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Modification of AMPA receptor clustering regulates cerebellar synaptic plasticity
Neuroscience Research 39 (2001) 261– 267 www.elsevier.com/locate/neures
Update Article
Modification of AMPA receptor clustering regulates cerebellar synaptic plasticity Hirokazu Hirai * Laboratory for Memory and Learning, RIKEN Brain Science Institute, Wako, Saitama 351 -0198, Japan Received 26 October 2000; accepted 7 December 2000
Abstract Cerebellar long-term depression (LTD) induced at parallel fiber-Purkinje neuron synapses is proposed to underlie certain types of motor learning. a-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, which mediate chemical transmission in these synapses, are clustered on the postsynaptic membrane. By increasing local density of the receptors, clustering is believed to increase synaptic efficacy. This article focuses on molecular mechanisms regulating the synaptic AMPA receptor clustering in Purkinje cells, which could underlie the expression of cerebellar LTD. Synaptic AMPA receptor clusters in dendritic spines of Purkinje cells are disrupted upon protein kinase C (PKC)-mediated phosphorylation of serine 880 in the C-terminal domain of GluR2. Phosphorylation of this residue causes significant reduction in the affinity of GluR2 C-terminal tail for glutamate receptor interacting protein (GRIP), a molecule known to be crucial for AMPA receptor clustering. Consequently, AMPA receptors on the synaptic membrane are destabilized and internalized by endocytosis. Based on these findings, a model for the expression of cerebellar LTD is proposed, in which a decrease in the number of postsynaptic AMPA receptors, initiated by phosphorylation of GluR2 serine 880, is the major mechanism underlying cerebellar LTD. © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: AMPA receptor; GluR2; Clustering; Phosphorylation; LTD; Purkinje cell; GRIP; PDZ domain
1. Introduction Molecular mechanisms regulating clusters of neurotransmitter receptors have recently drawn particular attention, because modulation of the clusters alters the postsynaptic functional receptor density and consequently, could affect the synaptic efficacy and plasticity (Carroll et al., 1999b; Kneussel et al., 1999). To localize neurotransmitter receptors on the postsynaptic membrane, cytoplasmic adapter proteins are essential to anchor receptor proteins to the cytoskeleton (Kirsch and Betz, 1995; Wyszynski et al., 1997; Allison et al., 1998; Matsuda and Hirai, 1999; Allison et al., 2000; Hirai, 2000). These adapter proteins can be divided into two groups, those with and without PDZ domains. The latter group includes proteins, which associate with * Tel.: +81-48-4621111/Ext. 6429; fax: + 81-48-4624697. E-mail address: [email protected] (H. Hirai).
cytoskeletal components such as a-actinin (Wyszynski et al., 1997) and spectrin (Hirai and Matsuda, 1999) for glutamate receptors or gephyrin for inhibitory glycine or GABAA receptors (Kneussel and Betz, 2000). The PDZ domain is a protein –protein interaction motif of approximately 90 amino acids, which in most cases, binds their target proteins at the C-terminal ends (Saras and Heldin, 1996). Recently, many PDZ domain-containing proteins interacting with various subtypes of glutamate receptors have been found using the yeast two-hybrid system. Each of the proteins has discrete binding partners of glutamate receptor subunits. For example, PSD95, the first PDZ domain-containing protein that was identified at vertebrate CNS synapses, binds to NR2 subunits of NMDA receptors (Kornau et al., 1995; Niethammer et al., 1996) and Homer/vesl associates with mGluR1 and mGluR5 of the metabotropic glutamate receptors (Brakeman et al., 1997; Kato et al., 1997). The a-amino-3-hydroxy-5-
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methylisoxazole-4-propionate (AMPA) receptor GluR2 and GluR3 subunits interact with several different PDZ domain-containing proteins such as protein interacting with C kinase (PICKI) (Xia et al., 1999), glutamate receptor interacting protein (GRIP) (Dong et al., 1997) and its analogue, AMPA receptor binding protein (ABP) (Srivastava et al., 1998). The expression of these PDZ domain-containing proteins, as well as the subunit composition of glutamate receptors, varies in different brain regions. Therefore, mechanisms for glutamate receptor anchoring and clustering can vary in different brain areas. Cerebellar Purkinje neurons exhibit high expression levels of GluR2/3 (Zhao et al., 1998) and a corresponding PDZ domain-containing protein, GRIP, both of which colocalize precisely in the dendritic arborizations (Wyszynski et al., 1998). These findings suggest that the interaction of GluR2/3 with GRIP has an important role in the localization and clustering of postsynaptic AMPA receptors in Purkinje cells. In this minireview, I present our recent data on the molecular mechanisms of GRIP-mediated AMPA receptor clustering in the dendritic spines of cerebellar Purkinje cells and further discuss its contribution to cerebellar long-term depression (LTD).
2. Phosphorylation of AMPA receptors
2.1. Phosphorylation of GluR1 subunit modulates channel kinetics of AMPA receptors The membrane topology of glutamate receptors consists of an extracellular N-terminal domain, three membrane-spanning regions, one membrane re-entrant loop and an intracellular C-terminal domain (Hollmann et al., 1994; Hirai et al., 1996). The C-terminal domain of a glutamate receptor, which occupies most of the intracellular region of the receptor protein, can be modulated by phosphorylation or interaction with various cytoskeletal or signaling molecules; thereby, the function of the receptor channel is altered (Ehlers et al., 1996; Kornau et al., 1997; Wyszynski et al., 1997; Zhao et al., 1998; Braithwaite et al., 1999). The C-terminal domain of the AMPA receptor GluR1 subunit is known to be phosphorylated by protein kinase C (PKC) and Ca2 + /calmodulin-dependent protein kinase II (CaM-KII) at serine 831 and by cyclic AMP-dependent protein kinase (PKA) at serine 845 (Roche et al., 1996; Barria et al., 1997). Earlier electrophysiological studies, using recombinant GluR1 channels, show that phosphorylation of serine 831 in GluR1 by CaM-KII increases the single-channel conductance (Derkach et al., 1999), while PKA-mediated phosphorylation of serine 845 in GluR1 increases the open probability of GluR1 receptors (Banke et al., 2000). These results
suggest that phosphorylation of AMPA receptors modulates synaptic efficacy, which may underlie long-term potentiation (LTP) or LTD of synaptic transmission.
2.2. Serine at position 880 (Ser880) in GluR2 is phosphorylated by PKC Our main interest has been the molecular mechanisms underlying cerebellar LTD induced at parallel fiber-Purkinje cell synapses (Ito, 1989). We focused our attention to two earlier findings. First, neurotransmission at this synapse is mediated by AMPA receptors with a high proportion of GluR2/3 and little of GluR1 (Kano and Kato, 1987; Zhao et al., 1998). Second, PKC activation is essential for cerebellar LTD induction (Crepel and Krupa, 1988; Linden and Connor, 1991; De Zeeuw et al., 1998). Therefore, we hypothesized that phosphorylation of GluR2/3 by PKC should play a key role in the induction of cerebellar LTD by directly altering AMPA receptor properties as indicated in GluR1 (Derkach et al., 1999; Banke et al., 2000). Since phosphorylation of GluR2 subunit had not yet been clarified, we first attempted to verify whether the GluR2 subunit could be a substrate for PKC by in vitro phosphorylation of glutathione S-transferase (GST)GluR2 C-terminal fusion protein in the presence of 32 P-ATP (Matsuda et al., 1999). The results showed that a significant amount of 32P was incorporated into the GluR2 C-terminal fusion protein incubated with PKC. Subsequent experiments using 31P-NMR (Hirai et al., 2000) and phosphorylation specific antibodies (Matsuda et al., 1999, 2000) revealed that Ser880 in the intracellular C-terminal domain of GluR2 is a major PKC-phosphorylation site in cultured Purkinje cells, as well as in transfected cells.
3. Regulation of AMPA receptor clusters
3.1. PKC phosphorylation of Ser880 in GluR2 disrupts GluR2 –GRIP interaction Ser880, the identified GluR2 PKC-phosphorylation site, is localized in the sequence (IESVKI) critical for binding to PDZ domain-containing proteins, GRIP (Dong et al., 1997), ABP (Srivastava et al., 1998) and PICK1 (Dev et al., 1999; Xia et al., 1999). Since these proteins are suggested to be important for clustering of synaptic AMPA receptors containing GluR2/3, one would expect that phosphorylation of GluR2 Ser880 localized in the PDZ-binding motif should modulate the interaction of GluR2 with these PDZ domain-containing proteins. Among the PDZ domain-containing proteins, the distribution of GRIP, in relation to the localization of GluR2, has been well characterized. Wyszynski et al. (1999) have shown that most of GRIP
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was localized in nonsynaptic areas and only limited amount of GRIP was colocalized with GluR2 in dendritic regions of hippocampal neurons. In the cerebral cortex, most GluR2-positive cells have been shown to exhibit only weak GRIP immunoreactivity (Burette et al., 1999). In contrast, perfect colocalization of GRIP and GluR2 was demonstrated in dendritic regions of Purkinje cells (Burette et al., 1999). These results suggest a functional implication of GluR2 – GRIP interaction in Purkinje cells. We, therefore, examined the effect of GluR2 Ser880 phosphorylation on GluR2 binding to GRIP. Our immunoprecipitation experiments (Matsuda et al., 1999) showed that the GSTGluR2 C-terminal fusion protein was efficiently coimmunoprecipitated with GluR2-binding domains of GRIP (PDZ 4-5, amino acids 419 – 673: GRIP419 – 673) (Fig. 1A). However, the binding of GluR2 carboxyl terminus to GRIP419 – 673 was almost completely inhibited when using either PKC-phosphorylated fusion protein (Fig. 1B) or mutant fusion protein whose Ser880 was replaced with alanine (Fig. 1C). Reduction in the affinity of phosphorylated GluR2 for GRIP has also been shown in transfected cells and cultured Purkinje cells stimulated by a PKC activator (Matsuda et al., 1999, 2000). Thus, phosphorylation of GluR2 Ser880 is considered to be a critical factor for the interaction of GluR2 with GRIP. Interestingly, a very recent study has shown that interaction of GluR2 with PICK1 was not affected by phosphorylation of Ser880 in GluR2 (Chung et al., 2000). Moreover, they showed that in hippocampal neurons, Ser880-phosphorylated GluR2 colocalized with PICK1 mostly in dendritic shafts and that PKC activation caused redistribution of PICK1 with Ser880phosphorylated GluR2 to dendritic spines. The physiological significance of PICK1 redistribution is still unclear. However, considering that only a small amount of GRIP is colocalized with GluR2 in hippocampal neurons (Wyszynski et al., 1999), it can be
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assumed that the interaction of GluR2 with PICK1 rather than with GRIP or the association of GluR1 with its binding proteins such as SAP97 (Leonard et al., 1999) is important for the localization and clustering of AMPA receptors at the excitatory synapses of hippocampal neurons.
3.2. PKC-phosphorylation of Ser880 in GluR2 disrupts clustering of AMPA receptors in Purkinje cells Earlier, Dong et al. (1997) showed that clusters of AMPA receptors in spinal cord neurons were extensively disrupted when GluR2 –GRIP interaction was interfered by overexpression of the C-terminal domain of GluR2. No such effects were observed with GluR2 lacking the last seven amino acids essential for GRIP binding, suggesting that interaction of the GluR2 C-terminal tail with GRIP was crucial for synaptic clustering of AMPA receptors. Therefore, it is reasonable to suppose that phosphorylation of GluR2 Ser880, which significantly decreases the affinity of GluR2 C-terminal tail for GRIP, would disrupt AMPA receptor clusters in Purkinje cells. We tested this hypothesis by immunocytochemistry using a heterologous expression system (Matsuda et al., 2000). In human embryonic kidney (HEK) 293 cells, wild-type GluR2, but not mutant GluR2 (Ser880 to aspartate), form clusters when expressed together with GRIP419 – 673 (Fig. 2A –C). The GluR2 clusters are significantly disrupted upon activation of endogenous PKC without affecting GRIP419 – 673 clusters (Fig. 2D). Loss of GluR2 clusters (Fig. 2C, D) is considered to be due to inhibition of the GluR2 – GRIP interaction by either phosphorylation of GluR2 Ser880 (Fig. 2D) or the mutation of Ser880 to aspartate, which mimics a phosphorylated residue (Fig. 2C). Subsequent experiments using cultured Purkinje cells show that PKC activation causes a significant disruption of postsynaptic AMPA receptor clusters (Fig. 2E, F), concomitant with both phosphorylation of Ser880
Fig. 1. A schematic depicting in vitro interaction of GRIP with a GST-GluR2 C-terminal fusion protein. His-tagged GRIP419 – 673 (GluR2-binding domains) is mixed with GST-fusion protein containing wild type (A, B) or mutant (Ser880Asp) (C) GluR2 C-terminus, treated (B) or not treated (A, C) with PKC. The probes are immunoprecitated with anti-His-tagged antibodies.
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and clustering of AMPA receptors containing GluR2/3 on the synaptic membrane. 4. Internalization of AMPA receptors upon phosphorylation of Ser880 in GluR2
Fig. 2. Disruption of GluR2 clusters in transfected cells and Purkinje neurons upon activation of endogenous PKC. (A–D) GluR2 (FITC labeled) is expressed alone (A) or together with GRIP419 – 673 (rhodamine labeled) (B – D) in HEK 293 cells. In (C), mutant GluR2 whose Ser880 is substituted with aspartate is used. Treatment of cells with 12-tetradecanoyl phorbol 13-acetate (TPA; 200 nM, 20 min) caused redistribution of GluR2 protein (D). (E, F) GluR2 localization on dendrites of cultured Purkinje cells in control (E) or treated with TPA (200 nM, 20 min) (F) is visualized by immunolabeling of cells with anti-GluR2 N-terminal antibodies. Activation of endogenous PKC causes significant disruption of postsynaptic GluR2 clusters. Bar, 5 mm.
in GluR2 and a reduction in the affinity of GluR2 to GRIP (Matsuda et al., 2000). An essential role of the GRIP – GluR2 interaction in stabilization of AMPA receptors on the postsynaptic membrane was also recently reported by Osten et al. (2000). Using virus-mediated expression of the GluR2 protein in hippocampal neurons, they showed that while mutant GluR2 lacking the GRIP-binding site (last 10 amino acids of GluR2 C-terminus) was efficiently targeted to synaptic sites, surface localization of mutant GluR2 was significantly reduced. In addition, similar results were obtained by expressing mutant GluR2 with Ser880 substituted with alanine, which cannot bind to GRIP. Their results are consistent with our conclusion that binding of the GluR2 C-terminal tail to GRIP plays a significant role in the stabilization
As shown in Fig. 3A, the AMPA receptor protein is targeted to and inserted into the postsynaptic membrane where the receptor is stabilized. The AMPA receptor in the postsynaptic membrane is removed by endocytosis (Carroll et al., 1999a; Luscher et al., 1999). Thus, synaptic AMPA receptors are considered to constitutively undergo recycling using exocytotic/endocytotic pathways. We, therefore, thought that removal of synaptic AMPA receptors by endocytosis might be facilitated after disruption of GluR2 –GRIP interaction. This hypothesis was verified by three different experiments (Matsuda et al., 2000). The first experiment was conducted to evaluate intradendritic GluR2 immunoreactivity in cultured Purkinje cells by confocal microscopy. We found that GluR2 immunoreactivity inside the dendrites was significantly increased upon PKC activation. In the second experiment, we measured the amount of surface-expressed GluR2 in cerebellar neuronal culture incubated with or without phorbor ester by cell-surface biotinylation. The results showed a significant reduction in the biotinylated GluR2, namely, extracellularly-expressed GluR2 by 40% of the control level upon PKC activation. The third experiment was performed in an attempt to examine if internalization of GluR2 is mediated by clathrincoated vesicles. A fraction containing endocytosed clathrin-coated vesicles was obtained by fractionation of cells (van Delft et al., 1997) from cerebellar neuronal culture with or without phorbor ester treatment. The amount of GluR2 protein in this fraction, determined by immunoblotting for GluR2, significantly increased to more than four-fold following phorbor ester treatment. These results strongly suggest that a significant amount of GluR2 protein molecules that lost their anchorage are internalized via clathrin-mediated endocytosis in Purkinje cells.
Fig. 3. A schematic showing a model of cerebellar LTD induction based on modulation of recycling of synaptic AMPA receptors. GRIP serves as a scaffold for the localization and clustering of AMPA receptors containing GluR2/3 subunits (A). PKC-phosphorylation of Ser880 in GluR2 disrupts the association of GluR2 C-terminal tail with GRIP, leading to internalization of synaptic AMPA receptors by endocytosis (B). Consequently, the number of functional AMPA receptors on the postsynaptic membrane is reduced.
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5. Disruption of synaptic AMPA receptor clusters and cerebellar LTD Activation of endogenous PKC by phorbor ester, which causes LTD of glutamate responses in cultured Purkinje cells (Linden and Connor, 1991), significantly disrupts AMPA receptor clusters at synapses (Fig. 2E, F). In addition, a similar disruption of AMPA receptor clusters in cultured Purkinje cells is induced following glutamate+KCl stimulation (Matsuda et al., 2000), a different protocol for the induction of cerebellar LTD (Kawasaki et al., 1999). These results suggest that disruption of synaptic AMPA receptor clusters is involved in the expression of cerebellar LTD. Recent studies show that synaptic plasticity is regulated by modification of AMPA receptor recycling at the steps of (1) intracellular trafficking, (2) plasma membrane expression by exocytosis, (3) stabilization on the membrane, (4) removal by endocytosis and (5) recycling of the receptor protein (Fig. 3A). For example, in hippocampal neurons, tetanic synaptic stimulation inducing LTP was shown to cause a rapid delivery of GFP-tagged GluR1 to synaptic sites (Shi et al., 1999; Hayashi et al., 2000) (promotion of steps 1 and 2), suggesting that hippocampal LTP is caused by an increase in the number of postsynaptic AMPA receptors. During the process of membrane expression of AMPA receptors involving GluR2 (step 2), interaction of an N-ethylmaleimide-sensitive fusion protein (NSF) with GluR2 is suggested to be crucial. Pharmacological disruption of the NSF interaction (disruption of step 2) causes reduction in the surface expression of AMPA receptors, concomitant with a decrease in AMPA receptor-mediated synaptic transmission (Song et al., 1998; Luscher et al., 1999; Noel et al., 1999). Moreover, expression of hippocampal LTD is shown to share with the blockade of the NSF-mediated AMPA receptor expression process (Luthi et al., 1999). On the other hand, inhibition of clathrin-dependent endocytosis (disruption of step 4) is also shown to prevent the expression of hippocampal and cerebellar LTD (Man et al., 2000; Wang and Linden, 2000). These results suggest that modification of AMPA receptor cycling at synapses regulate synaptic plasticity including LTP and LTD. From this point of view, the following model for the induction of cerebellar LTD is proposed (Fig. 3B). GRIP-mediated AMPA receptor stabilization on the postsynaptic membrane (step 3) is disrupted by PKC phosphorylation of GluR2 Ser880. Consequently, removal of the receptors by clathrin-mediated endocytosis (step 4) is facilitated, leading to LTD of the synaptic transmission because of a decreased number of functional AMPA receptors on the synaptic membrane. The regulatory mechanism of synaptic AMPA receptor clusters illustrated in Fig. 3 is considered to be
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unique to the cerebellum for the following reasons. (1) The AMPA receptor in Purkinje cells consists mostly of GRIP-binding subunits, GluR2 and GluR3 (Zhao et al., 1998), whereas those in hippocampal and cortical neurons contain other subunits of the AMPA receptor (Wenthold et al., 1992). (2) Distribution of GRIP is closely correlated with the localization of GluR2 in Purkinje cells, but not in hippocampal and cortical neurons (Wyszynski et al., 1998; Burette et al., 1999). These results suggest that mechanisms different from those in the cerebellum are involved in the postsynaptic stabilization of AMPA receptors and modification of synaptic plasticity in the hippocampus and cerebral cortex, which might include modulation of the interaction of GluR2 or GluR1 with PICK1 or SAP97, respectively, or with as yet unknown proteins. Alternatively, as mentioned in Section 2.1, modulation of AMPA receptor kinetics by phosphorylation of the GluR1 subunit of the AMPA receptor may contribute to hippocampal or cerebral synaptic plasticity. Likewise, although no data are currently available, it cannot be excluded that changes in the AMPA receptor channel kinetics upon phosphorylation of GluR2/3 contribute to expression of cerebellar LTD.
6. Concluding remarks Recent advances in molecular and biochemical approaches to the study of synaptic specialization have revealed a significant contribution of PDZ domain-containing proteins to the synaptic organization including synaptic plasticity. Taken together, our studies have provided some clues that lead to the understanding of cerebellar memory formation. However, our results were obtained using a cell culture system; it should be confirmed using in vivo preparations whether the regulation of synaptic AMPA receptor clusters in cerebellar Purkinje cells indeed underlies the expression of cerebellar LTD and the resultant motor learning. A promising approach would be to use genetically modified mice in which GluR2 –GRIP interaction is not affected by phosphorylation. Such experiments designed to establish a link between molecular and behavioral events will help elucidate further the physiological significance of cerebellar LTD in motor learning, including adaptation of vestibulo-ocular reflex (Ito, 1982) and associative eyeblink conditioning (Raymond et al., 1996).
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Update Article
Modification of AMPA receptor clustering regulates cerebellar synaptic plasticity Hirokazu Hirai * Laboratory for Memory and Learning, RIKEN Brain Science Institute, Wako, Saitama 351 -0198, Japan Received 26 October 2000; accepted 7 December 2000
Abstract Cerebellar long-term depression (LTD) induced at parallel fiber-Purkinje neuron synapses is proposed to underlie certain types of motor learning. a-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, which mediate chemical transmission in these synapses, are clustered on the postsynaptic membrane. By increasing local density of the receptors, clustering is believed to increase synaptic efficacy. This article focuses on molecular mechanisms regulating the synaptic AMPA receptor clustering in Purkinje cells, which could underlie the expression of cerebellar LTD. Synaptic AMPA receptor clusters in dendritic spines of Purkinje cells are disrupted upon protein kinase C (PKC)-mediated phosphorylation of serine 880 in the C-terminal domain of GluR2. Phosphorylation of this residue causes significant reduction in the affinity of GluR2 C-terminal tail for glutamate receptor interacting protein (GRIP), a molecule known to be crucial for AMPA receptor clustering. Consequently, AMPA receptors on the synaptic membrane are destabilized and internalized by endocytosis. Based on these findings, a model for the expression of cerebellar LTD is proposed, in which a decrease in the number of postsynaptic AMPA receptors, initiated by phosphorylation of GluR2 serine 880, is the major mechanism underlying cerebellar LTD. © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: AMPA receptor; GluR2; Clustering; Phosphorylation; LTD; Purkinje cell; GRIP; PDZ domain
1. Introduction Molecular mechanisms regulating clusters of neurotransmitter receptors have recently drawn particular attention, because modulation of the clusters alters the postsynaptic functional receptor density and consequently, could affect the synaptic efficacy and plasticity (Carroll et al., 1999b; Kneussel et al., 1999). To localize neurotransmitter receptors on the postsynaptic membrane, cytoplasmic adapter proteins are essential to anchor receptor proteins to the cytoskeleton (Kirsch and Betz, 1995; Wyszynski et al., 1997; Allison et al., 1998; Matsuda and Hirai, 1999; Allison et al., 2000; Hirai, 2000). These adapter proteins can be divided into two groups, those with and without PDZ domains. The latter group includes proteins, which associate with * Tel.: +81-48-4621111/Ext. 6429; fax: + 81-48-4624697. E-mail address: [email protected] (H. Hirai).
cytoskeletal components such as a-actinin (Wyszynski et al., 1997) and spectrin (Hirai and Matsuda, 1999) for glutamate receptors or gephyrin for inhibitory glycine or GABAA receptors (Kneussel and Betz, 2000). The PDZ domain is a protein –protein interaction motif of approximately 90 amino acids, which in most cases, binds their target proteins at the C-terminal ends (Saras and Heldin, 1996). Recently, many PDZ domain-containing proteins interacting with various subtypes of glutamate receptors have been found using the yeast two-hybrid system. Each of the proteins has discrete binding partners of glutamate receptor subunits. For example, PSD95, the first PDZ domain-containing protein that was identified at vertebrate CNS synapses, binds to NR2 subunits of NMDA receptors (Kornau et al., 1995; Niethammer et al., 1996) and Homer/vesl associates with mGluR1 and mGluR5 of the metabotropic glutamate receptors (Brakeman et al., 1997; Kato et al., 1997). The a-amino-3-hydroxy-5-
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methylisoxazole-4-propionate (AMPA) receptor GluR2 and GluR3 subunits interact with several different PDZ domain-containing proteins such as protein interacting with C kinase (PICKI) (Xia et al., 1999), glutamate receptor interacting protein (GRIP) (Dong et al., 1997) and its analogue, AMPA receptor binding protein (ABP) (Srivastava et al., 1998). The expression of these PDZ domain-containing proteins, as well as the subunit composition of glutamate receptors, varies in different brain regions. Therefore, mechanisms for glutamate receptor anchoring and clustering can vary in different brain areas. Cerebellar Purkinje neurons exhibit high expression levels of GluR2/3 (Zhao et al., 1998) and a corresponding PDZ domain-containing protein, GRIP, both of which colocalize precisely in the dendritic arborizations (Wyszynski et al., 1998). These findings suggest that the interaction of GluR2/3 with GRIP has an important role in the localization and clustering of postsynaptic AMPA receptors in Purkinje cells. In this minireview, I present our recent data on the molecular mechanisms of GRIP-mediated AMPA receptor clustering in the dendritic spines of cerebellar Purkinje cells and further discuss its contribution to cerebellar long-term depression (LTD).
2. Phosphorylation of AMPA receptors
2.1. Phosphorylation of GluR1 subunit modulates channel kinetics of AMPA receptors The membrane topology of glutamate receptors consists of an extracellular N-terminal domain, three membrane-spanning regions, one membrane re-entrant loop and an intracellular C-terminal domain (Hollmann et al., 1994; Hirai et al., 1996). The C-terminal domain of a glutamate receptor, which occupies most of the intracellular region of the receptor protein, can be modulated by phosphorylation or interaction with various cytoskeletal or signaling molecules; thereby, the function of the receptor channel is altered (Ehlers et al., 1996; Kornau et al., 1997; Wyszynski et al., 1997; Zhao et al., 1998; Braithwaite et al., 1999). The C-terminal domain of the AMPA receptor GluR1 subunit is known to be phosphorylated by protein kinase C (PKC) and Ca2 + /calmodulin-dependent protein kinase II (CaM-KII) at serine 831 and by cyclic AMP-dependent protein kinase (PKA) at serine 845 (Roche et al., 1996; Barria et al., 1997). Earlier electrophysiological studies, using recombinant GluR1 channels, show that phosphorylation of serine 831 in GluR1 by CaM-KII increases the single-channel conductance (Derkach et al., 1999), while PKA-mediated phosphorylation of serine 845 in GluR1 increases the open probability of GluR1 receptors (Banke et al., 2000). These results
suggest that phosphorylation of AMPA receptors modulates synaptic efficacy, which may underlie long-term potentiation (LTP) or LTD of synaptic transmission.
2.2. Serine at position 880 (Ser880) in GluR2 is phosphorylated by PKC Our main interest has been the molecular mechanisms underlying cerebellar LTD induced at parallel fiber-Purkinje cell synapses (Ito, 1989). We focused our attention to two earlier findings. First, neurotransmission at this synapse is mediated by AMPA receptors with a high proportion of GluR2/3 and little of GluR1 (Kano and Kato, 1987; Zhao et al., 1998). Second, PKC activation is essential for cerebellar LTD induction (Crepel and Krupa, 1988; Linden and Connor, 1991; De Zeeuw et al., 1998). Therefore, we hypothesized that phosphorylation of GluR2/3 by PKC should play a key role in the induction of cerebellar LTD by directly altering AMPA receptor properties as indicated in GluR1 (Derkach et al., 1999; Banke et al., 2000). Since phosphorylation of GluR2 subunit had not yet been clarified, we first attempted to verify whether the GluR2 subunit could be a substrate for PKC by in vitro phosphorylation of glutathione S-transferase (GST)GluR2 C-terminal fusion protein in the presence of 32 P-ATP (Matsuda et al., 1999). The results showed that a significant amount of 32P was incorporated into the GluR2 C-terminal fusion protein incubated with PKC. Subsequent experiments using 31P-NMR (Hirai et al., 2000) and phosphorylation specific antibodies (Matsuda et al., 1999, 2000) revealed that Ser880 in the intracellular C-terminal domain of GluR2 is a major PKC-phosphorylation site in cultured Purkinje cells, as well as in transfected cells.
3. Regulation of AMPA receptor clusters
3.1. PKC phosphorylation of Ser880 in GluR2 disrupts GluR2 –GRIP interaction Ser880, the identified GluR2 PKC-phosphorylation site, is localized in the sequence (IESVKI) critical for binding to PDZ domain-containing proteins, GRIP (Dong et al., 1997), ABP (Srivastava et al., 1998) and PICK1 (Dev et al., 1999; Xia et al., 1999). Since these proteins are suggested to be important for clustering of synaptic AMPA receptors containing GluR2/3, one would expect that phosphorylation of GluR2 Ser880 localized in the PDZ-binding motif should modulate the interaction of GluR2 with these PDZ domain-containing proteins. Among the PDZ domain-containing proteins, the distribution of GRIP, in relation to the localization of GluR2, has been well characterized. Wyszynski et al. (1999) have shown that most of GRIP
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was localized in nonsynaptic areas and only limited amount of GRIP was colocalized with GluR2 in dendritic regions of hippocampal neurons. In the cerebral cortex, most GluR2-positive cells have been shown to exhibit only weak GRIP immunoreactivity (Burette et al., 1999). In contrast, perfect colocalization of GRIP and GluR2 was demonstrated in dendritic regions of Purkinje cells (Burette et al., 1999). These results suggest a functional implication of GluR2 – GRIP interaction in Purkinje cells. We, therefore, examined the effect of GluR2 Ser880 phosphorylation on GluR2 binding to GRIP. Our immunoprecipitation experiments (Matsuda et al., 1999) showed that the GSTGluR2 C-terminal fusion protein was efficiently coimmunoprecipitated with GluR2-binding domains of GRIP (PDZ 4-5, amino acids 419 – 673: GRIP419 – 673) (Fig. 1A). However, the binding of GluR2 carboxyl terminus to GRIP419 – 673 was almost completely inhibited when using either PKC-phosphorylated fusion protein (Fig. 1B) or mutant fusion protein whose Ser880 was replaced with alanine (Fig. 1C). Reduction in the affinity of phosphorylated GluR2 for GRIP has also been shown in transfected cells and cultured Purkinje cells stimulated by a PKC activator (Matsuda et al., 1999, 2000). Thus, phosphorylation of GluR2 Ser880 is considered to be a critical factor for the interaction of GluR2 with GRIP. Interestingly, a very recent study has shown that interaction of GluR2 with PICK1 was not affected by phosphorylation of Ser880 in GluR2 (Chung et al., 2000). Moreover, they showed that in hippocampal neurons, Ser880-phosphorylated GluR2 colocalized with PICK1 mostly in dendritic shafts and that PKC activation caused redistribution of PICK1 with Ser880phosphorylated GluR2 to dendritic spines. The physiological significance of PICK1 redistribution is still unclear. However, considering that only a small amount of GRIP is colocalized with GluR2 in hippocampal neurons (Wyszynski et al., 1999), it can be
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assumed that the interaction of GluR2 with PICK1 rather than with GRIP or the association of GluR1 with its binding proteins such as SAP97 (Leonard et al., 1999) is important for the localization and clustering of AMPA receptors at the excitatory synapses of hippocampal neurons.
3.2. PKC-phosphorylation of Ser880 in GluR2 disrupts clustering of AMPA receptors in Purkinje cells Earlier, Dong et al. (1997) showed that clusters of AMPA receptors in spinal cord neurons were extensively disrupted when GluR2 –GRIP interaction was interfered by overexpression of the C-terminal domain of GluR2. No such effects were observed with GluR2 lacking the last seven amino acids essential for GRIP binding, suggesting that interaction of the GluR2 C-terminal tail with GRIP was crucial for synaptic clustering of AMPA receptors. Therefore, it is reasonable to suppose that phosphorylation of GluR2 Ser880, which significantly decreases the affinity of GluR2 C-terminal tail for GRIP, would disrupt AMPA receptor clusters in Purkinje cells. We tested this hypothesis by immunocytochemistry using a heterologous expression system (Matsuda et al., 2000). In human embryonic kidney (HEK) 293 cells, wild-type GluR2, but not mutant GluR2 (Ser880 to aspartate), form clusters when expressed together with GRIP419 – 673 (Fig. 2A –C). The GluR2 clusters are significantly disrupted upon activation of endogenous PKC without affecting GRIP419 – 673 clusters (Fig. 2D). Loss of GluR2 clusters (Fig. 2C, D) is considered to be due to inhibition of the GluR2 – GRIP interaction by either phosphorylation of GluR2 Ser880 (Fig. 2D) or the mutation of Ser880 to aspartate, which mimics a phosphorylated residue (Fig. 2C). Subsequent experiments using cultured Purkinje cells show that PKC activation causes a significant disruption of postsynaptic AMPA receptor clusters (Fig. 2E, F), concomitant with both phosphorylation of Ser880
Fig. 1. A schematic depicting in vitro interaction of GRIP with a GST-GluR2 C-terminal fusion protein. His-tagged GRIP419 – 673 (GluR2-binding domains) is mixed with GST-fusion protein containing wild type (A, B) or mutant (Ser880Asp) (C) GluR2 C-terminus, treated (B) or not treated (A, C) with PKC. The probes are immunoprecitated with anti-His-tagged antibodies.
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and clustering of AMPA receptors containing GluR2/3 on the synaptic membrane. 4. Internalization of AMPA receptors upon phosphorylation of Ser880 in GluR2
Fig. 2. Disruption of GluR2 clusters in transfected cells and Purkinje neurons upon activation of endogenous PKC. (A–D) GluR2 (FITC labeled) is expressed alone (A) or together with GRIP419 – 673 (rhodamine labeled) (B – D) in HEK 293 cells. In (C), mutant GluR2 whose Ser880 is substituted with aspartate is used. Treatment of cells with 12-tetradecanoyl phorbol 13-acetate (TPA; 200 nM, 20 min) caused redistribution of GluR2 protein (D). (E, F) GluR2 localization on dendrites of cultured Purkinje cells in control (E) or treated with TPA (200 nM, 20 min) (F) is visualized by immunolabeling of cells with anti-GluR2 N-terminal antibodies. Activation of endogenous PKC causes significant disruption of postsynaptic GluR2 clusters. Bar, 5 mm.
in GluR2 and a reduction in the affinity of GluR2 to GRIP (Matsuda et al., 2000). An essential role of the GRIP – GluR2 interaction in stabilization of AMPA receptors on the postsynaptic membrane was also recently reported by Osten et al. (2000). Using virus-mediated expression of the GluR2 protein in hippocampal neurons, they showed that while mutant GluR2 lacking the GRIP-binding site (last 10 amino acids of GluR2 C-terminus) was efficiently targeted to synaptic sites, surface localization of mutant GluR2 was significantly reduced. In addition, similar results were obtained by expressing mutant GluR2 with Ser880 substituted with alanine, which cannot bind to GRIP. Their results are consistent with our conclusion that binding of the GluR2 C-terminal tail to GRIP plays a significant role in the stabilization
As shown in Fig. 3A, the AMPA receptor protein is targeted to and inserted into the postsynaptic membrane where the receptor is stabilized. The AMPA receptor in the postsynaptic membrane is removed by endocytosis (Carroll et al., 1999a; Luscher et al., 1999). Thus, synaptic AMPA receptors are considered to constitutively undergo recycling using exocytotic/endocytotic pathways. We, therefore, thought that removal of synaptic AMPA receptors by endocytosis might be facilitated after disruption of GluR2 –GRIP interaction. This hypothesis was verified by three different experiments (Matsuda et al., 2000). The first experiment was conducted to evaluate intradendritic GluR2 immunoreactivity in cultured Purkinje cells by confocal microscopy. We found that GluR2 immunoreactivity inside the dendrites was significantly increased upon PKC activation. In the second experiment, we measured the amount of surface-expressed GluR2 in cerebellar neuronal culture incubated with or without phorbor ester by cell-surface biotinylation. The results showed a significant reduction in the biotinylated GluR2, namely, extracellularly-expressed GluR2 by 40% of the control level upon PKC activation. The third experiment was performed in an attempt to examine if internalization of GluR2 is mediated by clathrincoated vesicles. A fraction containing endocytosed clathrin-coated vesicles was obtained by fractionation of cells (van Delft et al., 1997) from cerebellar neuronal culture with or without phorbor ester treatment. The amount of GluR2 protein in this fraction, determined by immunoblotting for GluR2, significantly increased to more than four-fold following phorbor ester treatment. These results strongly suggest that a significant amount of GluR2 protein molecules that lost their anchorage are internalized via clathrin-mediated endocytosis in Purkinje cells.
Fig. 3. A schematic showing a model of cerebellar LTD induction based on modulation of recycling of synaptic AMPA receptors. GRIP serves as a scaffold for the localization and clustering of AMPA receptors containing GluR2/3 subunits (A). PKC-phosphorylation of Ser880 in GluR2 disrupts the association of GluR2 C-terminal tail with GRIP, leading to internalization of synaptic AMPA receptors by endocytosis (B). Consequently, the number of functional AMPA receptors on the postsynaptic membrane is reduced.
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5. Disruption of synaptic AMPA receptor clusters and cerebellar LTD Activation of endogenous PKC by phorbor ester, which causes LTD of glutamate responses in cultured Purkinje cells (Linden and Connor, 1991), significantly disrupts AMPA receptor clusters at synapses (Fig. 2E, F). In addition, a similar disruption of AMPA receptor clusters in cultured Purkinje cells is induced following glutamate+KCl stimulation (Matsuda et al., 2000), a different protocol for the induction of cerebellar LTD (Kawasaki et al., 1999). These results suggest that disruption of synaptic AMPA receptor clusters is involved in the expression of cerebellar LTD. Recent studies show that synaptic plasticity is regulated by modification of AMPA receptor recycling at the steps of (1) intracellular trafficking, (2) plasma membrane expression by exocytosis, (3) stabilization on the membrane, (4) removal by endocytosis and (5) recycling of the receptor protein (Fig. 3A). For example, in hippocampal neurons, tetanic synaptic stimulation inducing LTP was shown to cause a rapid delivery of GFP-tagged GluR1 to synaptic sites (Shi et al., 1999; Hayashi et al., 2000) (promotion of steps 1 and 2), suggesting that hippocampal LTP is caused by an increase in the number of postsynaptic AMPA receptors. During the process of membrane expression of AMPA receptors involving GluR2 (step 2), interaction of an N-ethylmaleimide-sensitive fusion protein (NSF) with GluR2 is suggested to be crucial. Pharmacological disruption of the NSF interaction (disruption of step 2) causes reduction in the surface expression of AMPA receptors, concomitant with a decrease in AMPA receptor-mediated synaptic transmission (Song et al., 1998; Luscher et al., 1999; Noel et al., 1999). Moreover, expression of hippocampal LTD is shown to share with the blockade of the NSF-mediated AMPA receptor expression process (Luthi et al., 1999). On the other hand, inhibition of clathrin-dependent endocytosis (disruption of step 4) is also shown to prevent the expression of hippocampal and cerebellar LTD (Man et al., 2000; Wang and Linden, 2000). These results suggest that modification of AMPA receptor cycling at synapses regulate synaptic plasticity including LTP and LTD. From this point of view, the following model for the induction of cerebellar LTD is proposed (Fig. 3B). GRIP-mediated AMPA receptor stabilization on the postsynaptic membrane (step 3) is disrupted by PKC phosphorylation of GluR2 Ser880. Consequently, removal of the receptors by clathrin-mediated endocytosis (step 4) is facilitated, leading to LTD of the synaptic transmission because of a decreased number of functional AMPA receptors on the synaptic membrane. The regulatory mechanism of synaptic AMPA receptor clusters illustrated in Fig. 3 is considered to be
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unique to the cerebellum for the following reasons. (1) The AMPA receptor in Purkinje cells consists mostly of GRIP-binding subunits, GluR2 and GluR3 (Zhao et al., 1998), whereas those in hippocampal and cortical neurons contain other subunits of the AMPA receptor (Wenthold et al., 1992). (2) Distribution of GRIP is closely correlated with the localization of GluR2 in Purkinje cells, but not in hippocampal and cortical neurons (Wyszynski et al., 1998; Burette et al., 1999). These results suggest that mechanisms different from those in the cerebellum are involved in the postsynaptic stabilization of AMPA receptors and modification of synaptic plasticity in the hippocampus and cerebral cortex, which might include modulation of the interaction of GluR2 or GluR1 with PICK1 or SAP97, respectively, or with as yet unknown proteins. Alternatively, as mentioned in Section 2.1, modulation of AMPA receptor kinetics by phosphorylation of the GluR1 subunit of the AMPA receptor may contribute to hippocampal or cerebral synaptic plasticity. Likewise, although no data are currently available, it cannot be excluded that changes in the AMPA receptor channel kinetics upon phosphorylation of GluR2/3 contribute to expression of cerebellar LTD.
6. Concluding remarks Recent advances in molecular and biochemical approaches to the study of synaptic specialization have revealed a significant contribution of PDZ domain-containing proteins to the synaptic organization including synaptic plasticity. Taken together, our studies have provided some clues that lead to the understanding of cerebellar memory formation. However, our results were obtained using a cell culture system; it should be confirmed using in vivo preparations whether the regulation of synaptic AMPA receptor clusters in cerebellar Purkinje cells indeed underlies the expression of cerebellar LTD and the resultant motor learning. A promising approach would be to use genetically modified mice in which GluR2 –GRIP interaction is not affected by phosphorylation. Such experiments designed to establish a link between molecular and behavioral events will help elucidate further the physiological significance of cerebellar LTD in motor learning, including adaptation of vestibulo-ocular reflex (Ito, 1982) and associative eyeblink conditioning (Raymond et al., 1996).
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