Central to organization of signaling pathways are scaffolding and anchoring proteins that mediate localized assembly of protein complexes containing receptors, second messenger enzymes, kinases, phosphatases, and substrates. AKAP79/150 is an excitatory postsynaptic PKA and protein phosphatase 2B/Calcineurin (CaN) anchoring protein linked to NMDA and AMPA glutamate receptors through PSD-95 family MAGUK scaffolds. These assemblies are thought to play central roles in regulating receptor activity, localization, and synaptic structure in synapse formation during development, synaptic plasticity in learning and memory, and neuropathologies such as excitotoxicity in stroke, neurodegeneration, epilepsy, chronic pain, schizophrenia and drug addiction. In particular, postsynaptic signaling functions of AKAP79/150-anchored PKA and CaN may regulate AMPA receptor phosphorylation and synaptic localization in NMDA receptor-dependent LTP and LTD plasticity and its modulation by norepinephrine and dopamine. Postsynaptic targeting of AKAP79/150 is mediated by an N-terminal region that binds PIP2, F-actin, and cadherin adhesion molecules. Localization of AKAP79/150 with MAGUKs and cadherins is stabilized by F-actin and disrupted by NMDA receptor-CaN signaling pathways implicated in AMPA receptor regulation in LTD. Thus, PKA and CaN anhcoring as well as linkage of the AKAP to cadherin-cytoskeletal and MAGUK-receptor complexes could regulate synaptic structure and function. These issues will be investigated through Specific Aim 1: NMDAR regulation of AKAP79/150, AMPAR, and F-actin postsynaptic localization through PLC activation. Hypothesis: NMDAR activation of PLC-mediated PIP2 hydrolysis is necessary for loss of AKAP79/150 and AMPARs from synapses, remodeling of F-actin, and induction of LTD. This hypothesis will be tested using cellular transfection, fluorescence imaging, biochemical and electrophysiological methods in cultured hippocampal neurons and acute hippocampal slices.
Specific Aim 2 : Role of anchored-PKA and CaN in NMDAR regulation of AKAP79/150 and AMPAR postsynaptic localization and activity. Hypothesis: NMDAR regulation of anchored-CaN and AKAP79/150-PKA loss from synapses control AMPAR localization and activity.
Specific Aim 3 : Regulation of synapse development by AKAP79/150 targeting domain and MAGUK interactions. Hypothesis: AKAP79 expression increases excitatory synapse number through the N-terminal targeting domain and synaptic size and strength through interactions with MAGUKs and AMPAR recruitment. The hypotheses in aims 2 and 3 will be tested in hippocampal neurons using RNAi knock-down of AKAP150, transfection of wild-type and mutant AKAP79 proteins, fluorescence imaging and electrophysiology. ? ?
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