Towards the Quantal Nature of Receptor/cAMP Signal.. (TOPAS)
Towards the Quantal Nature of Receptor/cAMP Signals
Start date: Jan 1, 2009,
End date: Dec 31, 2013
"Most drugs act via cell surface receptors. Some receptors signal to ion channels, while others regulate 2nd messengers, most importantly cAMP. Ion channels can be resolved at the single channel level using patch clamp analysis; they switch between discrete ""on"" and ""off"" states. In contrast, current techniques to analyze cAMP-signals are quite global and generally require destruction of the sample. Therefore, the activation mechanisms and switching behaviour of receptors signalling to cAMP are largely unknown. This project postulates minimal quantal cAMP-signals that are triggered by individual receptors. We aim to discover these quantal cAMP-signals, to characterize their temporal and spatial patterns, and to determine factors that influence them. To do so, we will develop a microscopic technology to image cAMP-signals with high sensitivity and resolution. We have already developed a first generation of cAMP-sensors that can be expressed in cells and respond to cAMP with a change in fluorescence resonance energy transfer (FRET). We will carry this technology to the sensitivity required for the postulated quantal cAMP-signals and - develop new FRET-sensors for cAMP with much greater brightness, amplitude and sensitivity, - build a specially designed total internal reflection fluorescence (TIRF) microscope to image the sub-membrane region of intact cells, - apply the technology to systems of increasing biological complexity: (a) isolated thyroid cells, where cAMP-signals can be compartmentalized, (b) intact thyroid follicles, where cAMP-signals can be triggered from various cellular sites, and (c) individual neurons in Drosophila brain, which are involved in a defined learning process. We believe that the ability to analyze 2nd messenger responses from individual receptors will open the way to a molecular understanding of receptor function and cAMP-signalling and provide a widely applicable technology and a mechanistic basis for receptor-directed drug development."
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