This renewal, requesting yr 30-34, focuses on establishing a molecular description of AKAP scaffolds that interact with and regulate G protein-coupled receptors. We seek to delineate structural, temporal and spatial characters of AKAP5 vs AKAP12 catalyzing downstream signaling.
Aim #1 tests the hypothesis: functional dissimilarities between AKAP5 and AKAP12 with respect to GPCR resensitization/recycling versus activation of Erk1/2 are structural and can be ascertained by detailed, comparative analysis of structure/function of each scaffold. We shall exploit the observation that A431 cells natively express AKAP12, but little AKAP5, whereas for HEK293 cells the opposite applies. Using knock-down (KD)/overexpression (OE), we shall probe beta2-AR resensitization/recycling versus Erk1/2 activation. Localization of AKAP5- and AKAP12-eGFP fusion proteins will be established as will effects of KD/OE of one AKAP on the dynamic localization of the other. The ability of AKAP5 to interact with both beta1- and beta2-AR provokes a parallel analysis for AKAP12. Oligomerization (homo- vs. hetero-) of AKAP5 and AKAP12 will be investigated.
Aim #2 tests the hypothesis: functional character of AKAP12 (and AKAP5) can be best understood through analysis of the protein-lipid and protein-protein interactions driving dynamic association with the membrane, GPCR, kinases, PPTases, PDEs, and adaptors. Interactions of AKAP5/12 essential in signaling, receptor recycling vs. Erk activation (i.e., N-myrisotylation, PCDs, and PIP2/PIP3 sequestration) will be analyzed in vitro by biophysical means and in vivo. The RBD of AKAP5 will be defined by mutagenesis and compared to a more detailed molecular analysis of the AKAP12 RBD. Protein docking sites for PP2B (other PPTases), PKC, and PDEs will be probed/characterized for AKAP12 vs AKAP5. Novel partners for the C-terminal ~800 aa reach of AKAP12 will be sought by proteomics. BiFC enables identification direct protein docking in live cells.
Aim #3 tests the hypothesis: phosphorylation drives the docking, trafficking, and signaling events catalyzed by GPCR-based AKAP scaffolds. Analysis of 3rd and 4th dimensions of AKAP function will be probed by FRET-based AKAR2- (a phosphorylation- dependent biosensor);FRET-based BiFC;and Fluorescence Correlation Spectroscopy to intergate analysis of phosphorylation, docking, and mass. AKAR2 substrate moiety will be engineered to provide a cis-acting reporter, signaling phosphorylation events of AKAP12. Public Health Relevance: Essential to growth and Health;dysregulation leads to diseases (e.g. cancer, diabetes, obesity). Cell signaling operates on a complex set of scaffolds (e.g. AKAPs) that localize, traffic and dock important partners essential for normal signaling to occur. Alterations in scaffolds (i.e., abundance and structure) lead to disease and our research seeks to understand how these scaffolds perform their multifaceted functions.
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