The Orphan G Protein-Coupled Receptor GPR52 is a Novel Regulator of Breast Cancer Multicellular Organization.

G protein-coupled receptors (GPCRs) are the largest class of membrane-bound receptors and transmit critical signals from the extracellular to the intracellular spaces. Transcriptomic data of resected breast tumors shows that low mRNA expression of the orphan GPCR GPR52 correlates with reduced overall survival in breast cancer patients, leading to the hypothesis that loss of GPR52 supports breast cancer progression. CRISPR-Cas9 was used to knockout GPR52 in human triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, and in the non-cancerous breast epithelial cell line, MCF10A. Loss of GPR52 was found to be associated with increased cell-cell interaction in 2D cultures, altered 3D spheroid morphology, and increased propensity to organize and invade collectively in Matrigel. Furthermore, GPR52 loss was associated with features of EMT in MDA-MB-468 cells. To determine the in vivo impact of GPR52 loss, MDA-MB-468 cells were injected into zebrafish and loss of GPR52 was associated with a greater total cancer area compared to control cells. RNA-sequencing and proteomic analyses of GPR52-null breast cancer cells reveal an increased cAMP signaling signature. Consistently, we found that treatment of wild-type (WT) cells with forskolin, which stimulates production of cAMP, induces some phenotypic changes associated with GPR52 loss, and inhibition of cAMP production rescued some of the GPR52 KO phenotypes. Overall, our results reveal GPR52 loss as a potential mechanism by which breast cancer progression may occur and support the investigation of GPR52 agonism as a therapeutic option in breast cancer.

An inflammation resolution-promoting intervention prevents atrial fibrillation caused by left ventricular dysfunction.

AIMS: Recent studies suggest that bioactive mediators called resolvins promote an active resolution of inflammation. Inflammatory signalling is involved in the development of the substrate for atrial fibrillation (AF). The aim of this study is to evaluate the effects of resolvin-D1 on atrial arrhythmogenic remodelling resulting from left ventricular (LV) dysfunction induced by myocardial infarction (MI) in rats. METHODS AND RESULTS: MI was produced by left anterior descending coronary artery ligation. Intervention groups received daily intraperitoneal resolvin-D1, beginning before MI surgery (early-RvD1) or Day 7 post-MI (late-RvD1) and continued until Day 21 post-MI. AF vulnerability was evaluated by performing an electrophysiological study. Atrial conduction was analysed by using optical mapping. Fibrosis was quantified by Masson's trichrome staining and gene expression by quantitative polymerase chain reaction and RNA sequencing. Investigators were blinded to group identity. Early-RvD1 significantly reduced MI size (17 ± 6%, vs. 39 ± 6% in vehicle-MI) and preserved LV ejection fraction; these were unaffected by late-RvD1. Transoesophageal pacing induced atrial tachyarrhythmia in 2/18 (11%) sham-operated rats, vs. 18/18 (100%) MI-only rats, in 5/18 (28%, P < 0.001 vs. MI) early-RvD1 MI rats, and in 7/12 (58%, P < 0.01) late-RvD1 MI rats. Atrial conduction velocity significantly decreased post-MI, an effect suppressed by RvD1 treatment. Both early-RvD1 and late-RvD1 limited MI-induced atrial fibrosis and prevented MI-induced increases in the atrial expression of inflammation-related and fibrosis-related biomarkers and pathways. CONCLUSIONS: RvD1 suppressed MI-related atrial arrhythmogenic remodelling. Early-RvD1 had MI sparing and atrial remodelling suppressant effects, whereas late-RvD1 attenuated atrial remodelling and AF promotion without ventricular protection, revealing atrial-protective actions unrelated to ventricular function changes. These results point to inflammation resolution-promoting compounds as novel cardio-protective interventions with a particular interest in attenuating AF substrate development.

Urotensin II Receptor Modulation with 1,3,4-Benzotriazepin-2-one Tetrapeptide Mimics.

Urotensin II receptor (UT) modulators that differentiate the effects of the endogenous cyclic peptide ligands urotensin II (UII) and urotensin II-related peptide (URP) offer potential for dissecting their respective biological roles in disease etiology. Selective modulators of hUII and URP activities were obtained using 1,3,4-benzotriazepin-2-one mimics of a purported bioactive γ-turn conformation about the Bip-Lys-Tyr tripeptide sequence of urocontrin ([Bip4]URP). Considering an active ß-turn conformer about the shared Phe-Trp-Lys-Tyr sequence of UII and URP, 8-substituted 1,3,4-benzotriazepin-2-ones were designed to mimic the Phe-Bip-Lys-Tyr tetrapeptide sequence of urocontrin, synthesized, and examined for biological activity. Subtle 5- and 8-position modifications resulted in biased signaling and selective modulation of hUII- or URP-induced vasoconstriction. For example, p-hydroxyphenethyl analogs 17b-d were strong Gα13 and ßarr1 activators devoid of Gαq-mediated signaling. Tertiary amides 15d and 17d negatively modulated hUII-induced vasoconstriction without affecting URP-mediated responses. Benzotriazepinone carboxamides proved to be exceptional tools for elucidating the pharmacological complexity of UT.

Exploration of the urocontrin A scaffold yields new urotensinergic system allosteric modulator and competitive antagonists.

The urotensinergic system, involved in the development and/or progression of numerous pathological conditions, is composed of one G protein-coupled receptor (UT) and two endogenous ligands known as urotensin II (UII) and urotensin II-related peptide (URP). These two structurally related hormones, which exert common and divergent effects, are thought to play specific biological roles. In recent years, we have characterized an analog termed urocontrin A (UCA), i.e. [Pep4]URP, which is capable of discriminating the effects of UII from URP. Such an action could allow the delineation of the respective functions of these two endogenous ligands. In an effort to define the molecular determinants involved in this behavior and to improve the pharmacological profile of UCA, we introduced modifications from urantide, considered for some time as a lead compound for the development of UT antagonists, into UCA and assessed the binding, contractile activity and G protein signaling of these newly developed compounds. Our results show that UCA and its derivatives exert probe-dependent effects on UT antagonism, and we have further identified [Pen2, Pep4]URP as a Gq biased ligand with an insurmountable antagonism in our aortic ring contraction assay.

In vivo detection of GPCR-dependent signaling using fiber photometry and FRET-based biosensors.

Genetically encoded fluorescent biosensors allow intracellular signaling dynamics to be tracked in live cells and tissues using optical detection. Many such biosensors are based on the principle of Förster resonance energy transfer (FRET), and we have recently developed a simple approach for in vivo detection of FRET-based biosensor signals using fiber photometry. By combining fiber photometry with FRET-based biosensors, we were able to track GPCR-dependent signaling pathways over time, and in response to drug treatments in freely-moving adult rats. Recording from specific neuronal populations, we can quantify intracellular signaling while simultaneously measuring behavioral responses. Our approach, described in detail here, uses adeno-associated viruses infused intracerebrally in order to express genetically-encoded FRET-based biosensors. After several weeks to allow biosensor expression, fiber photometry is used in order to record drug responses in real time from freely-moving adult rats. This methodology would be compatible with other mammalian species and with many biosensors. Hence, it has wide applicability across a spectrum of neuroscience research, ranging from the study of neural circuits and behavior, to preclinical drug development and screening.

High-Content Single-Cell Förster Resonance Energy Transfer Imaging of Cultured Striatal Neurons Reveals Novel Cross-Talk in the Regulation of Nuclear Signaling by Protein Kinase A and Extracellular Signal-Regulated Kinase 1/2.

Genetically encoded biosensors can be used to track signaling events in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. Here, we describe the use of genetically encoded biosensors based on Förster resonance energy transfer (FRET), combined with high-content microscopy, to image dynamic signaling events simultaneously in thousands of neurons in response to drug treatments. We first applied this approach to examine intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. Using high-content FRET imaging and immunofluorescence, we identified neuronal subpopulations with unique responses to pharmacological manipulation and used nuclear morphology to identify medium spiny neurons within these heterogeneous striatal cultures. Focusing on protein kinase A (PKA) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in the cytoplasm and nucleus, we noted pronounced intercellular differences among putative medium spiny neurons, in both the magnitude and kinetics of signaling responses to drug application. Importantly, a conventional "bulk" analysis that pooled all cells in culture yielded a different rank order of drug potency than that revealed by single-cell analysis. Using a single-cell analytical approach, we dissected the relative contributions of PKA and ERK1/2 signaling in striatal neurons and unexpectedly identified a novel role for ERK1/2 in promoting nuclear activation of PKA in striatal neurons. This finding adds a new dimension of signaling crosstalk between PKA and ERK1/2 with relevance to dopamine D1 receptor signaling in striatal neurons. In conclusion, high-content single-cell imaging can complement and extend traditional population-level analyses and provides a novel vantage point from which to study cellular signaling. SIGNIFICANCE STATEMENT: High-content imaging revealed substantial intercellular variation in the magnitude and pattern of intracellular signaling events driven by receptor stimulation. Since individual neurons within the same population can respond differently to a given agonist, interpreting measures of intracellular signaling derived from the averaged response of entire neuronal populations may not always reflect what happened at the single-cell level. This study uses this approach to identify a new form of cross-talk between PKA and ERK1/2 signaling in the nucleus of striatal neurons.

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands.

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp1, Leu2]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and, to a lesser extent, IP1 production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors.

As with many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell type-specific, and can change in the face of disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinson's disease (PD), alterations in this pathway lead to functional upregulation of extracellular regulated kinases 1/2 (ERK1/2), contributing to L-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Förster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with L-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.

Therapeutic Targeting of the General RNA Polymerase II Transcription Machinery.

Inhibitors targeting the general RNA polymerase II (RNAPII) transcription machinery are candidate therapeutics in cancer and other complex diseases. Here, we review the molecular targets and mechanisms of action of these compounds, framing them within the steps of RNAPII transcription. We discuss the effects of transcription inhibitors in vitro and in cellular models (with an emphasis on cancer), as well as their efficacy in preclinical and clinical studies. We also discuss the rationale for inhibiting broadly acting transcriptional regulators or RNAPII itself in complex diseases.

Propranolol Attenuates Proangiogenic Activity of Mononuclear Phagocytes: Implication in Choroidal Neovascularization.

Purpose: Targeting ß-adrenergic receptor signaling with propranolol has emerged as a potential candidate to counteract choroidal neovascularization (CNV). Little is known of its effect on macrophages, which play a critical role in CNV. We investigated the effect of propranolol on angiogenic response of mononuclear phagocytes (MPs). Methods: The angiogenic effect of propranolol was evaluated in laser-induced CNV model. Mice received intraperitoneal injections of propranolol (6 mg/kg/d) or vehicle. CNV area and inflammatory cells were determined respectively by using lectin staining and an anti-IBA-1 antibody on RPE/choroid flat mounts. Inflammatory gene expression was evaluated by quantitative (q) PCR analysis. Mechanisms of propranolol was studied in MP cell lines J774 and RAW264.7 and in primary peritoneal macrophages. Expression of pro- and antiangiogenic mediators was studied. In addition, effects of propranolol treatment of MPs was assessed on choroidal explant. Results: CNV was attenuated by propranolol and concomitantly associated with decreased inflammatory mediators IL-6 and TNFα, albeit with accumulation of (ß-adrenoceptor harboring) MPs in the CNV area. Conditioned media from MPs preincubated with propranolol exerted antiangiogenic effects. Treatment of J774 confirmed the attenuation of inflammatory response to propranolol and increased cleaved caspase-3 on choroidal explant. We found that propranolol increased pigment epithelium-derived factor (PEDF) expression in MPs. Trapping of PEDF with an antibody abrogated antiangiogenic effects of propranolol. PEDF was also detected in CNV-associated MPs. Conclusions: We hereby show that propranolol confers on MPs antiangiogenic properties by increasing PEDF expression, which complements its effects on vascular tissue resulting in inhibition of choroidal vasoproliferation in inflammatory conditions. The study supports possible use of propranolol as a therapeutic modality for CNV.

Design and biological assessment of membrane-tethering neuroprotective peptides derived from the pituitary adenylate cyclase-activating polypeptide type 1 receptor.

BACKGROUND: The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1), a class B G protein-coupled receptor (GPCR), has emerged as a promising target for treating neurodegenerative conditions. Unfortunately, despite years of research, no PAC1-specific agonist has been discovered, as activity on two other GPCRs, VPAC1 and VPAC2, is retained with current analogs. Cell signaling is related to structural modifications in the intracellular loops (ICLs) of GPCRs. Thus, we hypothesized that peptides derived from the ICLs (called pepducins) of PAC1 might initiate, as allosteric ligands, signaling cascades after recognition of the parent receptor and modulation of its conformational landscape. METHODS: Three pepducins were synthesized and evaluated for their ability to 1) promote cell survival; 2) stimulate various signaling pathways associated with PAC1 activation; 3) modulate selectively PAC1, VPAC1 or VPAC2 activation; and 4) sustain mobility and prevent death of dopaminergic neurons in a zebrafish model of neurodegeneration. RESULTS: Assays demonstrated that these molecules promote SH-SY5Y cell survival, a human neuroblastoma cell line expressing PAC1, and activate signaling via Gαs and Gαq, with distinct potencies and efficacies. Also, PAC1-Pep1 and PAC1-Pep2 activated selectively PAC1-mediated Gαs stimulation. Finally, experiments, using a zebrafish neurodegeneration model, showed a neuroprotective action with all three pepducins and in particular, revealed the ability of PAC1-Pep1 and PAC1-Pep3 to preserve fish mobility and tyrosine hydroxylase expression in the brain. CONCLUSION: We have developed the first neuroprotective pepducins derived from PAC1, a class B GPCR. GENERAL SIGNIFICANCE: PAC1-derived pepducins represent attractive templates for the development of innovative neuroprotecting molecules.

Functional Selectivity Revealed by N-Methylation Scanning of Human Urotensin II and Related Peptides.

In accordance with their common but also divergent physiological actions, human urotensin II (1) and urotensin II-related peptide (2) could stabilize specific urotensin II receptor (UTR) conformations, thereby activating different signaling pathways, a feature referred to as biased agonism or functional selectivity. Sequential N-methylation of the amides in the conserved core sequence of 1, 2, and fragment U-II4-11 (3) shed light on structural requirements involved in their functional selectivity. Thus, 18 N-methylated UTR ligands were synthesized and their biological profiles evaluated using in vitro competition binding assays, ex vivo rat aortic ring bioassays and BRET-based biosensor experiments. Biological activity diverged from that of the parent structures contingent on the location of amide methylation, indicating relevant hydrogen-bond interactions for the function of the endogenous peptides. Conformational analysis of selected N-methyl analogs indicated the importance of specific amide residues of 2 for the distinct pharmacology relative to 1 and 3.

BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes.

The type 1 angiotensin II (AngII) receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR. In a variety of cell lines, both epidermal growth factor (EGF) and AngII stimulated Grb2 recruitment to EGFR. The BRET assay was used to screen a panel of 9 G protein-coupled receptors (GPCRs) and further developed for other EGFR family members (HER2 and HER3); the AT1R was able to transactivate HER2, but not HER3. Mechanistically, AT1R-mediated ERK1/2 activation was dependent on Gq/11 and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of Gq/11 and only partially dependent on EGFR tyrosine kinase activity. This Gq/11 independence of EGFR transactivation was confirmed using AT1R mutants and in CRISPR cell lines lacking Gq/11. EGFR transactivation was also apparently independent of ß-arrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII- and EGF-stimulation promoted AT1R-EGFR heteromerization. In summary, we report an alternative approach to monitoring AT1R-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the AT1R and EGFR.

Discovery of New Allosteric Modulators of the Urotensinergic System through Substitution of the Urotensin II-Related Peptide (URP) Phenylalanine Residue.

Urotensin II (UII) and urotensin II-related peptide (URP) are functionally selective, suggesting that these two hormones might play distinct physiological role through different interactions with their cognate receptor UT. Hypothesizing that the Phe3 residue of URP, which is also present in UII, is a key-element of its specific UT activation, we evaluated the impact of its replacement by non-natural amino acids in URP. Each compound was evaluated for its ability to bind UT, induce rat aortic ring contraction, and activate Gq, G12, and ß-arrestin 1 signaling pathways. Such modifications impaired contractile efficacy, reflected by a reduced aptitude to activate G12 in URP but not in the truncated but equipotent UII4-11. Moreover, we have identified two structurally different UT modulators: [d-Phe(pI)3]URP and [Bip3]URP, which exert a probe-dependent action against UII and URP. These compounds should help us understand the specific roles of these hormones as well as guide further therapeutic development.

New insights about the peculiar role of the 28-38 C-terminal segment and some selected residues in PACAP for signaling and neuroprotection.

The pituitary adenylate cyclase-activating polypeptide (PACAP), which exists in two isoforms of 27 and 38 amino acids, can induce neuronal protection in vitro and in vivo following the activation of PAC1, a class B G protein-coupled receptor (GPCR). With its potent neuroprotective and anti-inflammatory effects, this peptide represents a promising avenue for the development of therapeutic strategies to potentially cure or at least slow the progression of neurodegenerative disorders. Beyond the canonical G protein signal effectors, GPCRs are also coupled to a multitude of intracellular signaling pathways that can be independently activated by biased ligands, thereby expanding vastly the potential for discovering new drugs. Interestingly, some studies have demonstrated distinct signaling features for the PACAP isoforms. With this observation in mind, we assessed the impact of chemical and structural modifications introduced into specific regions of the PACAP isoforms on their neuroprotective effects, and determined the role played by these physico-chemical and structural features on their signaling signatures. Each compound was also evaluated for its ability to bind the PACAP receptors, promote cell survival in a cellular model of Parkinson's disease and stimulate the signaling partners associated with PAC1 activation, including Gs and Gq, as well as ß-arrestin 1 and 2. Our results demonstrate that PACAP38 and its related analogs exert a more potent neuroprotective action than their 27-amino acid counterparts and that this neuroprotective effect is dependent on both Gq and Gs-dependent signaling. This study will definitely improve our understanding of the molecular and cellular mechanisms associated with PACAP neuroprotection.

Receptor- and cellular compartment-specific activation of the cAMP/PKA pathway by α1-adrenergic and ETA endothelin receptors.

The signalling functions of many G protein-coupled receptors (GPCRs) expressed in the myocardium are incompletely understood. Among these are the endothelin receptor (ETR) family and α1-adrenergic receptor (α1-AR), which are thought to couple to the G protein Gαq. In this study, we used transcriptome analysis to compare the signalling networks downstream of these receptors in primary neonatal rat cardiomyocytes. This analysis indicated increased expression of target genes of cAMP responsive element modulator (CREM) after 24 h treatment with the α1-AR agonist phenylephrine, but not the ETR agonist endothelin-1, suggesting a specific role for the α1-AR in promoting cAMP production in cardiomyocytes. To validate the difference observed between these two GPCRs, we used heterologous expression of the receptors and genetically encoded biosensors in HEK 293 cell lines. We validated that both α1A- and α1B-AR subtypes were able to lead to the accumulation of cAMP in response to phenylephrine in both the nucleus and cytoplasm in a Gαs-dependent manner. However, the ETR subtype ETA did not affect cAMP levels in either compartment. All three receptors were coupled to Gαq signalling as expected. Further, we showed that activation of PKA in different compartments was α1-AR subtype specific, with α1B-AR able to activate PKA in the cytoplasm and nucleus and α1A-AR only able to in the nucleus. We provide evidence for a pathway downstream of the α1-AR, and show that distinct pools of a receptor lead to differential activation of downstream effector proteins dependent on their cellular compartment.

Design, Synthesis, and Biological Assessment of Biased Allosteric Modulation of the Urotensin II Receptor Using Achiral 1,3,4-Benzotriazepin-2-one Turn Mimics.

Benzotriazepin-2-ones were designed to mimic the suggested bioactive γ-turn conformation of the Bip-Lys-Tyr tripeptide in Urocontrin ([Bip4]URP), which modulates the urotensin II receptor (UT) and differentiates the effects of the endogenous ligands urotensin II (UII) and urotensin II-related peptide (URP). Twenty-six benzotriazepin-2-ones were synthesized by acylation of anthranilate-derived amino ketones with an aza-glycine equivalent, chemoselective nitrogen functionalization, and ring closure. Several mimics exhibited selective modulatory effects on hUII- and URP-associated vasoconstriction in an ex vivo rat aortic ring bioassay. The C5 p-hydroxyphenethenyl benzotriazepin-2-one 20g decreased hUII potency and efficacy without changing URP induced vasoconstriction. Its saturated phenethyl counterpart 23g decreased URP potency without influencing hUII-mediated contraction. To our knowledge, 20g and 23g represent the first achiral molecules that modulate selectively hUII and URP biological activities. Effectively synthesized, benzotriaepin-2-one turn mimics offer the potential to differentiate the respective roles, signaling pathways, and phenotypic outcomes of hUII and URP in the UT system.

Insight into the role of urotensin II-related peptide tyrosine residue in UT activation.

While sharing common biological activity, the two endogenous ligands of the G protein-coupled receptor UT, e.g. urotensin II (UII) and urotensin II-related peptide (URP), also exhibit distinct effects that could be explained by distinct interactions with their cognate receptor (UT). Accordingly, introduction of a similar substitution at the intracyclic Tyr residue in UII and URP led to compounds with divergent pharmacologic profiles. Hypothesizing that the Tyr6 residue of URP is a key-element to understand the specific activation of UT by URP, we undertook a study of the structure-activity relationship in which this particular residue was replaced by non-natural and constrained amino acids. Each compound was evaluated for its ability to bind UT, to induce rat aortic ring contraction and to activate Gq and G12 signaling pathways. We identified [Pep6]URP, that binds UT with an affinity similar to that of URP, but behaves as a biased ligand. Used as an antagonist, this peptide is also able to selectively reduce the maximal aortic contraction of URP but not UII. Our results suggest that the orientation of the Tyr residue can stabilize at least two different conformations of UT, leading to biased signaling and a probe-dependent allosteric effect.

Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex.

Mechanisms that prevent aggregation and promote folding of nascent G protein-coupled receptors (GPCRs) remain poorly understood. We identified chaperonin containing TCP-1 subunit eta (CCT7) as an interacting partner of the ß-isoform of thromboxane A2 receptor (TPß) by yeast two-hybrid screening. CCT7 coimmunoprecipitated with overexpressed TPß and ß2-adrenergic receptor (ß2AR) in HEK 293 cells, but also with endogenous ß2AR. CCT7 depletion by small interfering RNA reduced total and cell-surface expression of both receptors and caused redistribution of the receptors to juxtanuclear aggresomes, significantly more so for TPß than ß2AR. Interestingly, Hsp90 coimmunoprecipitated with ß2AR but virtually not with TPß, indicating that nascent GPCRs can adopt alternative folding pathways. In vitro pull-down assays showed that both receptors can interact directly with CCT7 through their third intracellular loops and C-termini. We demonstrate that Trp334 in the TPß C-terminus is critical for the CCT7 interaction and plays an important role in TPß maturation and cell-surface expression. Of note, introducing a tryptophan in the corresponding position of the TPα isoform confers the CCT7-binding and maturation properties of TPß. We show that an interaction with a subunit of the CCT/TCP-1 ring complex (TRiC) chaperonin complex is involved in regulating aggregation of nascent GPCRs and in promoting their proper maturation and expression.

Designing BRET-based conformational biosensors for G protein-coupled receptors.

Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures. Despite our growing ability to use such signaling signatures, we remain hampered by the fact that signaling signatures may be particular to an individual cell type and thus our platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. Here, we provide a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in intracellular loop 3 of FP, the receptor for PGF2α. We discuss the design and optimization of such sensors for orthosteric and allosteric ligands.