Postsynaptic Signaling by Norepinephrine and cAMP Abstract Norepinephrine (NE) regulates attention and alertness. The β2AR adrenergic receptor (β2AR) is the prevalent postsynaptic NE effector at glutamatergic synapses, where it interacts with AMPARs and the postsynaptic L- type Ca2+ channel (LTCC) CaV1.2. These complexes also contain all downstream effectors of β2AR (Gs, adenylyl cyclases (ACs) and the cAMP-dependent protein kinase PKA) for highly localized signaling by cAMP (see our pioneering work in Science 293, 98; Science 293, 2205; EMBO J 29, 482; EMBO J 35, 1330; Science Signaling10, eaaf9659 and eaaf9647). Our exciting preliminary data indicate that 1) NE enters neurons and endosomes via the NE transporters OCT3 and PMAT to stimulate intracellular β2AR signaling and drive AMPARs to the cell surface; 2) the cAMP degrading phosphodiesterase PDE4A5, which is increased during sleep deprivation and mediates the resulting memory impairment, is linked to the β2AR - AMPAR complex via PSD- 95; 3) CaV1.2 forms a supercomplex with AMPARs, which allows efficient activation of CaV1.2 during synaptic transmission at postsynaptic sites with NE present. Aim 1 is to test whether activation of intracellular β2ARs upon NE uptake by OCT3 & PMAT triggers AMPAR phosphorylation and surface delivery, thereby promoting LTP as PKA is known to stimulate AMPAR surface insertion. We will test whether NE-induced phosphorylation (immunoblotting; IB) and surface insertion of GluA1 (immunofluorescent microscopy; IF) and NE-dependent LTP are abrogated by inhibitors and KO of OCT3 &PMAT. This will provide for the first time lacking but important evidence for intracellular signaling by NE in neurons. Aim 2 is to test whether the cAMP-hydrolyzing PDE4A5 curbs upregulation of AMPARs by β2AR - cAMP - PKA signaling. We will test whether displacing PDE4A5 from GluA1 with polypeptides elevates GluA1 phosphorylation by PKA and surface expression of GluA1 in HCs (IF) and postsynaptic AMPARs responses (mEPSC, EPSC). Aim 3 is to determine the AMPAR - CaV1.2 interaction sites and test the functional relevance of the AMPAR - CaV1.2 supercomplex formation. We will disrupt the interaction by mutations and acute peptide application to determine its role in Ca2+ influx into dendritic spines and GluA1 surface insertion by live imaging. This work will define unexplored fundamental molecular mechanisms of how NE regulates postsynaptic functions. It will elucidate new molecular details that might be affected in neurological diseases such as Alzheimer’s disease, which is at least in part due to dysregulation of CaV1.2 and glutamate receptors by β2AR signaling. NE signaling is also relevant for ADHD and PTSD. The postsynaptic assembly of specific signaling components that control PKA-mediated phosphorylation of AMPARs and CaV1.2 and their functional interactions constitute potentially effective and specific targets for drugs that disrupt some of these interactions while not affecting others (e.g...