Spatiotemporal visualization of adenylyl cyclase signaling

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PROJECT SUMMARY/ABSTRACT Cyclic AMP (cAMP) is a critical secondary messenger that modulates many cell signaling pathways throughout physiology. Adenylyl cyclase (AC) catalytically converts ATP to cAMP in response to G protein- coupled receptor signaling, thus acting as an essential relay and integration center for cell signaling. Currently, ACs represent an unexploited target for treating a range of diseases including cancer, chronic obstructive pulmonary disease, neuropsychiatric disorders, diabetes, heart disease, and drug abuse. Within a cellular context, differential expression, compartmentalization, and microdomain localization of nine transmembrane AC isoforms give rise to a broad range of possible signaling outcomes, complicating our ability to therapeutically target this critical rheostat of cell signaling. Our mechanistic understanding of AC function is limited, with only a recent first glimpse of a full-length AC-Gαs structure revealed by cryo-electron microscopy (cryoEM). However, the static snapshot of AC in isolation forms an incomplete picture of the spatiotemporal signaling that occurs in vivo. The lack of molecular and mechanistic details of AC signaling assemblies in native environments leaves a gap in our understanding, further restricting the development of pharmaceuticals targeting AC and cAMP pathways. To address this gap, the overarching goal of this proposal is to capture physiologically relevant complexes between adenylyl cyclase and signaling partners in near-native and cellular environments. This will be achieved through the following specific aims: (1) structurally and biochemically characterize a functional signaling assembly of adenylyl cyclase, and (2) visualize adenylyl cyclase microdomain topography and its dynamics in response to cyclase activation in situ. Completing these aims will represent a substantial leap forward in adenylyl cyclase biology, providing the framework for further adenylyl cyclase study. This work builds upon my G protein biochemistry background and experience with cell signaling assays, protein purification, and fluorescence microscopy. Taking advantage of the world-class training and resources available in the laboratory of my mentor, Dr. Georgios Skiniotis, the proposed studies also provide an opportunity to acquire expertise in structural biology, cryo-electron tomography, proteomic approaches, sharpen my abilities as a researcher, and develop as an emerging leader in the adenylyl cyclase field. As a result, I will be well-positioned to establish a program of successfully funded independent research.