The conformational signature of ß-arrestin2 predicts its trafficking and signalling functions.

Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based 'signalsomes'. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters to monitor conformational changes in ß-arrestin2, here we show that GPCRs impose distinctive arrestin 'conformational signatures' that reflect the stability of the receptor-arrestin complex and role of ß-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in ß-arrestin2 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average ß-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands and in identifying factors that dictate arrestin conformation and function.

PD-1/PD-L1 checkpoint inhibitors in combination with olaparib display antitumor activity in ovarian cancer patient-derived three-dimensional spheroid cultures.

Immune checkpoint inhibitors (ICIs) that target programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) have shown modest activity as monotherapies for the treatment of ovarian cancer (OC). The rationale for using these therapies in combination with poly (ADP-ribose) polymerase inhibitors (PARP-Is) has been described, and their in vivo application will benefit from ex vivo platforms that aid in the prediction of patient response or resistance to therapy. This study examined the effectiveness of detecting patient-specific immune-related activity in OC using three-dimensional (3D) spheroids. Immune-related cell composition and PD-1/PD-L1 expression status were evaluated using cells dissociated from fresh OC tissue from two patients prior to and following 3D culture. The patient sample with the greatest increase in the proportion of PD-L1 + cells also possessed more activated cytotoxic T cells and mature DCs compared to the other patient sample. Upon cytokine stimulation, patient samples demonstrated increases in cytotoxic T cell activation and DC major histocompatibility complex (MHC) class-II expression. Pembrolizumab increased cytokine secretion, enhanced olaparib cytotoxicity, and reduced spheroid viability in a T cell-dependent manner. Furthermore, durvalumab and olaparib combination treatment increased cell death in a synergistic manner. This work demonstrates that immune cell activity and functional modulation can be accurately detected using our ex vivo 3D spheroid platform, and it presents evidence for their utility to demonstrate sensitivity to ICIs alone or in combination with PARP-Is in a preclinical setting.

S1P in HDL promotes interaction between SR-BI and S1PR1 and activates S1PR1-mediated biological functions: calcium flux and S1PR1 internalization.

HDL normally transports about 50-70% of plasma sphingosine 1-phosphate (S1P), and the S1P in HDL reportedly mediates several HDL-associated biological effects and signaling pathways. The HDL receptor, SR-BI, as well as the cell surface receptors for S1P (S1PRs) may be involved partially and/or completely in these HDL-induced processes. Here we investigate the nature of the HDL-stimulated interaction between the HDL receptor, SR-BI, and S1PR1 using a protein-fragment complementation assay and confocal microscopy. In both primary rat aortic vascular smooth muscle cells and HEK293 cells, the S1P content in HDL particles increased intracellular calcium concentration, which was mediated by S1PR1. Mechanistic studies performed in HEK293 cells showed that incubation of cells with HDL led to an increase in the physical interaction between the SR-BI and S1PR1 receptors that mainly occurred on the plasma membrane. Model recombinant HDL (rHDL) particles formed in vitro with S1P incorporated into the particle initiated the internalization of S1PR1, whereas rHDL without supplemented S1P did not, suggesting that S1P transported in HDL can selectively activate S1PR1. In conclusion, these data suggest that S1P in HDL stimulates the transient interaction between SR-BI and S1PRs that can activate S1PRs and induce an elevation in intracellular calcium concentration.

The arrestin-selective angiotensin AT1 receptor agonist [Sar1,Ile4,Ile8]-AngII negatively regulates bradykinin B2 receptor signaling via AT1-B2 receptor heterodimers.

The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar(1),Ile(4),Ile(8)]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar(1),Ile(4), Ile(8)]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar(1),Ile(4),Ile(8)]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar(1),Ile(4),Ile(8)]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar(1),Ile(4),Ile(8)]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar(1),Ile(4),Ile(8)]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic actions.

Development of inhibitors of heterotrimeric Gαi subunits.

Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gαi by competing for the GPCR and Gßγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α2-adrenoceptor (α2AR) mediated decreases in cAMP in HEK293 cells at 100nM. We then sought to discover selective small molecule inhibitors for Gαi. Molecular docking was used to identify potential molecules that bind to and stabilize the Gαi-GDP complex by directly interacting with both Gαi and GDP. Gαi-GTP and Gαq-GDP were used as a computational counter screen and Gαq-GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gαi. Several compounds showed Gαi specific inhibition and were able to block α2AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.

Emergent biological properties of arrestin pathway-selective biased agonism.

Our growing appreciation of the pluridimensionality of G protein-coupled receptor (GPCR) signaling, combined with the phenomenon of orthosteric ligand "bias", has created the possibility of drugs that selectively modulate different aspects of GPCR function for therapeutic benefit. When viewed from the short-term perspective, e.g. changes in receptor conformation, effector coupling or second messenger generation, biased ligands appear to activate a subset of the response profile produced by a conventional agonist. Yet when examined in vivo, the limited data available suggest that biased ligand effects can diverge from their conventional counterparts in ways that cannot be predicted from their in vitro efficacy profile. What is currently missing, at least with respect to G protein and arrestin pathway-selective ligands, is a rational framework for relating the in vitro efficacy of a "biased" agonist to its in vivo actions that will enable drug screening programs to identify ligands with the desired biological effects.

A high-content, live-cell, and real-time approach to the quantitation of ligand-induced ß-Arrestin2 and Class A/Class B GPCR mobilization.

We report the development of a method to analyze receptor and ß-arrestin2 mobilization between Class A and B GPCRs via time-resolved fluorescent microscopy coupled with semiautomated high-content multiparametric analysis. Using transiently expressed, tagged ß2-adrenergic receptor (ß2-AR) or parathyroid hormone receptor type 1 (PTH1R), we quantified trafficking of the receptors along with the mobilization and colocalization of coexpressed tagged ß-arrestin2. This classification system allows for exclusion of cells with nonoptimal characteristics and calculation of multiple morphological and spatial parameters including receptor endosome formation, ß-arrestin mobilization, colocalization, areas, and shape. Stimulated Class A and B receptors demonstrate dramatically different patterns with regard to ß-arrestin interactions. The method provides high kinetic resolution measurement of receptor translocation, which allows for the identification of the fleeting ß-arrestin interaction found with ß2-AR agonist stimulation, in contrast to stronger mobilization and receptor colocalization with agonist stimulation of the PTH1R. Though especially appropriate for receptor kinetic studies, this method is generalizable to any dual fluorescence probe system in which quantification of object formation and movement is desired. These methodologies allow for quantitative, unbiased measurement of microscopy data and are further enhanced by providing real-time kinetics.

Inhibition of the Myocardin-Related Transcription Factor Pathway Increases Efficacy of Trametinib in NRAS-Mutant Melanoma Cell Lines.

The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma; it is aberrantly activated in almost 80% of human cutaneous melanomas (≈50% BRAFV600 mutations and ≈30% NRAS mutations). While drugs targeting the MAPK pathway have yielded success in BRAFV600 mutant melanoma patients, such therapies have been ineffective in patients with NRAS mutant melanomas in part due to their cytostatic effects and primary resistance. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to the MEK inhibitor, trametinib, in a panel of NRAS mutant melanoma cell lines. A combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in SK-Mel-147 cells, which are highly resistant to trametinib. These findings suggest a role of the Rho/MRTF-pathway in intrinsic trametinib resistance in a subset of NRAS mutant melanoma cell lines and highlight the therapeutic potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

Pharmacological Inhibition of Myocardin-related Transcription Factor Pathway Blocks Lung Metastases of RhoC-Overexpressing Melanoma.

Melanoma is the most dangerous form of skin cancer with the majority of deaths arising from metastatic disease. Evidence implicates Rho-activated gene transcription in melanoma metastasis mediated by the nuclear localization of the transcriptional coactivator, myocardin-related transcription factor (MRTF). Here, we highlight a role for Rho and MRTF signaling and its reversal by pharmacologic inhibition using in vitro and in vivo models of human melanoma growth and metastasis. Using two cellular models of melanoma, we clearly show that one cell type, SK-Mel-147, is highly metastatic, has high RhoC expression, and MRTF nuclear localization and activity. Conversely, SK-Mel-19 melanoma cells have low RhoC expression, and decreased levels of MRTF-regulated genes. To probe the dependence of melanoma aggressiveness to MRTF transcription, we use a previously developed small-molecule inhibitor, CCG-203971, which at low micromolar concentrations blocks nuclear localization and activity of MRTF-A. In SK-Mel-147 cells, CCG-203971 inhibits cellular migration and invasion, and decreases MRTF target gene expression. In addition, CCG-203971-mediated inhibition of the Rho/MRTF pathway significantly reduces cell growth and clonogenicity and causes G1 cell-cycle arrest. In an experimental model of melanoma lung metastasis, the RhoC-overexpressing melanoma cells (SK-Mel-147) exhibited pronounced lung colonization compared with the low RhoC-expressing SK-Mel-19. Furthermore, pharmacologic inhibition of the MRTF pathway reduced both the number and size of lung metastasis resulting in a marked reduction of total lung tumor burden. These data link Rho and MRTF-mediated signaling with aggressive phenotypes and support targeting the MRTF transcriptional pathway as a novel approach to melanoma therapeutics. Mol Cancer Ther; 16(1); 193-204. ©2016 AACR.

Suppression of GNAI2 message in ovarian cancer.

BACKGROUND: Understanding the integration of hormone signaling and how it impacts oncogenesis is critical for improved cancer treatments. Here we elucidate GNAI2 message alterations in ovarian cancer (OvCa). GNAI2 is a heterotrimeric G protein which couples cell surface hormone receptors to intracellular enzymes, and is best characterized for its direct role in regulating cAMP response element-binding protein (CREB) function by decreasing intracellular cAMP through inhibiting adenylyl cyclase. METHODS: We probed the Origene human OvCa array for the presence of polymorphisms and gene expression alterations of GNAI2 using directing sequencing and qPCR. These data were supported by database mining of the [NCBI NIH GSE:6008, GSE:14764, GSE:29450, GDS:4066, GDS:3297, GSE:32474, and GSE:2003] datasets. RESULTS: No significant polymorphisms were found, including an absence of the gip2 oncogene. However, 85.9% of (506 of 589) OvCa patients had decreased GNAI2 message. Further characterization demonstrated that the GNAI2 message was on average decreased 54% and maximally decreased by 2.8 fold in clear cell carcinoma. GNAI2 message decreased in early stage cancer while message was increased compared to normal in advanced cancers. The changes in GNAI2 also correlated to deregulation of CREB, Fos, Myc, cyclins, Arf, the transition from estrogen dependence to independence, and metastatic potential. CONCLUSION: These data strongly implicate GNAI2 as a critical regulator of oncogenesis and an upstream driver of cancer progression in OvCa.

Biasing the parathyroid hormone receptor: relating in vitro ligand efficacy to in vivo biological activity.

Recent advances in our understanding of the pluridimensional nature of GPCR signaling have provided new insights into how orthosteric ligands regulate receptors, and how the phenomenon of functional selectivity or ligand "bias" might be exploited in pharmaceutical design. In contrast to the predictions of simple two-state models of GPCR function, where ligands affect all aspects of GPCR signaling proportionally, current models assume that receptors exist in multiple "active" conformations that differ in their ability to couple to different downstream effectors, and that structurally distinct ligands can bias signaling by preferentially stabilizing different active states. The type 1 parathyroid hormone receptor (PTH(1)R) offers unique insight into both the opportunities and challenges of exploiting ligand bias in pharmaceutical design, not only because numerous "biased" PTH analogs have been described but also because many of them have been characterized for biological activity in vivo. The PTH(1)R has pleiotropic signaling capacity, coupling to G(s), G(q/11), and G(i/o) family heterotrimeric G proteins, and binding arrestins, which mediate receptor desensitization and arrestin-dependent signaling. Here, we compare the activity of six different PTH(1)R ligands in a common HEK293 cell background using three readouts of receptor activation, cAMP production, intracellular calcium influx, and ERK1/2 activation, demonstrating the range of signal bias that can be experimentally observed in a "typical" screening program. When the in vitro activity profiles of these ligands are compared to their reported effects on bone mass in murine models, it is apparent that ligands activating cAMP production produce an anabolic response that does not correlate with the ability to also elicit calcium signaling. Paradoxically, one ligand that exhibits inverse agonism for cAMP production and arrestin-dependent ERK1/2 activation in vitro, (D-Trp(12), Tyr(34))-bPTH(7-34), reportedly produces an anabolic bone response in vivo despite an activity profile that is dramatically different from that of other active ligands. This underscores a major challenge facing efforts to rationally design "biased" GPCR ligands for therapeutic application. While it is clearly plausible to identify functionally selective ligands, the ability to predict how bias will affect drug response in vivo, is often lacking, especially in complex disorders.