Neurons and epithelial cells are polarized cells that target proteins to both structural and functional compartments within the cell. Because of this compartmentalization, the localization of specific signaling components is necessary to achieve ligand-specific cellular responses. At present, little is known about the compartmentalization of signaling components such as G proteins. In this proposal, we hypothesize that localization of signaling molecules into discrete microdomains prevents signal cross-talk and contributes to specific cellular responses. This renewal application proposes to extend this hypothesis by defining the mechanisms of Ga regulation by two different modulatory proteins important in epithelial cells and neurons. As work in progress, we have demonstrated direct interactions between Galpha12 and Galpha12 with the epithelial cell tight junction (TJ) protein Zona Occludens-1 (ZO-1), and between Galpha-0 and the neuronal protein, Purkinje cell protein-2 (Pcp2). Pcp2 activates Galpha 0 in-vitro by stimulating GDP release. Galpha-12 and Galpha12 are localized within the TJ where they regulate fundamental TJ properties including its assembly and the barrier to paracellular particle movement.
Aim 1 will focus on ZO-1/Galpha12 and ZO-1/Galpha-12 interactions to determine whether there is direct modulation of Galpha by ZO-1, and/or ZO-1 is a scaffold that organizes signaling complexes important for regulating the junction. Utilizing baculovirus expressed proteins, glutathione-S-transferase (GST) fusion proteins, MDCK cell culture systems, and enzymatic assays, we aim to: (1) define the domains of Galpha12 and Galpha-12 interacting with ZO- 1 (2) determine if ZO- 1 modulates k-cat and k-off of Galpha12 and Galpha-12; (3) test whether phosphorylation of Ga12 or ZO-l affects the interaction, and (4) characterize the Ga12/Src signaling pathway regulating the TJ in MDCK cells.
Aim 2 will define the mechanism of Pcp2 activation of Ga0 and the function of this interaction in neurons. Pcp2 contains a Goloco motif, a conserved domain found in several other proteins that regulate Ga subunits. We hypothesize that Pcp2 regulates Ga0 through the Goloco motif to stimulate nucleotide exchange, and this interaction regulates a basic neuronal process such as neurite formation. Here, we will; (1) identify the contact sites and regulatory domains of Ga0 and Pcp2; (2) determine role of serine/threonine phosphorylation (MAP Kinasel) of Pcp2 in regulating the interaction with Ga0, and (3) characterize Ga0/Pcp2 localization in adult and new born cerebellar tissue, and utilize inducible Pcp2 expression in cultured neuronal cells (PC 12) to determine localization, phosphorylation, and the influence on neurite formation. Such experiments will yield insight into the regulation of compartmentalized cellular responses, and could provide the basis for development of novel therapeutics for disorders influencing the epithelial cell TJ as well as those impacting neuronal function.
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