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 generation of distinct classes of neurons at defined positions within the developing vertebrate nervous system depends on inductive signals provided by local cell groups that act as organizing centers. Genetic and embryological studies have begun to elucidate the processes that control the pattern and identity of neuronal cell types. Here we discuss the cellular interactions and molecular mechanisms that direct neuronal cell fates in the dorsal half of the vertebrate central nervous system. The specification of dorsal neuronal cell fates appears to depend on a cascade of inductive signals initiated by cells of the epidermal ectoderm that flank the neural plate and propagated by roof plate cells within the neural tube. Members of the transforming growth factor-beta (TGF beta) family of secreted proteins have a prominent role in mediating these dorsalizing signals. Additional signals involving members of the Wnt and fibroblast growth factor (FGF) families may also contribute to the proliferation and differentiation of dorsal neuronal cell types.

Inductive factors are known to direct the regional differentiation of the vertebrate central nervous system (CNS) but their role in the specification of individual neuronal cell types is less clear. We have examined the function of GDF7, a BMP family member expressed selectively by roof plate cells, in the generation of neuronal cell types in the dorsal spinal cord. We find that GDF7 can promote the differentiation in vitro of two dorsal sensory interneuron classes, D1A and D1B neurons. In Gdf7-null mutant embryos, the generation of D1A neurons is eliminated but D1B neurons and other identified dorsal interneurons are unaffected. These findings show that GDF7 is an inductive signal from the roof plate required for the specification of neuronal identity in the dorsal spinal cord and that GDF7 and other BMP family members expressed by the roof plate have non-redundant functions in vivo. More generally, these results suggest that BMP signaling may have a prominent role in the assignment of neuronal identity within the mammalian CNS.

During neural development in vertebrates, a spatially ordered array of neurons is generated in response to inductive signals derived from localized organizing centres. One organizing centre that has been proposed to have a role in the control of neural patterning is the roof plate. To define the contribution of signals derived from the roof plate to the specification of neuronal cell types in the dorsal neural tube, we devised a genetic strategy to ablate the roof plate selectively in mouse embryos. Embryos without a roof plate lack all the interneuron subtypes that are normally generated in the dorsal third of the neural tube. Using a genetically based lineage analysis and in vitro assays, we show that the loss of these neurons results from the elimination of non-autonomous signals provided by the roof plate. These results reveal that the roof plate is essential for specifying multiple classes of neurons in the mammalian central nervous system.