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.

Dosage-dependent regulation of cell proliferation and adhesion through dual ß2-adrenergic receptor/cAMP signals.

The role of ß-adrenergic receptors (ß-ARs) remains controversial in normal and tumor breast. Herein we explore the cAMP signaling involved in ß-AR-dependent control of proliferation and adhesion of nontumor human breast cell line MCF-10A. Low concentrations of a ß-agonist, isoproterenol (ISO), promote cell adhesion (87.5% cells remaining adherent to the plastic dishes following specific detachment vs. 35.0% in control, P<0.001), while increasing concentrations further engages an additional 36% inhibition of Erk1/2 phosphorylation (p-Erk1/2)-dependent cell proliferation (P<0.01). Isoproterenol dose response on cell adhesion was fitted to a 2-site curve (EC50(1): 16.5±11.5 fM, EC50(2): 4.08±3.09 nM), while ISO significantly inhibited p-Erk1/2 according to a 1-site model (EC50: 0.25±0.13 nM). Using ß-AR-selective agonist/antagonists and cAMP analogs/inhibitors, we identified a dosage-dependent signaling in which low ISO concentrations target a ß2-AR population localized in raft microdomains and stimulate a Gs/cAMP/Epac/adhesion-signaling module, while higher concentrations engage a concomitant activation of another ß2-AR population outside rafts and inhibit the proliferation by a Gs/cAMP/PKA-dependent signaling module. Our data provide a new molecular basis for the dose-dependent switch of ß-AR signaling. This study also sheds light on a new cAMP pathway core mechanism with a single receptor triggering dual cAMP signaling controlled by PKA or Epac but with different cellular outputs.

Deciphering biased-agonism complexity reveals a new active AT1 receptor entity.

Functional selectivity of G protein-coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer-based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein-coupling activity of [(1)Sar(4)Ile(8)Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT(1)) receptor-biased agonist that is G protein independent but ß-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein-dependent AT(1A) receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.

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.

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.

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.

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.