Agonists of the orphan human G2A receptor identified from inducible G2A expression and beta-lactamase reporter screen.

The G protein-coupled receptor (GPCR) G2A (for G2 accumulation) was identified as a stress-inducible antiproliferative cell cycle regulator. Targeted G2A gene deletion in mice resulted in systemic lupus erythematosus-like and atherosclerotic lesion phenotypes. These findings suggested that G2A may be a therapeutic target for cancers and autoimmune and cardiovascular diseases. The G2A receptor is cytotoxic upon ectopic expression, and its cognate ligand has not been identified, making it difficult to generate a cell line for screening using a conventional approach. The function of human G2A remains obscure. Here we show that by using an inducible T-REx (Invitrogen, Carlsbad, CA) expression system an inducible G2A functional cell-based beta-lactamase reporter assay could be developed using the constitutive activity of the receptor. Furthermore, G2A expression levels can be controlled under this inducible system to avoid the expression artifacts of conventional approaches using constitutive expression vectors. This stable cell line expressing the human G2A receptor was screened against a chemical library containing 740,000 compounds, and small molecules showing selective agonistic activity on G2A were identified. We believe the strategy employed here for G2A should be applicable to other "intractable" GPCRs where target gene expression results in cytotoxic and/or high constitutive activities.

A homogeneous fluorescent live-cell assay for measuring 7-transmembrane receptor activity and agonist functional selectivity through beta-arrestin recruitment.

Seven-transmembrane (7TM) receptors play an essential role in the regulation of a wide variety of physiological processes, making them one of the top target classes for pharmaceuticals. 7TM receptor function is mediated and modulated through 2 primary processes: G-protein and beta-arrestin signaling. Classically, it has been recognized that these 2 processes can interact with one another during 7TM receptor desensitization, but it has more recently been recognized that these 2 processes can also act independently of one another and can activate parallel signaling pathways. As such, the methods used to interrogate 7TM receptor signaling, both from a biological and a pharmaceutical perspective, may need to be reevaluated and the question of whether functionally selective compounds (compounds that selectively activate one pathway over another) can be rationally developed must be raised. Although numerous high-throughput screening (HTS) compatible assays exist for studying second messengers arising from G-protein signaling, far fewer HTS compatible assays exist for studying beta-arrestin recruitment. The authors report on the Tango 7TM receptor assay technology, a high-throughput homogeneous assay method for monitoring beta-arrestin recruitment that uses a live-cell fluorescent readout. This assay format is broadly applicable to 7TM receptors, independent of G-protein coupling and, as such, has been used to produce assays for over 70 7TM receptor targets. The authors also show how flow cytometry can be used to select clones with desired pharmacological profiles and how an inducible expression system can increase the assay window for targets with high levels of constitutive activity. Finally, they demonstrate how the Tango system can be used in parallel with assays aimed at second-messenger signaling to enable functional selectivity studies.

High-throughput cellular assays for regulated posttranslational modifications.

We have developed a set of high-throughput screening (HTS)-compatible assays capable of measuring regulated, target-specific posttranslational modifications in a mammalian cell-based format. We chose the NFkappaB signal transduction cascade as a model system to validate this approach because specific target proteins in this signaling pathway undergo a multitude of posttranslational modifications in response to pathway stimulation. In this pathway, TNFalpha induces the phosphorylation, ubiquitination, and proteasomal degradation of IkappaBalpha, which leads to the release and translocation of the NFkappaB transcriptional complex into the nucleus. To measure these cellular processes, we describe the use of a stable cell line expressing a fusion of green fluorescent protein (GFP) with IkappaBalpha that can be interrogated for either ubiquitination or phosphorylation using a unique set of terbium-labeled antibodies in a time-resolved Förster resonance energy transfer (TR-FRET)-based readout. Concurrently, we have engineered a beta-lactamase-IkappaBalpha reporter cell line that can be used to quantify proteasomal degradation of IkappaBalpha in living cells. Both TR-FRET and beta-lactamase reporter technologies provide a convenient, sensitive, and robust means to interrogate the chronological steps in NFkappaB signaling in a physiologically relevant cellular context without the need to overexpress any enzyme involved in this pathway. Cellular HTS assays that interrogate such processes will provide a unique integrated approach to dissecting intermediate steps in NFkappaB activation and could serve as examples of broadly applicable pathway analysis tools for target-based drug discovery.

mua-6, a gene required for tissue integrity in Caenorhabditis elegans, encodes a cytoplasmic intermediate filament.

Locomotion in Caenorhabditis elegans requires force transmission through a network of proteins linking the skeletal muscle, via an intervening basal lamina and epidermis (hypodermis), to the cuticle. Mutations in mua-6 result in hypodermal rupture, muscle detachment from the bodywall, and progressive paralysis. It is shown that mua-6 encodes the cytoplasmic intermediate filament (cIF) A2 protein and that a MUA-6/IFA-2::GFP fusion protein that rescues the presumptive mua-6 null allele localizes to hypodermal hemidesmosomes. This result is consistent with what is known about the function of cIFs in vertebrates. Although MUA-6/IFA-2 is expressed embryonically, and plays an essential postembryonic role in tissue integrity, it is not required for embryonic development of muscle-cuticle linkages nor for the localization of other cIFs or hemidesmosome-associated proteins in the embryo. Finally, the molecular lesion in the mua-6(rh85) allele suggests that the head domain of the MUA-6/IFA-2 is dispensable for its function.