Probe dependency in the determination of ligand binding kinetics at a prototypical G protein-coupled receptor.

Drug-target binding kinetics are suggested to be important parameters for the prediction of in vivo drug-efficacy. For G protein-coupled receptors (GPCRs), the binding kinetics of ligands are typically determined using association binding experiments in competition with radiolabelled probes, followed by analysis with the widely used competitive binding kinetics theory developed by Motulsky and Mahan. Despite this, the influence of the radioligand binding kinetics on the kinetic parameters derived for the ligands tested is often overlooked. To address this, binding rate constants for a series of histamine H1 receptor (H1R) antagonists were determined using radioligands with either slow (low koff) or fast (high koff) dissociation characteristics. A correlation was observed between the probe-specific datasets for the kinetic binding affinities, association rate constants and dissociation rate constants. However, the magnitude and accuracy of the binding rate constant-values was highly dependent on the used radioligand probe. Further analysis using recently developed fluorescent binding methods corroborates the finding that the Motulsky-Mahan methodology is limited by the employed assay conditions. The presented data suggest that kinetic parameters of GPCR ligands depend largely on the characteristics of the probe used and results should therefore be viewed within the experimental context and limitations of the applied methodology.

A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor.

There is a growing interest in understanding the binding kinetics of compounds that bind to G protein-coupled receptors prior to progressing a lead compound into clinical trials. The widely expressed adenosine A3 receptor (A3AR) has been implicated in a range of diseases including immune conditions, and compounds that aim to selectively target this receptor are currently under development for arthritis. Kinetic studies at the A3AR have been performed using a radiolabelled antagonist, but due to the kinetics of this probe, they have been carried out at 10 °C in membrane preparations. In this study, we have developed a live cell NanoBRET ligand binding assay using fluorescent A3AR antagonists to measure kinetic parameters of labelled and unlabelled compounds at the A3AR at physiological temperatures. The kinetic profiles of four fluorescent antagonists were determined in kinetic association assays, and it was found that XAC-ser-tyr-X-BY630 had the longest residence time (RT = 288 ± 62 min) at the A3AR. The association and dissociation rate constants of three antagonists PSB-11, compound 5, and LUF7565 were also determined using two fluorescent ligands (XAC-ser-tyr-X-BY630 or AV039, RT = 6.8 ± 0.8 min) as the labelled probe and compared to those obtained using a radiolabelled antagonist ([3H]PSB-11, RT = 44.6 ± 3.9 min). There was close agreement in the kinetic parameters measured with AV039 and [3H]PSB-11 but significant differences to those obtained using XAC-S-ser-S-tyr-X-BY630. These data indicate that selecting a probe with the appropriate kinetics is important to accurately determine the kinetics of unlabelled ligands with markedly different kinetic profiles.

Development of novel fluorescent histamine H1-receptor antagonists to study ligand-binding kinetics in living cells.

The histamine H1-receptor (H1R) is an important mediator of allergy and inflammation. H1R antagonists have particular clinical utility in allergic rhinitis and urticaria. Here we have developed six novel fluorescent probes for this receptor that are very effective for high resolution confocal imaging, alongside bioluminescence resonance energy transfer approaches to monitor H1R ligand binding kinetics in living cells. The latter technology exploits the opportunities provided by the recently described bright bioluminescent protein NanoLuc when it is fused to the N-terminus of a receptor. Two different pharmacophores (mepyramine or the fragment VUF13816) were used to generate fluorescent H1R antagonists conjugated via peptide linkers to the fluorophore BODIPY630/650. Kinetic properties of the probes showed wide variation, with the VUF13816 analogues having much longer H1R residence times relative to their mepyramine-based counterparts. The kinetics of these fluorescent ligands could also be monitored in membrane preparations providing new opportunities for future drug discovery applications.

Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestins.

The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser(362), Ser(363) and Thr(366) residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr(350) and Ser(349) are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output.

ß-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells.

Obestatin/GPR39 signaling stimulates skeletal muscle repair by inducing the expansion of satellite stem cells as well as myofiber hypertrophy. Here, we describe that the obestatin/GPR39 system acts as autocrine/paracrine factor on human myogenesis. Obestatin regulated multiple steps of myogenesis: myoblast proliferation, cell cycle exit, differentiation and recruitment to fuse and form multinucleated hypertrophic myotubes. Obestatin-induced mitogenic action was mediated by ERK1/2 and JunD activity, being orchestrated by a G-dependent mechanism. At a later stage of myogenesis, scaffolding proteins ß-arrestin 1 and 2 were essential for the activation of cell cycle exit and differentiation through the transactivation of the epidermal growth factor receptor (EGFR). Upon obestatin stimulus, ß-arrestins are recruited to the membrane, where they functionally interact with GPR39 leading to Src activation and signalplex formation to EGFR transactivation by matrix metalloproteinases. This signalplex regulated the mitotic arrest by p21 and p57 expression and the mid- to late stages of differentiation through JNK/c-Jun, CAMKII, Akt and p38 pathways. This finding not only provides the first functional activity for ß-arrestins in myogenesis but also identify potential targets for therapeutic approaches by triggering specific signaling arms of the GPR39 signaling involved in myogenesis.

ß-Arrestin signal complex plays a critical role in adipose differentiation.

ß-Arrestins were identified as scaffold-proteins that have the capacity to desensitize G protein-coupled receptors. However, it has been found that ß-arrestins activate signaling pathways independent of G protein activation. The diversity of these signaling pathways has also been recognized for receptor tyrosine kinase. The aim of the present study was to validate the ß-arrestin-dependent signaling mechanism(s) responsible for regulation of adipogenesis. Two signal models were selected, ghrelin and insulin, based on its ß-arrestin-associated Akt activity. Herein, we found that ß-arrestin 1 and 2 were essential molecules for adipocyte differentiation. More specifically, the role of these scaffolding proteins was demonstrated by depletion of ß-arrestin 1 and 2 during ghrelin-induced adipogenesis in 3T3-L1 cells, which decreased the adipocyte differentiation and the expression levels of master regulators of early, the CCAAT/enhancer-binding protein ß (C/EBPß) and the CCAAT/enhancer-binding protein δ (C/EBPδ), and terminal, the peroxisome proliferator-activated receptor (PPARγ) and the CCAAT/enhancer-binding protein α (C/EBPα), adipogenesis. Accordingly ghrelin-induced Akt activity and its downstream targets, the mammalian target of rapamycin complex 1 (mTORC1) and the ribosomal protein S6 kinase beta-1 (S6K1), were inhibited by ß-arrestin 1 and 2 siRNAs. By contrast, assays performed during insulin-activated adipogenesis showed an intensifying effect on the adipocyte differentiation as well as on the expression of C/EBPß, C/EBPδ, PPARγ and C/EBPα. The increase in insulin-induced adipogenesis by ß-arrestin knock-down was concomitant to a decrease in the insulin receptor susbtrate-1 (IRS-1) serine phosphorylation, proving the loss of the negative feedback loop on IRS-1/phosphoinositide 3-kinase (PI3K)/Akt. Therefore, ß-arrestins control the extent and intensity of the lipogenic and adipogenic factors associated to Akt signaling, although the mechanistic and functional principles that underlie the connection between signaling and ß-arrestins are specifically associated to each receptor type.