The computational power of the nervous system relies on the ability of individual cells to integrate multiple inputs and generate an output targeted to the appropriate partner(s). These functions would be impossible without the asymmetric localization of proteins within the cell. For example, many of the proteins that are in the axon are specialized for functions like transport and electrical conduction. In contrast, many of the proteins in the dendrites are specialized for receiving information from other cells. Restriction of localization is important for limiting the activity of a particular protein to a specific cellular compartment, but it can also be an important regulator of biochemical reactions. Many proteins behave differently when localized to a multiprotein complex due to the ability of direct binding partners to alter their conformation. This function of localization has important implications for how neurons process information that is relevant to many therapeutic interventions since it means that the behavior or activity of a protein cannot always be predicted from in vitro studies. This proposal has the goal of understanding how CaMKII and two proteins that directly bind to it, dCASK, a scaffold protein, and Eag, a potassium channel, are regulated by localization and cellular context. To achieve an understanding of how cellular context can influence the activity of these proteins we propose two sets of experiments.
In Aim #1 we will use a transfected cell system to understand how dCASK and CaMKII interact to change the location, activity and potential substrates of CaMKII in response to calcium influx. We will do a parallel set of experiments in dCASK mutant transgenic flies that express a YFP tagged dCASK transgene that restores normal behavior. We will determine where in the cell dCASK has to reside and what proteins it has to complex with to carry out its behavioral function.
Aim #2 addresses how Eag, a voltage-gated potassium channel, is localized and regulated by other proteins that bind to it. We have discovered a novel role for RNA editing in localization that has implications for many other genes that undergo this type of modification. We will also investigate the function of an alternative splice product of the eag gene that does not conduct ions, but instead functions as a scaffold for CaMKII and other signaling proteins. We address these issues by using homologous recombination to create mutations in the eag gene that selectively alter each of these processes and assess the impact on neuronal gene expression, excitability and behavior.
The vast majority of drug targets are proteins, many of which have enzymatic activity. While in many cases we have extensive information about structure at the atomic level or kinetic parameters in solution, a critical gap exists in our understanding of how proteins actually work within the cell. Understanding how the context of a protein's location can affect or regulate activity is critical to rational drug design and may even provide new drug targets.
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