description): Adenylyl cyclases generate the ubiquitous second messenger cAMP. The importance of this messenger in regulating a large number of cellular processes is acknowledged. However, the methods for measuring cAMP lack both temporal and spatial resolution, leading to the widespread belief that these signals are simple. Against this notion, the large number of interactions between signaling pathways for Ca2+ and cAMP have been demonstrated and the dynamic nature of Ca2+ makes it likely that cAMP signals are also complex. In particular, Ca2+-sensitive adenylyl cyclases are regulated discreetly by physiological modes of Ca2+ entry and cAMP modulates Ca2+ entry by a variety of mechanisms. The present application proposes to develop adenylyl cyclase/ aequorin and adenylyl cyclase/ cameleon chimeras as localized sensors of Ca2+ and to develop olfactory cyclic nucleotide gated (oCNG) channels as rapid membrane-bound sensors of cAMP. Once these sensors are optimized, the applicant intends to examine dynamic and interdependent changes in the two signals in pituitary-derived GH4 cells, an excitable cell type. He will also use the cAMP sensor to study the rapid kinetics of adenylyl cyclase regulation in its native environment. These studies represent a first attempt to associate cAMP and Ca2+ signaling on the same temporal and spatial scale. In the long-term, such information will lead to greater generalized understanding of how the coordinated activity of these two second messengers control cellular function.
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