An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters.

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

G protein-coupled receptors can control the Hippo/YAP pathway through Gq signaling.

The Hippo pathway is an evolutionarily conserved kinase cascade involved in the control of tissue homeostasis, cellular differentiation, proliferation, and organ size, and is regulated by cell-cell contact, apical cell polarity, and mechanical signals. Miss-regulation of this pathway can lead to cancer. The Hippo pathway acts through the inhibition of the transcriptional coactivators YAP and TAZ through phosphorylation. Among the various signaling mechanisms controlling the hippo pathway, activation of G12/13 by G protein-coupled receptors (GPCR) recently emerged. Here we show that a GPCR, the ghrelin receptor, that activates several types of G proteins, including G12/13, Gi/o, and Gq, can activate YAP through Gq/11 exclusively, independently of G12/13. We revealed that a strong basal YAP activation results from the high constitutive activity of this receptor, which can be further increased upon agonist activation. Thus, acting on ghrelin receptor allowed to modulate up-and-down YAP activity, as activating the receptor increased YAP activity and blocking constitutive activity reduced YAP activity. Our results demonstrate that GPCRs can be used as molecular switches to finely up- or down-regulate YAP activity through a pure Gq pathway.

HTRF® Total and Phospho-YAP (Ser127) Cellular Assays.

The YAP protein is a co-transcription factor increasing the expression of genes involved in cell proliferation and repressing the expression of genes important for cell differentiation and apoptosis. It is regulated by several inputs, like the Hippo pathway, through the action of kinases that phosphorylate YAP on several residues. The level of phosphorylation of the residues serine 127 (S127) of YAP is generally assessed in cellular models, native tissues, and organs, as a marker of YAP activity and location, and is regulated by numerous partners. This phosphorylation event is classically detected using a western blot technical approach. Here, we describe a novel approach to detect both the relative amount of total YAP (T-YAP assay) and the phosphorylation of the residue S127 of YAP (S127-P-YAP assay) using a HTRF®-based method. This easy-to-run method can easily be miniaturized and allows for a high-throughput analysis in 96/384-well plate format, requiring less cellular material and being more rapid than other approaches.

Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling.

The parathyroid hormone receptor 1 (PTH1R) is a member of family B of G-protein-coupled receptors (GPCRs), predominantly expressed in bone and kidney where it modulates extracellular Ca2+ homeostasis and bone turnover. It is well established that phosphorylation of GPCRs constitutes a key event in regulating receptor function by promoting arrestin recruitment and coupling to G-protein-independent signaling pathways. Mapping phosphorylation sites on PTH1R would provide insights into how phosphorylation at specific sites regulates cell signaling responses and also open the possibility of developing therapeutic agents that could target specific receptor functions. Here, we have used mass spectrometry to identify nine sites of phosphorylation in the C-terminal tail of PTH1R. Mutational analysis revealed identified two clusters of serine and threonine residues (Ser489-Ser495 and Ser501-Thr506) specifically responsible for the majority of PTH(1-34)-induced receptor phosphorylation. Mutation of these residues to alanine did not affect negatively on the ability of the receptor to couple to G-proteins or activate extracellular-signal-regulated kinase 1/2. Using fluorescence resonance energy transfer and bioluminescence resonance energy transfer to monitor PTH(1-34)-induced interaction of PTH1R with arrestin3, we show that the first cluster Ser489-Ser495 and the second cluster Ser501-Thr506 operated in concert to mediate both the efficacy and potency of ligand-induced arrestin3 recruitment. We further demonstrate that Ser503 and Thr504 in the second cluster are responsible for 70% of arrestin3 recruitment and are key determinants for interaction of arrestin with the receptor. Our data are consistent with the hypothesis that the pattern of C-terminal tail phosphorylation on PTH1R may determine the signaling outcome following receptor activation.

Engineered hyperphosphorylation of the ß2-adrenoceptor prolongs arrestin-3 binding and induces arrestin internalization.

G protein-coupled receptor phosphorylation plays a major role in receptor desensitization and arrestin binding. It is, however, unclear how distinct receptor phosphorylation patterns may influence arrestin binding and subsequent trafficking. Here we engineer phosphorylation sites into the C-terminal tail of the ß2-adrenoceptor (ß2AR) and demonstrate that this mutant, termed ß2AR(SSS), showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. By measuring arrestin-3 recruitment and the stability of arrestin-3 receptor complexes in real time using fluorescence resonance energy transfer and fluorescence recovery after photobleaching, we demonstrate that arrestin-3 dissociated quickly and almost completely from the ß2AR, whereas the interaction with ß2AR(SSS) was 2- to 4-fold prolonged. In contrast, arrestin-3 interaction with a ß2-adrenoceptor fused to the carboxyl-terminal tail of the vasopressin type 2 receptor was nearly irreversible. Further analysis of arrestin-3 localization revealed that by engineering phosphorylation sites into the ß2-adrenoceptor the receptor showed prolonged interaction with arrestin-3 and colocalization with arrestin in endosomes after internalization. This is in contrast to the wild-type receptor that interacts transiently with arrestin-3 at the plasma membrane. Furthermore, ß2AR(SSS) internalized more efficiently than the wild-type receptor, whereas recycling was very similar for both receptors. Thus, we show how the interaction between arrestins and receptors can be increased with minimal receptor modification and that relatively modest increases in receptor-arrestin affinity are sufficient to alter arrestin trafficking.