Innovative Bioluminescence Resonance Energy Transfer Assay Reveals Differential Agonist-Induced D2 Receptor Intracellular Trafficking and Arrestin-3 Recruitment.

The dopamine D2 receptor (D2R) mediates ligand-biased signaling with potential therapeutic implications. However, internalization, choice of endocytic routes, and degradation of the D2R in lysosomes may also participate in agonist-directed trafficking. We developed bioluminescence resonance energy transfer (BRET) assays that measure relative distances between Renilla luciferase8-tagged D2R and green fluorescent protein 2 (GFP2)-tagged K-Ras (plasma membrane marker), and between luciferase8-tagged D2R and GFP2-Rab5 (early), GFP2-Rab4 (recycling), or GFP2-Rab7 (late) endosomal markers. The BRET signal between D2R-Luc and GFP2-K-Ras was robustly diminished after receptor internalization induced by dopamine, with subsequent BRET signals increasing when luciferase8-tagged D2R approached GFP2-Rab proteins in endosomal compartments. All BRET signals were blocked by the selective D2R antagonist haloperidol and were decreased by low temperature and high sucrose blocks, two parameters interfering with internalization. Some antipsychotic drugs, such as aripiprazole, are less efficacious in internalizing D2R than most of the antiparkinsonian agents. However, antipsychotics were nearly as efficacious as antiparkinsonians in directing the D2R toward early and recycling endosomes. The Rab7 marker for the late endosome/lysosome route was also capable of discriminating between D2R compounds. We could show that some drugs engaged the D2R either to interact preferentially with arrestin-3 or to internalize. Our study revealed that D2R trafficking in cells was differentially regulated by antipsychotic and antiparkinsonian drugs. Taken together, the BRET assays reported here could further help decipher D2R ligand-induced arrestin-3 recruitment and trafficking, with potentially more selective therapeutic profiles and fewer undesired side effects.

Pharmacological evaluation of a series of smoothened antagonists in signaling pathways and after topical application in a depilated mouse model.

The Hedgehog (HH) pathway has been linked to the formation of basal cell carcinoma (BCC), medulloblastoma, and other cancers. The recently approved orally active drugs vismodegib (GDC-0449) and sonidegib (LDE-225) were not only efficacious for the treatment of advanced or metastatic BCC by antagonizing the smoothened (SMO) receptor, but also produced important side effects, limiting their use for less invasive BCC. Herein, we compared a large series of SMO antagonists, including GDC-0449 and LDE-225, the clinically tested BMS-833923, CUR-61414, cyclopamine, IPI-926 (saridegib), itraconazole, LEQ-506, LY-2940680 (taladegib), PF-04449913 (glasdegib), and TAK-441 as well as preclinical candidates (PF-5274857, MRT-83) in two SMO-dependent cellular assays and for G-protein activation. We report marked differences in inhibitor potencies between compounds as well as a notable disparity between the G-protein assay and the cellular tests, suggesting that classification of drugs is assay dependent. Furthermore, we explored topical efficacies of SMO antagonists on depilated mice using Gli1 and Ptch1 mRNA quantification in skin as biomarkers of the HH signaling inhibition. This topical model rapidly discriminated drugs in terms of efficacies and potencies for inhibition of both biomarkers. SMO antagonists showed also a large variation in their blood and skin partition, suggesting that some drugs are more favorable for topical application. Overall, our data suggested that in vitro and in vivo efficacious drugs such as LEQ-506 and TAK-441 may be of interest for topical treatment of less invasive BCC with minimal side effects.

Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2'-deoxycytidine in human leukemic KG1 cells.

5-azacytidine and 5-aza-2'-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2'-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2'-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2'-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2'-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.

µ-Opioid and 5-HT1A receptors heterodimerize and show signalling crosstalk via G protein and MAP-kinase pathways.

µ-opioid receptors have been shown to form heterodimers with several G protein coupled receptors involved in pain regulation such as α(2A)-adrenergic and neurokinin 1 receptors. Because the 5-HT(1A) receptor is also involved in pain control, we investigated whether it can interact with the µ-opioid receptor in cell lines. Using epitope-tagged µ-opioid and 5-HT(1A) receptors, we show that both receptors can co-immunoprecipate when expressed in the same cells. This physical interaction was corroborated by a Bioluminescence Resonance Energy Transfer signal between the µ-opioid receptor fused to Renilla luciferase and the 5-HT(1A) receptor fused to the Green Fluorescent Protein. Consistent with the presence of functional heterodimers, the µ-opioid receptor activated a Gα(o) protein covalently fused to the 5-HT(1A) receptor in membrane preparations as well as a Gα(15) protein fused to the 5-HT(1A) receptor in living cells. We demonstrate that both receptors can coexerce control of the ERK1/2 pathway: for example, µ-opioid receptor-induced ERK1/2 phosphorylation was selectively desensitized by 5-HT(1A) receptor activation. Although 5-HT(1A) and µ-opioid receptors were capable to internalize in response to their own activation, they were ineffective to induce the co-internalization of their partners. Thus, we show a functional heterodimerization of µ-opioid and 5-HT(1A) receptors in cell lines, a complex that might play a role in the control of pain in vivo. These results also support the potential therapeutic action of 5-HT(1A) agonists against nociceptive processes.

Cellular BRET assay suggests a conformational rearrangement of preformed TrkB/Shc complexes following BDNF-dependent activation.

We developed a cellular Bioluminescent Resonance Energy Transfer (BRET) assay based on the interaction of TrkB fused to Renilla luciferase with the intracellular adaptor protein Shc fused to Enhanced Yellow Fluorescent Protein (EYFP). The TrkB agonist Brain Derived Neurotrophic Factor (BDNF) induced a maximum BRET signal as of 10 min with an EC(50) value of 1.4 nM, similar to the other endogenous agonists NT-3 and NT-4/5, 1.5 nM and 0.34 nM, respectively. Interestingly, measure of the BRET signal with increasing expression of Shc-EYFP, in the presence or absence of BDNF, suggested a conformational change of preformed TrkB/Shc complexes rather than Shc recruitment. Furthermore, the Y516F TrkB mutant deficient to bind Shc as well as the kinase-dead K572R TrkB mutant was unable to respond to BDNF and exhibited a lower basal BRET signal than that of the wild-type TrkB receptor, again suggesting a preformed complex with constitutive activity. The double YY706/707FF TrkB mutant in the kinase activation loop also showed reduced basal activity but surprisingly kept its capacity to enhance BDNF-induced interaction with Shc, though with less efficacy. The Trk selective kinase inhibitors K252a and BMS-9 blocked BDNF-induced BRET signal with similar potency (100-150 nM), the preferential c-Met inhibitor PF-2341006 being one order of magnitude less potent. Remarkably, in the absence of BDNF, K252a and BMS-9 also reduced basal activity to the level of the Y516F TrkB mutant, suggesting that these compounds were able to reduce the TrkB constitutive activity. BRET responses of mutants and to kinase inhibitors thus reveal a complex level of interaction between TrkB and Shc and suggest that this BRET assay could be of great utility to test blockers of TrkB signalling in a physiologically relevant context.

Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo.

Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone 39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new class of antithrombotic agents.

Mutant 5-hydroxytryptamine 1A receptor D116A is a receptor activated solely by synthetic ligands with a rich pharmacology.

Like other biogenic amine G protein-coupled receptors, mutation of the conserved aspartatic residue into alanine at position 116 (D116A(3.32)) in the 5-hydroxytryptamine (5-HT)(1A) receptor greatly affects 5-HT binding and signal transduction. [(3)H]8-Hydroxy-2-dipropylaminotetralin (8-OH-DPAT) and [(3)H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) are capable to bind the 5-HT(1A)-D116A mutant and, using these radioligands, we show here that this mutation dramatically reduces the affinities of the selective 5-HT(1A) agonists N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methylpyridin-2-yl)-methylamino methyl]piperidine (F13640), 3-chloro-4-fluorophenyl-(4-fluorophenyl-4-{[(5-methyl-6 methylamino-pyridin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone (F13714), and 2-[5-[3-(4-methylsulfonylamino)benzyl-1,4-oxadiazol-5-yl]-1H-indole-3-yl]ethylamine (L694247) and that of 5-carboxamidotryptamine. Although to a lesser extent, the binding of buspirone, (+)-flesinoxan, (-)-pindolol, and (-)-8-OH-DPAT are also highly decreased. In contrast, affinities of the 5-HT(1A) ligands WAY100,635, spiperone, (-)-4-(dipropylamino)-1,3,4,5-tetrahydrobenz {c,d}indole-6-carboxamide (LY228,729), and 1-[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl) piperazine (S14506) and the prototypical 5-HT(1A) agonist (+)-8-OH-DPAT are only slightly affected by the mutation, suggesting a moderate contribution of Asp116 to the binding pocket for these latter. Furthermore, LY228,729, S14506, and (+)-8-OH-DPAT induce a potent and efficacious coupling of the 5-HT(1A)-D116A receptor to G protein activation as measured by Ca(2+) mobilization and guanosine 5'-O-(3-[(35)S]thio)triphosphate binding in Chinese hamster ovary cells as well as by G protein-coupled inwardly rectifying potassium channel current activation in Xenopus laevis oocytes. It is interesting that the selective 5-HT(1A) antagonist WAY100,635 shows potent partial agonist activity at the 5-HT(1A)-D116A mutant, whereas spiperone maintains its inverse agonist properties. The pharmacological approach reported here re-evaluates the binding and functional properties of the 5-HT(1A)-D116A receptor and describes for the first time this mutant as a receptor activated solely by synthetic ligands (RASSL), with a rich pharmacology. By bioengineering animal models incorporating this RASSL, one may further explore the role of 5-HT(1A) receptor signaling in the central nervous system as well as G(i) protein-mediated signaling pathways in other tissues.

Effects of a new PAR1 antagonist, F 16618, on smooth muscle cell contraction.

This study evaluated the effects of two PAR1 antagonists on vessels contracted by SFLLR. ER 121958 antagonized the SFLLR-induced contraction on rat denuded superior mesenteric artery and pig coronary artery in a non-competitive manner (IC(50) values were 22 [7.5-43.6] nM and 2.9 [2.09-4.02] nM, respectively). F 16618 inhibited the SFLLR-induced superior mesenteric arteries and coronary arteries contraction in a competitive manner (pA(2) values of 7.3 and 6.2, respectively). PAR1 antagonists do not affect vessel resting tension or haemodynamic parameters in anaesthetized rats. Thus, PAR1 antagonists could have beneficial effects against vasospasm due to vessel injury.

Pharmacological characterization of protease activated receptor-1 by a serum responsive element-dependent reporter gene assay: major role of calmodulin.

We studied the protease activated receptor-1 coupling to a serum response element (SRE)-dependent luciferase activity readout in transfected COS-7 cells. Thrombin, with a pEC50 of 10.5, was 3000-fold more potent than the peptide agonists SFLLR and its derived compound C721-40 in stimulating luciferase activity, although the three agonists exhibited similar efficacy at the maximal concentration tested. Interestingly, SFLLR- and C721-40-induced luciferase activity was biphasic, suggesting that at least two populations of G proteins couple to the receptor. Further pharmacological characterization of this system was performed using selective protease activated receptor-1 antagonists. SCH203099 and ER-112787 blocked SFLLR-induced luciferase activity with similar potencies (pK(B) of 7), slightly higher than that exhibited by an arylisoxazole derivative compound from Merck (pK(B) of 6.1). These values correlated with their affinities established by competition binding experiments using [3H]-C721-40 as radioligand for protease activated receptor-1. Transduction mechanisms of protease activated receptor-1 coupling to SRE-dependent luciferase activity were examined using specific inhibitors. The Ca2+ chelator BAPTA-AM, as well as the calmodulin inhibitors W-7 and ophiobolin A, robustly inhibited SFLLR-induced SRE activation. Overexpression of RGS2 and a dominant negative rhoA protein abolished the SFLLR signal in an additive manner, suggesting a major role of Gq and G12/13 proteins. Furthermore, inhibition of phospholipase C, MAP-kinases, phosphatidyl inositol-3 kinase, rho-kinase and Ca2+/calmodulin-dependent protein kinases, all downstream effectors of Gq and G12/13, partially blocked the SFLLR-induced luciferase signal. Taken together, this SRE-luciferase assay reveals a complex network of transduction pathways of protease activated receptor-1 in accordance with the pleiotrophic action of thrombin.

Differential profile of antipsychotics at serotonin 5-HT1A and dopamine D2S receptors coupled to extracellular signal-regulated kinase.

The effects of antipsychotics targeting dopamine D2 and serotonin 5-HT1A receptors were compared with conventional antipsychotics on phosphorylation of Extracellular signal-Regulated Kinase 1/2 (ERK 1/2) in CHO cell lines stably expressing either the human serotonin 5-HT1A or human dopamine D2S receptor. All antipsychotics except haloperidol and olanzapine exhibited agonist properties at serotonin 5-HT1A receptors. Emax values (% effect of 10 microM 5-HT) were: bifeprunox (74), SSR181507 (73), SLV313 (72), aripiprazole (60), ziprasidone (56), clozapine (33). At dopamine D2S receptors, partial agonist activity (% effect of 10 microM dopamine) was observed for bifeprunox (76), SSR181507 (66) and aripiprazole (59). Other antipsychotics attenuated dopamine-induced ERK phosphorylation, with pK(B) values of : SLV313 (8.5), haloperidol (8.1), olanzapine (7.8), ziprasidone (7.7), and clozapine (6.4). Amongst the dopamine D2/serotonin 5-HT1A receptor compounds, aripiprazole acts as a partial dopamine D2S and serotonin 5-HT1A receptor agonist. SSR181507 and bifeprunox possess a profile of action similar to each other, efficaciously stimulating both serotonin 5-HT1A and dopamine D2S receptors. In contrast, SLV313, also an efficacious serotonin 5-HT1A receptor agonist, acted as a high potency dopamine D2 receptor antagonist. Thus, antipsychotics display varying efficacies at serotonin 5-HT1A and dopamine D2S receptors which may play a major role in their differential functional profiles in blocking the diverse symptoms of schizophrenia.

Promotion of G alpha i3 subunit down-regulation by GIPN, a putative E3 ubiquitin ligase that interacts with RGS-GAIP.

We have isolated an RGS-GAIP interacting protein that links RGS proteins to protein degradation. GIPN (GAIP interacting protein N terminus) is a 38-kDa protein with an N-terminal leucine-rich region, a central RING finger-like domain, and a putative C-terminal transmembrane domain. GIPN binds exclusively to RGS proteins of subfamily A, RGS-GAIP, RGSZ1, and RGSZ2. The N-terminal leucine-rich region of GIPN interacts with the cysteine-rich motif of RGS-GAIP. GIPN mRNA is ubiquitously expressed, and GIPN is found on the plasma membrane of transfected HEK293 cells. Endogenous GIPN is concentrated along the basolateral plasma membrane of proximal and distal tubules in rat kidney, where many G protein-coupled receptors and some G proteins are also located. Two immunoreactive species are found in rat kidney, a 38-kDa cytosolic form and an approximately 94-kDa membrane form. GIPN shows Zn2+- and E1/E2-dependent autoubiquitination in vitro, suggesting that it has E3 ubiquitin ligase activity. Overexpression of GIPN stimulates proteasome-dependent reduction of endogenous G alpha i3 in HEK293 cells and reduces the half-life of overexpressed G alpha i3-YFP. Thus, our findings suggest that GIPN is involved in the degradation of G alpha i3 subunits via the proteasome pathway. RGS-GAIP functions as a bifunctional adaptor that binds to G alpha subunits through its RGS domain and to GIPN through its cysteine string motif.

The G-protein regulator AGS3 controls an early event during macroautophagy in human intestinal HT-29 cells.

AGS3 contains GoLoco or G-protein regulatory motifs in its COOH-terminal half that stabilize the GDP-bound conformation of the alpha-subunit of the trimeric Gi3 protein. The latter is part of a signaling pathway that controls the lysosomal-autophagic catabolism in human colon cancer HT-29 cells. In the present work we show that the mRNA encoding for AGS3 is expressed in human intestinal cell lines (Caco-2 and HT-29) whatever their state of differentiation. Together with the full-length form, minute amounts of the mRNA encoding a NH2-terminal truncated form of AGS3, previously characterized in cardiac tissues, were also detected. Both the endogenous form of AGS3 and a tagged expressed form have a localization compatible with a role in the Galphai3-dependent control of autophagy. Accordingly, expressing its non-Galphai3-interacting NH2-terminal domain or its Galphai3-interacting COOH-terminal domain reversed the stimulatory role of AGS3 on autophagy. On the basis of biochemical and morphometric analysis, we conclude that AGS3 is involved in an early event during the autophagic pathway probably prior to the formation of the autophagosome. These data demonstrate that AGS3 is a novel partner of the Galphai3 protein in the control of a major catabolic pathway.

Endogenous RGS proteins facilitate dopamine D(2S) receptor coupling to G(alphao) proteins and Ca2+ responses in CHO-K1 cells.

The role of RGS proteins on dopaminergic D2S receptor (D2SR) signalling was investigated in Chinese hamster ovary (CHO)-K1 cells, using recombinant RGS protein- and PTX-insensitive G alphao proteins. Dopamine-mediated [35S]GTPgammaS binding was attenuated by more than 60% in CHO-K1 D2SR cells coexpressing a RGS protein- and PTX-insensitive G(alphao)Gly184Ser:Cys351Ile protein versus cells coexpressing a similar amount of PTX-insensitive G alphaoCys351Ile protein. Dopamine-agonist-mediated Ca2+ responses were dependent on the coexpression with a G alphao Cys351Ile protein and were fully abolished upon coexpression with a G alphaoGly184Ser:Cys351Ile protein. These results suggest that interactions between the G alphao protein and RGS proteins are involved in efficient D2SR signalling.