Many important biological processes and responses are regulated by signaling pathways and interactions between signaling pathways that result in signaling networks. Understanding the mechanisms of networking an important component in developing a detailed picture of varied diseases such as cancers, cardiovascular and mental disorders. The focus in our laboratory has been on signaling through heterotrimeric G proteins to understand how such signaling regulates complex biological responses. In the coming, term we propose to study the pathways regulated by Galphao, how these pathways interact to form networks, and the biological consequences of networking in regulating neurite outgrowth. At the upstream level we will characterize how receptors regulate Galphao interactions with Rap 1GAPII to target it for degradation by the ubiquitin-proteasomal system and how this novel regulation leads to activation of Rap 1. We will use a combination of experimental (molecular biological and biochemical) and computational approaches to understand if and how Galphao regulation may lead to Rap1 functioning as a graded switch and the role of such graded switching property plays in Rap1 mediated neurite extension in Neuro-2A cells. All of these studies will conducted in a fully endogenous system in Neuro-2A cells using the CB-1 cannabinoid receptor. We will study the mechanisms by which a cascade of small GTPases including Rap 1 may be involved in the Go/i coupled cannabinoid receptor and Galphao regulation of c-Src and consequently Stat-3. We will also explore the role of JNK and p38 MAP-kinases in the cannabinoid receptor and Galphao induced neurite outgrowth. All of these studies will include experimental analysis and development of data-constrained computational models. We will then integrate the models for Galphao regulation of the multiple signaling pathways to develop a unified computational model of the Galphao signaling network, using a mixed modeling approach that involves both deterministic and stochastic regimes to define conditions that lead to simultaneous vs hierarchical activation of components within the network. From these studies we anticipate that we will develop a predictive understanding of how the cannabinoid and Galphao regulated signaling network triggers neurite extension.
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