Interactions between dopamine and N -methyl- d -aspartate receptors (NMDARs) in prefrontal cortex (PFC) and other brain regions are believed to play an important role in normal mental function and neuropsychiatric disorders. In this study, we examined the regulation of NMDAR currents by the dopamine D ₁ receptor in PFC pyramidal neurons. Application of the D ₁ receptor agonist SKF81297 caused a prominent increase of the steady-state NMDA-evoked current in acutely isolated PFC pyramidal neurons. The D ₁ effect on NMDARs was independent of protein kinase A or protein phosphatase 1, but was abolished by incubation of neurons in Ca ²⁺ -free medium. Intracellular application of the Ca ²⁺ chelator, calmodulin, or calmodulin inhibitors largely prevented the D ₁ modulation of NMDAR currents. Moreover, inhibiting PKC activity or disrupting PKC association with its anchoring protein also significantly reduced the D ₁ effect on NMDAR currents. This upregulation of NMDAR activity by dopamine D ₁ receptors and the previous finding on up-regulation of dopamine D ₁ receptors by NMDAR activation provide a cellular mechanism for the reciprocal interactions between D ₁ and NMDARs. These interactions may play an important role in modulating synaptic plasticity and thus in cognitive and emotional processes.

The efficacy of synaptic inhibition depends on the number of γ-aminobutyric acid type A receptors (GABA _A Rs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABA _A R endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABA _A R β subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the β3 subunit) incorporates the major sites of serine phosphorylation within receptor β subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pepβ3) corresponding to the AP2 binding motif in the GABA _A R β3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pepβ3 peptide, but not its phosphorylated equivalent (pepβ3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABA _A R current. These effects of pepβ3 on GABA _A R current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pepβ3 on GABA _A R endocytosis. Therefore phospho-dependent regulation of AP2 binding to GABA _A Rs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission.

The regulation of the number of γ2-subunit-containing GABA _A receptors (GABA _A Rs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface γ2-subunit-containing GABA _A Rs is regulated. Here, we identify a γ2-subunit-specific Yxxφ-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for γ2-subunit tyrosine phosphorylation. Blocking GABA _A R-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxφ motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that γ2-subunit-containing heteromeric GABA _A Rs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABA _A R surface levels and synaptic inhibition.

Gestational exposure to ethanol causes fetal alcohol syndrome, which is associated with cerebellar hypoplasia. Previous in vitro studies demonstrated ethanol-impaired neuronal survival with reduced signaling through the insulin receptor (IRβ). We examined insulin signaling in an experimental rat model of chronic gestational exposure to ethanol in which the pups exhibited striking cerebellar hypoplasia with increased apoptosis. Immunoprecipitation and Western blot analyses detected reduced levels of tyrosyl-phosphorylated IRβ, tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1), and p85-associated IRS-1 but no alterations in IRβ, IRS-1, or p85 protein expression in cerebellar tissue from ethanol-exposed pups. In addition, ethanol exposure significantly reduced the levels of total phosphoinositol 3-kinase, Akt kinase, phospho-BAD (inactive), and glyceraldehyde-3-phosphate dehydrogenase and increased the levels of glycogen synthase kinase-3 activity, activated BAD, phosphatase and tensin homolog deleted in chromosome 10 (PTEN) protein, and PTEN phosphatase activity in cerebellar tissue. Cerebellar neurons isolated from ethanol-exposed pups had reduced levels of insulin-stimulated phosphoinositol 3-kinase and Akt kinase activities and reduced insulin inhibition of PTEN and glycogen synthase kinase-3 activity. The results demonstrate that cerebellar hypoplasia produced by chronic gestational exposure to ethanol is associated with impaired survival signaling through insulin-regulated pathways, including failure to suppress PTEN function.

α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors undergo constitutive and ligand-induced internalization that requires dynamin and the clathrin adaptor complex AP-2. We report here that an atypical basic motif within the cytoplasmic tails of AMPA-type glutamate receptors directly associates with μ2-adaptin by a mechanism similar to the recognition of the presynaptic vesicle protein synaptotagmin 1 by AP-2. A synaptotagmin 1-derived AP-2 binding peptide competes the interaction of the AMPA receptor subunit GluR2 with AP-2μ and increases the number of surface active glutamate receptors in living neurons. Moreover, fusion of the GluR2-derived tail peptide with a synaptotagmin 1 truncation mutant restores clathrin/AP-2-dependent internalization of the chimeric reporter protein. These data suggest that common mechanisms regulate AP-2-dependent internalization of pre- and postsynaptic membrane proteins.