly cell- and tissue-dependent, emphasizing the impact of system bias on the functional outcome. Different proteins have been demonstrated to interact with melatonin receptors, and thus, we postulate that part of this system bias has its molecular basis in differences of the expression of receptor-associated proteins including heterodimerization partners. Finally, bias at the level of the receptor, by the expression of genetic receptor variants, will be discussed to show how a modified receptor function can have an effect on the risk for common diseases like type 2 diabetes in humans. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

Melatonin Receptor Signaling: Impact of Receptor Oligomerization on Receptor Function.

The melatonin receptor subfamily is composed of three members, MT1 and MT2, which are binding to melatonin, and GPR50, which shows high sequence homology to MT1 and MT2 but does not bind to melatonin or any other known ligand. An interesting feature of these receptors is their capacity to form homo- and heteromers between each other and also with other GPCRs. The following heteromers have been described: MT1/MT2, MT1/GPR50, and heteromers composed of MT2 and the serotonin 5-HT2c receptor or the orphan GPR61, GPR62, and GPR135 receptors. These heteromers represent novel pharmacological entities as they exhibit functional properties that are different from those of the corresponding homomers. Formation of several of these heteromers has been confirmed in tissues. MT2/5-HT2c heteromers are targeted by the clinically relevant antidepressant agomelatine, and MT1/MT2 heteromers regulate nocturnal retinal light sensitivity. Here, we resume our current knowledge on melatonin receptor heteromerization and discuss how it contributes to the diversification of the function of melatonin receptors.

Gain of affinity for VEGF165 binding within the VEGFR2/NRP1 cellular complex detected by an HTRF-based binding assay.

Neuroplin 1 (NRP1), a transmembrane protein interacting with Vascular Endothelial Growth Factor VEGF-A165 (called here VEGF165) and the tyrosine kinase Receptor 2 (VEGFR2) promote angiogenesis and vascular homeostasis. In a pathophysiological context, several studies suggested that VEGFR2 and NRP1 mediate tumor development and progression. Given the involvement of the VEGF165 network in promoting tumor angiogenesis, NRP1, VEGFR2 and VEGF165 have been identified as targets for anti-angiogenic therapy. No binding assay exists to monitor specifically the binding of VEGF165 to the VEGFR2/NRP1 complex in intact cells. We established a binding assay based on the homogenous time-resolved fluorescence (HTRF®) technology. This unique binding assay enables to assess the interaction of VEGF165 with VEGFR2 or NRP1 within the VEGFR2/NRP1 complex. Ligand binding saturation experiments revealed that VEGF165 binds the VEGFR2/NRP1 complex at the cell surface with a ten to twenty-fold higher affinity compared to SNAP-VEGFR2 or SNAP-NRP1 receptors alone not engaged in the heteromeric complex. The assay allows characterizing the impact of NRP1 ligands on VEGF165 to the complex. It shows high specificity, reproducibility and robustness, making it compatible with high throughput screening (HTS) applications for identifying new VEGF165 antagonists selective for NRP1 or the VEGFR2/NRP1 complex.

Orphan GPR61, GPR62 and GPR135 receptors and the melatonin MT2 receptor reciprocally modulate their signaling functions.

Understanding the function of orphan G protein-coupled receptors (GPCRs), whose cognate ligand is unknown, is of major importance as GPCRs are privileged drug targets for many diseases. Recent phylogenetic studies classified three orphan receptors, GPR61, GPR62 and GPR135 among the melatonin receptor subfamily, but their capacity to bind melatonin and their biochemical functions are not well characterized yet. We show here that GPR61, GPR62 and GPR135 do not bind [3H]-melatonin nor 2-[125I]iodomelatonin and do not respond to melatonin in several signaling assays. In contrast, the three receptors show extensive spontaneous ligand-independent activities on the cAMP, inositol phosphate and ß-arrestin pathways with distinct pathway-specific profiles. Spontaneous ß-arrestin recruitment internalizes all three GPRs in the endosomal compartment. Co-expression of the melatonin binding MT2 receptor with GPR61, GPR62 or GPR135 has several consequences such as (i) the formation of receptor heteromers, (ii) the inhibition of melatonin-induced ß-arrestin2 recruitment to MT2 and (iii) the decrease of elevated cAMP levels upon melatonin stimulation in cells expressing spontaneously active GPR61 and GPR62. Collectively, these data show that GPR61, GPR62 and GPR135 are unable to bind melatonin, but show a reciprocal regulatory interaction with MT2 receptors.

An acquired hypocalciuric hypercalcemia autoantibody induces allosteric transition among active human Ca-sensing receptor conformations.

The seven-spanning calcium-sensing receptor (CaSR) activates multiple G proteins including Gq and Gi, and thereby activates a variety of second messengers and inhibits parathyroid hormone (PTH) secretion. However, the exact signaling mechanisms underlying the functional activity of CaSR are not yet fully understood. The heterozygous inactivation of CaSR or its inhibition by antibody blocking results in either familial hypocalciuric hypercalcemia or acquired hypocalciuric hypercalcemia (AHH), respectively. Here, we report the identification of a unique CaSR autoantibody in an AHH patient. Paradoxically, we find that this autoantibody potentiates the Ca(2+)/Gq-dependent accumulation of inositol phosphates by slightly shifting the dose dependence curve of the Ca(2+) mediated activation of phosphatidylinositol turnover to the left, whereas it inhibits the Ca(2+)/Gi-dependent phosphorylation of ERK1/2 in HEK293 cells stably expressing human CaSR. Treatment of these same cells with a calcimimetic, NPS-R-568, augments the CaSR response to Ca(2+), increasing phosphatidylinositol turnover and ERK1/2 phosphorylation, and overcoming the autoantibody effects. Our observations thus indicate that a calcium-stimulated CaSR primed by a specific autoantibody adopts a unique conformation that activates Gq but not Gi. Our findings also suggest that CaSR signaling may act via both Gq and Gi to inhibit PTH secretion. This is the first report of a disease-related autoantibody that functions as an allosteric modulator and maintains G protein-coupled receptors (GPCRs) in a unique active conformation with its agonist. We thus speculate that physiological modulators may exist that enable an agonist to specifically activate only one signaling pathway via a GPCR that activates multiple signaling pathways.

Signal flare phenomenon as active bleeding in retroperitoneal hematoma with hematocrit effect on dynamic CT scan: three clinical cases and experimental study based on a specific gravity theory.

The contrast medium was observed as a thin line indicating active bleeding in the cellular component and stayed at the boundary between the plasma and cellular components in liquefied hematomas. We could also reproduce the same phenomenon in an experimental study using human blood. The characteristic dynamic motion of the contrast medium demonstrated in the liquefied hematoma, which we have called the "signal flare" phenomenon, is a significant sign indicating active bleeding.