Sang Hyoung Lee

Distinct molecular mechanisms and divergent endocytotic pathways of AMPA receptor internalization

Internalization of postsynaptic AMPA receptors depresses excitatory transmission, but the underlying dynamics and mechanisms of this process are unclear. Using immunofluorescence and surface biotinylation, we characterized and quantified basal and regulated AMPA receptor endocytosis in cultured hippocampal neurons, in response to synaptic activity, AMPA and insulin. AMPA-induced AMPA receptor internalization is mediated in part by secondary activation of voltage-dependent calcium channels, and in part by ligand binding independent of receptor activation. Although both require dynamin, insulin- and AMPA-induced AMPA receptor internalization are differentially dependent on protein phosphatases and sequence determinants within the cytoplasmic tails of GluR1 and GluR2 subunits. AMPA receptors internalized in response to AMPA stimulation enter a recycling endosome system, whereas those internalized in response to insulin diverge into a distinct compartment. Thus, the molecular mechanisms and intracellular sorting of AMPA receptors are diverse, and depend on the internalizing stimulus.

Clathrin Adaptor AP2 and NSF Interact with Overlapping Sites of GluR2 and Play Distinct Roles in AMPA Receptor Trafficking and Hippocampal LTD

Proteins that bind to the cytoplasmic tails of AMPA receptors control receptor trafficking and thus the strength of postsynaptic responses. Here we show that AP2, a clathrin adaptor complex important for endocytosis, associates with a region of GluR2 that overlaps the NSF binding site. Peptides used previously to interfere with NSF binding also antagonize GluR2-AP2 interaction. Using GluR2 mutants and peptide variants that dissociate NSF and AP2 interaction, we find that AP2 is involved specifically in NMDA receptor-induced (but not ligand-dependent) internalization of AMPA receptors, and is essential for hippocampal long-term depression (LTD). NSF function, on the other hand, is needed to maintain synaptic AMPA receptor responses, but is not directly required for NMDA receptor-mediated internalization and LTD.

Subunit Rules Governing the Sorting of Internalized AMPA Receptors in Hippocampal Neurons

Removal of synaptic AMPA receptors is important for synaptic depression. Here, we characterize the roles of individual subunits in the inducible redistribution of AMPA receptors from the cell surface to intracellular compartments in cultured hippocampal neurons. The intracellular accumulation of GluR2 and GluR3 but not GluR1 is enhanced by AMPA, NMDA, or synaptic activity. After AMPA-induced internalization, homomeric GluR2 enters the recycling pathway, but following NMDA, GluR2 is diverted to late endosomes/lysosomes. In contrast, GluR1 remains in the recycling pathway, and GluR3 is targeted to lysosomes regardless of NMDA receptor activation. Interaction with NSF plays a role in regulated lysosomal targeting of GluR2. GluR1/GluR2 heteromeric receptors behave like GluR2 homomers, and endogenous AMPA receptors show differential activity-dependent sorting similar to homomeric GluR2. Thus, GluR2 is a key subunit that controls recycling and degradation of AMPA receptors after internalization.

Tyrosine phosphorylation of GluR2 is required for insulin‐stimulated AMPA receptor endocytosis and LTD

The α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) subtype of glutamate receptors is subject to functionally distinct constitutive and regulated clathrin‐dependent endocytosis, contributing to various forms of synaptic plasticity. In HEK293 cells transiently expressing GluR1 or GluR2 mutants containing domain deletions or point mutations in their intracellular carboxyl termini (CT), we found that deletion of the first 10 amino acids (834–843) selectively reduced the rate of constitutive AMPA receptor endocytosis, whereas truncation of the last 15 amino acids of the GluR2 CT, or point mutation of the tyrosine residues in this region, only eliminated the regulated (insulin‐stimulated) endocytosis. Moreover, in hippocampal slices, both insulin treatment and low‐frequency stimulation (LFS) specifically stimulated tyrosine phosphorylation of the GluR2 subunits of native AMPA receptors, and the enhanced phosphorylation appears necessary for both insulin‐ and LFS‐induced long‐term depression of AMPA receptor‐mediated excitatory postsynaptic currents. Thus, our results demonstrate that constitutive and regulated AMPA receptor endocytosis requires different sequences within GluR CTs and tyrosine phosphorylation of GluR2 CT is required for the regulated AMPA receptor endocytosis and hence the expression of certain forms of synaptic plasticity.

The role of CaMKII as an F-actin-bundling protein crucial for maintenance of dendritic spine structure

Ca2+-calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine protein kinase critically involved in synaptic plasticity in the brain. It is highly concentrated in the postsynaptic density fraction, exceeding the amount of any other signal transduction molecules. Because kinase signaling can be amplified by catalytic reaction, why CaMKII exists in such a large quantity has been a mystery. Here, we provide biochemical evidence that CaMKII is capable of bundling F-actin through a stoichiometric interaction. Consistent with this evidence, in hippocampal neurons, RNAi-mediated down-regulation of CaMKII leads to a reduction in the volume of dendritic spine head that is mediated by F-actin dynamics. An overexpression of CaMKII slowed down the actin turnover in the spine head. This activity was associated with β subunit of CaMKII in a manner requiring its actin-binding and association domains but not the kinase domain. This finding indicates that CaMKII serves as a central signaling molecule in both functional and structural changes during synaptic plasticity.

AMPA Receptor Trafficking and the Control of Synaptic Transmission

meable to calcium and that controls synaptic plasticity. Activation of NMDA receptors leads to the appearance of functional AMPA receptors (“unsilencing”) in previously silent synapses, thereby potentiating synaptic transmission (Malenka and Nicoll, 1999; Malinow et al., Morgan Sheng1,2 and Sang Hyoung Lee2 Department of Neurobiology and Howard Hughes Medical Institute Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts 02114 2000). This postsynaptic potentiation could be due to the activation of nonfunctional AMPA receptors already existing in synapses (e.g., by phosphorylation), or by the delivery of new AMPA receptors to the postsynaptic Glutamate is the major excitatory neurotransmitter in membrane. Supporting the latter idea, the level of AMPA mammalian brain. After release from the presynaptic receptors in synapses is influenced by synaptic activity terminal, glutamate acts on specific receptors that are and varies greatly between different synapses, with clustered in the postsynaptic membrane. The AMPAsome synapses being devoid of AMPA receptors (Nustype glutamate receptor, a ligand-gated cation channel ser, 2000). that opens upon glutamate binding, mediates most of AMPA Receptor Delivery the excitatory (depolarizing) postsynaptic response in Recent studies have revealed that AMPA receptors can glutamatergic synapses. Thus, changing the activity of translocate from nonsynaptic to synaptic sites, providAMPA receptors is a powerful way to control the ing a cell biological basis for controlling the synaptic strength of synaptic transmission, which is important level of AMPA receptors and hence postsynaptic refor information storage in the brain. sponsiveness (Lüscher et al., 2000; Malinow et al., 2000). AMPA receptors are formed from heteromeric (probaThe first direct evidence for movement of AMPA recepbly tetrameric) combinations of subunits GluR1-4. GluR tors came when Shi et al. (1999) showed that following subunits can be divided into two groups, GluR1 and strong synaptic stimulation and NMDA receptor activaGluR4, and GluR2 and GluR3, based on sequence simition, GFP-tagged GluR1 translocated from the main larity of their C-terminal cytoplasmic domains. In the shaft of dendrites into spines, specialized dendritic prohippocampus, GluR2 and GluR3 have short cytoplasmic trusions on which excitatory synapses are formed. tails of around 50 amino acids with a conserved C-terminal sequence (-SVKI) that binds to cytoplasmic PDZ proteins GRIP/ABP and PICK-1 (Sheng and Pak, 2000; Scannevin and Huganir, 2000). The longer GluR1 cytoplasmic tail (terminating in -ATGL) binds to a distinct set of proteins (Figure 1). In the hippocampus (part of the brain important for learning and memory and where many experiments on synaptic transmission are conducted), endogenous AMPA receptors are composed mainly of GluR1/GluR2 and GluR2/GluR3 heteromers (Wenthold et al., 1996). Recent work from Roberto Malinow and colleagues, culminating in a paper in Cell (Shi et al., 2001), has defined an important set of subunitspecific rules governing the delivery of AMPA receptors to synapses. These rules provide new insights into the postsynaptic trafficking of AMPA receptors and open inroads into the molecular mechanisms that tune synaptic strength. Silent Synapses A simple way to modify synaptic responses is to change the number of postsynaptic AMPA receptors available for activation by released glutamate. Electrophysiological and morphological evidence for such a mechanism has accumulated over the past few years, driven by the discovery of ‘‘silent synapses.’’ A subset of glutamatergic synapses in many parts of the CNS lack AMPA receptor currents. These so-called “silent synapses” nevertheless contain functional NMDA receptors, another type of ionotropic glutamate receptor that is perFigure 1. Membrane Topology and Cytoplasmic Protein Interactions of AMPA Receptor Subunits

Identification of a Novel Divergent Calmodulin Isoform from Soybean Which Has Differential Ability to Activate Calmodulin-dependent Enzymes

Calmodulin plays pivotal roles in the transduction of various Ca-mediated signals and is one of the most highly conserved proteins in eukaryotic cells. In plants, multiple calmodulin isoforms with minor amino acid sequence differences were identified but their functional significances are unknown. To investigate the biological function of calmodulins in the regulation of calmodulin-dependent enzymes, we cloned cDNAs encoding calmodulins in soybean. Among the five cDNAs isolated from soybean, designated as SCaM-1 to −5, SCaM-4 and −5 encoded very divergent calmodulin isoforms which have 32 amino acid substitutions from the highly conserved calmodulin, SCaM-1 encoded by SCaM-1 and SCaM-3. SCaM-4 protein produced in Escherichia coli showed typical characteristics of calmodulin such as Ca-dependent electrophoretic mobility shift and the ability to activate phosphodiesterase. However, the extent of mobility shift and antigenicity of SCaM-4 were different from those of SCaM-1. Moreover, SCaM-4 did not activate NAD kinase at all in contrast to SCaM-1. Also there were differences in the expression pattern of SCaM-1 and SCaM-4. Expression levels of SCaM-4 were approximately 5-fold lower than those of SCaM-1 in apical and elongating regions of hypocotyls. In addition, SCaM-4 transcripts were barely detectable in root whereas SCaM-1 transcripts were as abundant as in apical and elongating regions of hypocotyls. In conclusion, the different biochemical properties together with differential expression of SCaM-4 suggest that this novel calmodulin may have different functions in plant cells.

Development of neuron-neuron synapses.

Our understanding of neuronal synapse development has advanced in recent years. The development of glycinergic synapses appears to depend on gephyrin and glycine receptor activity. Molecular characterization of the structure and development of glutamatergic synapses is in progress, but the underlying mechanisms remain unclear. Activity-dependent mechanisms and specific molecules that regulate the morphological development of dendritic spines have recently been identified.

Identification of a Calmodulin-Regulated Soybean Ca2+-ATPase (SCA1) That Is Located in the Plasma Membrane

Ca2+-ATPases are key regulators of Ca2+ ion efflux in all eukaryotes. Animal cells have two distinct families of Ca2+ pumps, with calmodulin-stimulated pumps (type IIB pumps) found exclusively at the plasma membrane. In plants, no equivalent type IIB pump located at the plasma membrane has been identified at the molecular level, although related isoforms have been identified in non–plasma membrane locations. Here, we identify a plant cDNA, designated SCA1 (for soybean Ca2+-ATPase 1), that encodes Ca2+-ATPase and is located at the plasma membrane. The plasma membrane localization was determined by sucrose gradient and aqueous two-phase membrane fractionations and was confirmed by the localization of SCA1p tagged with a green fluorescent protein. The Ca2+-ATPase activity of the SCA1p was increased approximately sixfold by calmodulin (K1/2 ∼10 nM). Two calmodulin binding sequences were identified in the N-terminal domain. An N-terminal truncation mutant that deletes sequence through the two calmodulin binding sites was able to complement a yeast mutant (K616) that was deficient in two endogenous Ca2+ pumps. Our results indicate that SCA1p is structurally distinct from the plasma membrane–localized Ca2+ pump in animal cells, belonging instead to a novel family of plant type IIB pumps found in multiple subcellular locations. In plant cells from soybean, expression of this plasma membrane pump was highly and rapidly induced by salt (NaCl) stress and a fungal elicitor but not by osmotic stress.

Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration.

Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients.