The yeast two-hybrid system has been adapted to study protein-protein, protein-RNA, protein-DNA, and protein-small molecule interactions [48]. A one-hybrid system has been developed that utilizes ds-acting sequences to identify DNA-binding proteins that can initiate transcription [49]. A yeast three-hybrid was developed to study RNA-protein interactions that are especially useful for developing screens against viruses [50]. In this system, the hybrid RNA containing sites recognized by the RNA interacting proteins links the two-hybrid proteins containing the DNA-binding and activation domains, respectively. The yeast two-hybrid system has been recently applied to find inhibitors of the N type calcium channel [51,52]. Alternative screening techniques use mammalian cells to measure calcium channel activity with electrophysiological and spectro-photometric methods to measure calcium influx. These methods are labor intensive, difficult, and not compatible with high-throughput screening. In the yeast two-hybrid system, the interacting, regulatory portion of the a1 subunit of the channel fused to the Gal4 activation domain and the full length P3 subunit fused to the yeast Gal4 DNA binding domain were expressed. The system could be adapted to find inhibitors of specific calcium channels by selecting the specific interacting domains.

E. coli Two-Hybrid System. The yeast two-hybrid system and its modifications have been widely used for studying protein-protein interactions. Although it is useful for identifying the interacting proteins, the interacting proteins are not easily accessible to inhibitors that must cross the cell wall and membrane and also the nuclear membrane where the two-hybrid constructs are located in the yeast cell. Simpler systems have been developed in E. coli that may be more effective for screening. An example of such a system is a protein dimerization system developed in E. coli. Dimerization and multimerization is required for the activation of many cell surface proteins, such as single transmembrane receptors and channels. A bacterial system called ToxR has been developed to detect dimerization of cell surface receptors and channels [53]. The Vibrio cholerae ToxR gene product is a Type 2 membrane protein that has an extracellular domain, a single transmembrane domain, and a cytoplasmic domain that acts as a transcription factor, binding directly to the Tox promoter to activate toxin secretion. In V. cholerae, the extracellular domain of ToxR activates in response to external stimulus and induces the cytoplasmic portion of the receptor to bind the Ctx promoter directly to induce transcription of the toxin gene. In the screening system, ToxR has been cloned into E. coli, and the extracellular domain of ToxR has been replaced by the extracellular domain of the TrkC, the receptor for the neurotropin, NT3. Dimerization of the extracellular domain of the TrkC receptor activates the ToxR promoter. In the E. coli system, a reporter, such as P-galactosi-dase or the antibiotic resistance gene chloramphenicol acetyl transferase, has been engineered instead of toxin, for obtaining an easily measured readout (Fig. 10).

The ToxR system has been also been used for expressing the influenza virus M2 protein. In contrast to the system described in the previous section, where the M2 protein induces membrane permeability, the M2-ToxR chimera can be used to identify compounds that bind the channel and alter the multimeri-zation state. The anti-influenza drug Amantadine is known to block the M2 channel, and in the M2 ToxR system, it was shown to alter the transcriptional signal produced by the M2-ToxR chimera. Although this screening system does not specifically find blockers of the M2 proton pump, the screen is designed to test a large number of compounds rapidly and identify those compounds that bind and affect aggregation. Some of these compounds could inhibit M2 function. The ToxR system has also been used for studying the formation of homodimers of Immunoglobulin VL domains [54]. Thus different functional systems can be used to develop screens for the same target.

More recently, a different system using the CadC protein in E. coli has been developed for developing protein dimerization screens (unpublished obser-

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