We have developed an experimental strategy to monitor protein interactions in a cell with a high degree of selectivity and sensitivity. A transcription factor is tethered to a membrane-bound receptor with a linker that contains a cleavage site for a specific protease. Activation of the receptor recruits a signaling protein fused to the protease that then cleaves and releases the transcription factor to activate reporter genes in the nucleus. This strategy converts a transient interaction into a stable and amplifiable reporter gene signal to record the activation of a receptor without interference from endogenous signaling pathways. We have developed this assay for three classes of receptors: G protein-coupled receptors, receptor tyrosine kinases, and steroid hormone receptors. Finally, we use the assay to identify a ligand for the orphan receptor GPR1, suggesting a role for this receptor in the regulation of inflammation.cellular assays ͉ G protein-coupled receptor ͉ protein interaction A ll cells have evolved mechanisms to respond to rapid changes in the environment. Extracellular signals are detected by transmembrane receptors that translate binding into intracellular signaling events. Most signaling systems that respond to environmental cues exhibit adaptation mechanisms that afford the cell a facile response to rapid changes in their surroundings. Mechanisms to assure the rapid but transient response to environmental cues are of obvious advantage to the cell but seriously limit most assays for receptor function. We have genetically modified receptors such that transient responses to ligand result in the stable transcription of a reporter gene. The transformation of a transient intracellular response to a stable amplifiable readout provides a sensitive and quantitative assay for receptor function.We have developed an assay for receptor activation and more generally for protein-protein interaction that involves the fusion of a membrane receptor with a transcriptional activator. The membrane-bound receptor and transcription factor sequences are separated by a cleavage site for a highly specific viral protease. A second gene encodes a fusion of the viral protease with a cellular protein that interacts only with activated receptor. Ligand binding to the receptor will stimulate this proteinprotein interaction, recruiting the protease to its cleavage site. Site-specific cleavage will release the transcriptional regulator that can now enter the nucleus and activate reporter genes. Recently, a similar principle, based on the complementation of split tobacco etch virus (TEV) protease fragments, has been used to monitor protein interactions (1). Our experimental scheme derives conceptually from the mechanism of action of the Notch receptor in which ligand binding elicits proteolytic cleavage events in the receptor to release a Notch intracellular domain that translocates to the nucleus and modulates transcription of downstream target genes (2, 3) (Fig. 1A).The assay we have developed relies solely on exogenous genes in...

The expression of a single odorant receptor (OR) gene from a large gene family in individual sensory neurons is an essential feature of the organization and function of the olfactory system. We have used chromosome conformation capture to demonstrate the specific association of an enhancer element, H, on chromosome 14 with multiple OR gene promoters on different chromosomes. DNA and RNA fluorescence in situ hybridization (FISH) experiments allow us to visualize the colocalization of the H enhancer with the single OR allele that is transcribed in a sensory neuron. In transgenic mice bearing additional H elements, sensory neurons that express OR pseudogenes also express a second functional receptor. These data suggest a model of receptor choice in which a single trans-acting enhancer element may allow the stochastic activation of only one OR allele in an olfactory sensory neuron.

We have developed a genetic approach to examine the role of spontaneous activity and synaptic release in the establishment and maintenance of an olfactory sensory map. Conditional expression of tetanus toxin light chain, a molecule that inhibits synaptic release, does not perturb targeting during development, but neurons that express this molecule in a competitive environment fail to maintain appropriate synaptic connections and disappear. Overexpression of the inward rectifying potassium channel, Kir2.1, diminishes the excitability of sensory neurons and more severely disrupts the formation of an olfactory map. These studies suggest that spontaneous neural activity is required for the establishment and maintenance of the precise connectivity inherent in an olfactory sensory map.