Taste cells, like other sensory cells, transduce environmental stimuli into membrane conductance changes, but definitive mechanisms are unclear. This project aims to clone a taste receptor and other gustatory signaling molecules in order to characterize the molecular mechanisms of taste. To isolate a sweetness receptor through expression cloning, a human genomic library and a cDNA expression library from taste receptor cells will be screened using a variety of affinity selection techniques involving sweet proteins. Since these receptors are highly tissue restricted, elucidation of their sequence and structure has proved difficult. To isolate gustatory signaling molecules, a taste cell specific expression library will be screened using a yeast two hybrid system. The second phase of the project, to study signal transduction initiated by activation of the sweetness receptor, will involve expression of mutated proteins. Though our specific goal is to clone the sweetness receptor, the proposal emphasizes the learning of molecular strategies generally useful in identifying and characterizing surface receptors and signaling molecules involved in brain processes and disorders: expression cloning, construction of chimeric fusion proteins, study of signal transduction, and application of the yeast two-hybrid system. A five year fellowship period will allow me to focus entirely on laboratory work, complete the proposed research program, and move towards an independent position. The diverse and innovative techniques utilized in the laboratory, many developed by Dr. Seed, will provide a strong foundation in molecular techniques necessary for independent work in the future. I plan a career in basic neuroscience research. I will focus on the cloning and elucidation of scarce neurosensory and neuronal receptors, isolating intracellular proteins involved in neuronal signaling, and studying mechanisms of signal transduction and their alteration in disease. I hope that the continuation of my proposed studies will provide me with sufficient experience and success to obtain the academic position and funding to pursue my research interests.
