J. Neurochem. (2011) 119, 27–39. Abstract α‐Amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionic acid receptors (AMPARs) are the primary mediators of excitatory synaptic transmission in the brain. Alterations in AMPAR localization and turnover have been considered critical mechanisms underpinning synaptic plasticity and higher brain functions, but the molecular processes that control AMPAR trafficking and stability are still not fully understood. Here, we report that mammalian AMPARs are subject to ubiquitination in neurons and in transfected heterologous cells. Ubiquitination facilitates AMPAR endocytosis, leading to a reduction in AMPAR cell‐surface localization and total receptor abundance. Mutation of lysine residues to arginine residues at the glutamate receptor subunit 1 (GluA1) C‐terminus dramatically reduces GluA1 ubiquitination and abolishes ubiquitin‐dependent GluA1 internalization and degradation, indicating that the lysine residues, particularly K868, are sites of ubiquitination. We also find that the E3 ligase neural precursor cell expressed, developmentally down‐regulated 4 (Nedd4) is enriched in synaptosomes and co‐localizes and associates with AMPARs in neurons. Nedd4 expression leads to AMPAR ubiquitination, leading to reduced AMPAR surface expression and suppressed excitatory synaptic transmission. Conversely, knockdown of Nedd4 by specific siRNAs abolishes AMPAR ubiquitination. These data indicate that Nedd4 is the E3 ubiquitin ligase responsible for AMPAR ubiquitination, a modification that regulates multiple aspects of AMPAR molecular biology including trafficking, localization and stability.

.1, homeostatic increase in GluR1 immunointensity (Upper) was completely blocked by coincubation with PhTx to specifically block the GluR2-lacking AMPARs (Lower). (B) Homeostatic response was blocked when PhTx was applied for only the first day or for 2 d after transfection. (Syn, 1.08 ± 0.06, n = 50; Kir2.1, 1.29 ± 0.08, n = 53; P < 0.05; Kir2.1 with PhTx-for 1 d, 1.09 ± 0.07, n = 61; P > 0.05; for 2 d 1.15 ± 0.06, n = 53; P > 0.05 compared with the Synapsin-YFP control). However, when supplemented only at the second day after transfection, PhTx failed to block the homeostatic response (1.24 ± 0.06, n = 56; P < 0.05 compared with Synapsin-YFP control), indicating a critical role of GluR2-lacking AMPARs in the early stages of homeostasis induction.

Neuronal activity largely depends on two key components on the membrane: the Na,K-ATPase (NKA) that maintains the ion gradients and sets the foundation of excitability, and the ionotropic glutamatergic AMPA receptors (AMPARs) through which sodium influx forms the driving force for excitation. Because the frequent sodium transients from glutamate receptor activity need to be efficiently extruded, a functional coupling between NKA and AMPARs should be a necessary cellular device for synapse physiology. We show that NKA is enriched at synapses and associates with AMPARs. NKA dysfunction induces a rapid reduction in AMPAR cell-surface expression as well as total protein abundance, leading to a long-lasting depression in synaptic transmission. AMPAR proteolysis requires sodium influx, proteasomal activity and receptor internalization. These data elucidate a novel mechanism by which NKA regulates AMPAR turnover and thereby synaptic strength and brain function.

In the process of membrane biogenesis several dozen proteins must operate in precise concert to generate Ϸ100 lipids at appropriate concentrations. To study the regulation of bilayer assembly in a cell cycle-independent manner, we have exploited the fact that phagocytes replenish membranes expended during particle engulfment in a rapid phase of lipid synthesis. In response to phagocytosis of latex beads, human embryonic kidney 293 cells synthesized cholesterol and phospholipids at amounts equivalent to the surface area of the internalized particles.

Fusion of phagosomes with late endocytic organelles is essential for cellular digestion of microbial pathogens, senescent cells, apoptotic bodies, and retinal outer segment fragments. To further elucidate the biochemistry of the targeting process, we developed a scintillation proximity assay to study the stepwise association of lysosomes and phagosomes in vitro. Incubation of tritium-labeled lysosomes with phagosomes containing scintillant latex beads led to light emission in a reaction requiring cytosol, ATP, and low Ca(2+) concentrations. The nascent complex was sensitive to disruption by alkaline carbonate, indicating that the organelles had "docked" but not fused. Through inhibitor studies and fluorescence microscopy we show that docking is preceded by a tethering step that requires actin polymerization and calmodulin. In the docked state ongoing actin polymerization and calmodulin are no longer necessary. The tethering/docking activity was purified to near homogeneity from rat liver cytosol. Major proteins in the active fractions included actin, calmodulin and IQGAP2. IQGAPs are known to bind calmodulin and cross-link F-actin, suggesting a key coordinating role during lysosome/phagosome attachment. The current results support the conclusion that lysosome/phagosome interactions proceed through distinct stages and provide a useful new approach for further experimental dissection.