At the postsynaptic density (PSD) of neuronal excitatory synapses, AMPA (AMPAR) and NMDA (NMDAR) glutamate receptors are linked to signaling proteins and the actin cytoskeleton in dendritic spines through a network of scaffolding proteins that play important roles during synaptic plasticity underlying learning and memory.  AMPARs are recruited to dendritic spines through NMDAR activation during induction of long term potentiation (LTP) in hippocampal neurons through pathways that also increase spine size and actin polymerization.  Phosphorylation of AMPAR-GluR1 subunits by the cAMP-dependent protein kinase (PKA) may promote surface expression of AMPARs recruited during LTP.  In contrast, induction of long-term depression (LTD) leads to calcineurin-protein phosphatase 2B (CaN) mediated dephosphorylation of PKA phosphorylated GluR1, removal of AMPARs from synapses and depolymerization of spine actin followed by spine shrinkage.  However, mechanisms for coordinately regulating AMPAR localization, phosphorylation, and spine structural plasticity are not well understood.  A-kinase-anchoring protein (AKAP) 79/150 (human79/rodent150) is a PKA and CaN anchoring protein linked to NMDARs and AMPARs through PSD-95 and SAP97 membrane-associated guanylate kinase (MAGUK) scaffolds.  AKAP79/150 is targeted to spines by an N-terminal basic region that binds phosphatidylinositol-4,5-bisphosphate (PIP2), F-actin, and cadherin adhesion molecules.  Importantly, findings from the last funding period and recent preliminary studies indicate that AKAP79 is recruited to spines in LTP through palmitoylation of its targeting domain and that AKAP79 overexpression enhances dendritic spine size and AMPAR activity through MAGUK binding.  In contrast, NMDAR-CaN signaling pathways implicated in AMPAR depression and spine shrinkage in LTD disrupt AKAP79/150 interactions with actin, MAGUKs and cadherins and lead to loss of the AKAP and anchored PKA from synapses.  This AKAP79/150 translocation from spines depends on actin reorganization and phospholipase C (PLC) cleavage of PIP2, and preliminary studies suggest additional modulation by palmitoylation.  Thus, AKAP79/150 is likely to play important structural and signaling roles in plasticity.  Due to the complexity of PKA and CaN signaling in neurons and the multi-functionality of scaffold proteins such as AKAP79/150, it is a considerable challenge to understand the specific postsynaptic functions served by these proteins using simple pharmacologic, knock-out or RNAi approaches because these methods eliminate all functions at once.  Thus, in this project we will pair RNAi knockdown with a mutant replacement approach in cultured rat neurons in addition to using a novel AKAP150 knock-in mutant mouse to probe the functions of specific AKAP79/150 membrane targeting motifs and protein-protein interactions in control of postsynaptic structure and function during induction of LTD and LTP.  The hypotheses that we will be testing are that regulation of AKAP79/150 postsynaptic targeting and signaling by palmitoylation (Aim 1), MAGUK scaffolding interactions (Aim 1), and CaN anchoring (Aims 2 &3) coordinately regulate dendritic spine structure and AMPAR function in plasticity.  PUBLIC HEALTH RELEVANCE: The AKAP79/150-organized neuronal excitatory postsynaptic signaling processes we are studying that control dendritic spine structure and glutamate receptor function are believed to be relevant for mechanisms of altered synaptic plasticity and cognition in neurological disorders such as Alzheimer's and epilepsy and mental health disorders such as Down syndrome and schizophrenia.  These same pathways also have relevance for understanding how excessive glutamate receptor activation leads to excitotoxic neuronal death in neurodegenerative diseases, brain injury and stroke.  In particular, regulation of glutamate receptor activity and dendritic spine structural changes have been implicated in both plasticity and excitoxicity, thus understanding the role of AKAP79/150 in controlling these events through both its structural interactions and signaling functions is important for understanding basic synaptic processes that are altered in human disease.