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...

Mammals have retained two functionally and anatomically independent collections of olfactory neurons located in the main olfactory epithelium and in the vomeronasal organ (VNO). Pheromones activate the VNO in order to elicit fixed action behaviors and neuroendocrine changes involved in animal reproduction and aggression. Differential screening of cDNA libraries constructed from individual rat VNO neurons has led to the isolation of a novel family of approximately 100 genes encoding seven transmembrane receptors with sequence similarity with Ca2+-sensing and metabotropic glutamate receptors. These genes are likely to encode a novel family of pheromone receptors. Patterns of receptor gene expression suggest that the VNO is organized into discrete and sexually dimorphic functional units that may permit segregation of pheromone signals leading to specific arrays of behaviors and neuroendocrine responses.

The use of a continuous Percoll gradient in a procedure for the rapid isolation of bacteroidcontaining vesicles from French bean nodules is described. The purified vesicles appeared to be free from contamination by naked bacteroids and plant mitochondria and suitable for physiological studies. The metabolite uptake activities of vesicles isolated by this method were compared with those of free bacteroids. Succinate was transported through both peribacteroid and bacteroid membranes: the K , values were 320 p .~ and 57 p~, respectively. For glucose K , values were 142 p .~ for the peribacteroid membrane and 102 p .~ for the bacteroid membrane, indicating that glucose could act as an energy-yielding substrate in functioning nodules. Experiments with inhibitors showed the involvement of ATPases in these transport processes and suggested that a proton-motive force was probably associated with them. The regulatory role of the peribacteroid membrane in the movement of the metabolites from the plant cytosol to bacteroids was demonstrated by its impermeability to glutamate, aspartate, citrate and benzoate.

Enriched peribacteroid membranes were prepared from Phaseolus vulgaris nodules and, in the presence of metleghemoglobin and H202, membranal lipid peroxidation was observed. The initial rate of the reaction was low and increased with time. Ferrous leghemoglobin was unable to induce this peroxidation with H202. Thus, it appears that leghemoglobin (IV) is not the activated species involved in this process. Heme plays a role in this peroxidation and the hydroxyl radical is not an intermediate of the reaction. Lipid peroxidation in peribacteroid membranes was also observed in the presence of iron ions. A mixture of iron (Ill) and iron (II) produced a maximal peroxidation. Senescing nodule extracts were able to provoke membranal lipid peroxidation; they contained nonprotein-bound iron. Peribacteroid membranes were more sensitive than microsomes to peroxidation, as measured by malonaldehyde formation.