G protein-coupled receptors (GPCRs) represent the largest family of proteins involved in signal transduction. Here we present a bioluminescence resonance energy transfer (BRET) assay that directly monitors in real time the early interactions between human GPCRs and their cognate G-protein subunits in living human cells. In addition to detecting basal precoupling of the receptors to Galpha-, Gbeta- and Ggamma-subunits, BRET measured very rapid ligand-induced increases in the interaction between receptor and Galphabetagamma-complexes (t(1/2) approximately 300 ms) followed by a slower (several minutes) decrease, reflecting receptor desensitization. The agonist-promoted increase in GPCR-Gbetagamma interaction was highly dependent on the identity of the Galpha-subunit present in the complex. Therefore, this G protein-activity biosensor provides a novel tool to directly probe the dynamics and selectivity of receptor-mediated, G-protein activation-deactivation cycles that could be advantageously used to identify ligands for orphan GPCRs.

These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.

The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein ␣ subunit function. However, using a peptide that binds G␤␥ (␤ARKct), we show that G␤␥ is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the ␤ 2 -adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, ␤ 2 AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.

Bioluminescence resonance energy transfer (BRET)and co-immunoprecipitation experiments revealed that heterotrimeric G proteins and their effectors were found in stable complexes that persisted during signal transduction. Adenylyl cyclase, Kir3.1 channel subunits and several Gprotein subunits (G␣ ␣ s , G␣ ␣ i , G␤ ␤ 1 and G␥ ␥ 2 ) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically G␤ ␤ 1 -YFP 1-158 and G␥ ␥ 2 -YFP 159-238 , which heterodimerize to produce fluorescent YFP-G␤ ␤ 1 ␥ ␥ 2 ). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-G␥ ␥ 2 , GFP-G␤ ␤ 1 or YFP-G␤ ␤ 1 ␥ ␥ 2 . G␣ ␣ subunits were also stably associated with both effectors regardless of whether or not signal transduction was initiated by a receptor agonist. Although BRET between effectors and G␤ ␤␥ ␥ was increased by receptor stimulation, our data indicate that these changes are likely to be conformational in nature. Furthermore, receptor-sensitive G-protein-effector complexes could be detected before being transported to the plasma membrane, providing the first direct evidence for an intracellular site of assembly.

Both β 1 -and β 3 -adrenergic receptors (β 1 ARs and β 3 ARs) are present on nuclear membranes in adult ventricular myocytes. These nuclear-localized receptors are functional with respect to ligand binding and effector activation. In isolated cardiac nuclei, the non-selective βAR agonist isoproterenol stimulated de novo RNA synthesis measured using assays of transcription initiation (Boivin et al., 2006 Cardiovasc Res. 71:69-78). In contrast, stimulation of endothelin receptors, another G protein-coupled receptor (GPCR) that localizes to the nuclear membrane, resulted in decreased RNA synthesis. To investigate the signalling pathway(s) involved in GPCR-mediated regulation of RNA synthesis, nuclei were isolated from intact adult rat hearts and treated with receptor agonists in the presence or absence of inhibitors of different mitogen-activated protein kinase (MAPK) and PI3K/PKB pathways. Components of p38, JNK, and ERK1/2 MAP kinase cascades as well as PKB were detected in nuclear preparations. Inhibition of PKB with triciribine, in the presence of isoproterenol, converted the activation of the βAR from stimulatory to inhibitory with regards to RNA synthesis, while ERK1/2, JNK and p38 inhibition reduced both basal and isoproterenol-stimulated activity. Analysis by qPCR indicated an increase in the expression of 18 S rRNA following isoproterenol treatment and a decrease in NFκB mRNA. Further qPCR experiments revealed that isoproterenol treatment also reduced the expression of several other genes involved in the activation of NFκB, while ERK1/2 and PKB inhibition substantially * Corresponding authors. Hébert is to be contacted

Cryptococcus neoformans ( C. neoformans var. grubii ) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype.

The vast burden of cryptococcal meningitis occurs in immunosuppressed patients, driven by HIV, and is caused by Cryptococcus neoformans var. grubii. We previously reported cryptococcal meningitis in Vietnam arising atypically in HIV uninfected, apparently immunocompetent patients, caused by a single amplified fragment length polymorphism (AFLP) cluster of C. neoformans var. grubii (VNIγ). This variant was less common in HIV infected individuals; it remains unclear why this lineage is associated with apparently immunocompetent patients. To study this host tropism we aimed to further our understanding of clinical phenotype and genomic variation within Vietnamese C. neoformans var. grubii. After performing MLST on C. neoformans clinical isolates we identified 14 sequence types (STs); ST5 correlated with the VNIγ cluster. We next compared clinical phenotype by lineage and found HIV infected patients with cryptococcal meningitis caused by ST5 organisms were significantly more likely to have lymphadenopathy (11% vs. 4%, p = 0.05 Fisher’s exact test) and higher blood lymphocyte count (median 0.76 versus 0.55 X109 cells/L, p = 0.001, Kruskal-Wallis test). Furthermore, survivors of ST5 infections had evidence of worse disability outcomes at 70 days (72.7% (40/55) in ST5 infections versus 57.1% (52/91) non-ST5 infections (OR 2.11, 95%CI 1.01 to 4.41), p = 0.046). To further investigate the relationship between strain and disease phenotype we performed genome sequencing on eight Vietnamese C. neoformans var. grubii. Eight genome assemblies exhibited >99% nucleotide sequence identity and we identified 165 kbp of lineage specific to Vietnamese isolates. ST5 genomes harbored several strain specific regions, incorporating 19 annotated coding sequences and eight hypothetical proteins. These regions included a phenolic acid decarboxylase, a DEAD-box ATP-dependent RNA helicase 26, oxoprolinases, a taurine catabolism dioxygenase, a zinc finger protein, membrane transport proteins and various drug transporters. Our work outlines the complexity of genomic pathogenicity in cryptococcal infections and identifies a number of gene candidates that may aid the disaggregation of the pathways associated with the pathogenesis of Cryptococcus neoformans var. grubii.

At the cell surface, βARs and endothelin receptors can regulate nitric oxide (NO) production. β-adrenergic receptors (βARs) and type B endothelin receptors (ETB) are present in cardiac nuclear membranes and regulate transcription. The present study investigated the role of the NO pathway in the regulation of gene transcription by these nuclear G protein-coupled receptors. Nitric oxide production and transcription initiation were measured in nuclei isolated from the adult rat heart. The cell-permeable fluorescent dye 4,5-diaminofluorescein diacetate (DAF2 DA) was used to provide a direct assessment of nitric oxide release. Both isoproterenol and endothelin increased NO production in isolated nuclei. Furthermore, a β3AR-selective agonist, BRL 37344, increased NO synthesis whereas the β1AR-selective agonist xamoterol did not. Isoproterenol increased, whereas ET-1 reduced, de novo transcription. The NO synthase inhibitor l-NAME prevented isoproterenol from increasing either NO production or de novo transcription. l-NAME also blocked ET-1-induced NO-production but did not alter the suppression of transcription initiation by ET-1. Inhibition of the cGMP-dependent protein kinase (PKG) using KT5823 also blocked the ability of isoproterenol to increase transcription initiation. Furthermore, immunoblotting revealed eNOS, but not nNOS, in isolated nuclei. Finally, caged, cell-permeable isoproterenol and endothelin-1 analogs were used to selectively activate intracellular β-adrenergic and endothelin receptors in intact adult cardiomyocytes. Intracellular release of caged ET-1 or isoproterenol analogs increased NO production in intact adult cardiomyocytes. Hence, activation of the NO synthase/guanylyl cyclase/PKG pathway is necessary for nuclear β3ARs to increase de novo transcription. Furthermore, we have demonstrated the potential utility of caged receptor ligands in selectively modulating signaling via endogenous intracellular G protein-coupled receptors.