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Polarized sorting of AMPA receptors to the somatodendritic domain is regulated by adaptor protein AP-4
Neuroscience Research 65 (2009) 1–5
Contents lists available at ScienceDirect
Neuroscience Research journal homepage: www.elsevier.com/locate/neures
Update article
Polarized sorting of AMPA receptors to the somatodendritic domain is regulated by adaptor protein AP-4 Shinji Matsuda *, Michisuke Yuzaki Department of Physiology, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 April 2009 Received in revised form 18 May 2009 Accepted 19 May 2009 Available online 27 May 2009
Neurons are highly polarized cells comprising somatodendritic and axonal domains. For proper neuronal function, such as neurotransmission and synaptic plasticity, membrane proteins must be transported to precise positions. a-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPA receptors) are membrane proteins localized in the somatodendritic domain. AMPA receptors mediate excitatory synaptic transmission; thus, regulation of the intracellular trafficking of AMPA receptors has a critical role in synaptic plasticity. An understanding of the molecular mechanisms that regulate AMPA receptor trafficking is essential for gaining further insight into neuronal function. Despite its importance, however, how neurons selectively transport AMPA receptors to the somatodendritic domain is largely unknown. In this Update Article, we discuss recent progress in studies of the mechanisms underlying the somatodendritic targeting of AMPA receptors in neurons. ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: AMPA receptor Glutamate receptor Stargazin Adaptor protein Membrane trafficking Polarized sorting Autophagy
1. Introduction Neurons are highly polarized cells comprising two morphologically and functionally distinct domains, the axonal and somatodendritic domains. The somatodendritic domain receives inputs from other neurons whereas the axonal domain transfers signals to other cells. The functional difference between the somatodendritic and axonal domains is determined by their resident proteins. For example, certain types of voltage-gated channels localize in the axonal domains to generate action potentials. On the other hand, certain neurotransmitter receptors localize in the somatodendritic domain to receive signals from other neurons. The a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor is an ionotropic glutamate receptor subtype that is selectively transported to the dendrites where it mediates the majority of excitatory synaptic transmission. Regulation of the number of AMPA receptors at the postsynaptic site plays a critical role in synaptic plasticity. Therefore, knowledge of the molecular mechanisms that regulate the intracellular trafficking of AMPA receptors is essential towards understanding neuronal function. How do neurons selectively transport certain kinds of membrane proteins, including AMPA receptors, to the somatoden-
dritic domain? During development, neurons extend several neurites, one of which becomes an axon whereas the others become dendrites. Once neuronal processes are destined to become axons or dendrites during development, neuronal polarity is maintained throughout adulthood by the selective sorting of membrane proteins such as ion channels, receptors, transporters, and adhesion molecules to their proper locations. Recent studies have begun to clarify the mechanisms by which neuronal polarity is established (Arimura and Kaibuchi, 2005), but the molecular mechanism that regulates polarized sorting in neurons is largely unknown. Polarized protein sorting has been studied mainly in epithelial cells. Epithelial cells have two distinct domains, the basolateral and apical domains. Interestingly, proteins sorted to the basolateral domain in epithelial cells are often (Dotti and Simons, 1990; Jareb and Banker, 1998), but not always (Silverman et al., 2005), selectively transported to the somatodendritic domain in neurons (Fig. 1). Therefore, neurons and epithelial cells may share some common polarized sorting machinery. We recently reported that an adaptor protein (AP) complex, AP-4, mediates dendritic sorting of the AMPA receptor (Matsuda et al., 2008). In this Update Article, we review AP-4-based dendriteselective sorting in neurons. 2. Tag sequences for dendrite-selective sorting
* Corresponding author. Tel.: +81 3 5363 3749; fax: +81 3 3359 0437. E-mail address: [email protected] (S. Matsuda).
Although a ‘tag’ sequence (an amino acid sequence that determines the subcellular localization of the protein) is required
0168-0102/$ – see front matter ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2009.05.007
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Fig. 1. Polarized sorting in neuronal cells and epithelial cells. Neurons and epithelial cells seem to have a common mechanism for the polarized sorting of membrane proteins. Vesicular stomatitis virus (VSV) G proteins are transported to the basolateral domains in epithelial cells and to the somatodendritic domains in neurons. Influenza hemagglutinin proteins are sorted to the apical membranes in epithelial cells and to the axonal domains in neurons (Dotti and Simons, 1990). A cell adhesion molecule, NgCAM, is transported to both the axonal (Wisco et al., 2003) and apical surfaces (Anderson et al., 2005). Epidermal growth factor (EGF) receptors (Silverman et al., 2005), transferrin (Tf) receptors, low-density lipoprotein (LDL) receptors (Jareb and Banker, 1998), and transmembrane AMPA receptor regulatory proteins (TARPs) (Bedoukian et al., 2008; Matsuda et al., 2008), are transported to the somatodendritic and basolateral domains.
for polarized sorting in neurons, the proteins that bind to these tag sequences and mediate polarized sorting are unclear. Dendriteselective sorting often requires a short stretch of amino acids, such as a dileucine, tyrosine-based motif, or phenylalanine motif within the cytosolic domain of the membrane protein that is to be sorted. For example, the dileucine motif has an essential role in the dendrite-specific sorting of Kv.4.2 K-channels and glycine transporters (GlyT1b) (Poyatos et al., 2000; Rivera et al., 2003). A tyrosine-based motif is required for dendrite targeting of the transferrin and low-density lipoprotein (LDL) receptors (Jareb and Banker, 1998). A phenylalanine-based motif transports telencephalin to the somatodendrite domain (Mitsui et al., 2005). Mutations of these motifs cause the mutant proteins to be transported to both the somatodendritic and axonal domains. Therefore, these motifs achieve dendrite-specific transport by inhibiting axonal transport rather than by actively facilitating dendritic transport. 3. AP-4-dependent polarized sorting of the AMPA receptor What kinds of proteins do the sorting motifs recognize? One candidate protein is the AP complex. Four AP complexes, AP-1, AP2, AP-3, and AP-4, control the sorting of membrane proteins in secretory and endocytic pathways (Boehm and Bonifacino, 2001). AP-1 and AP-2 are components of clathrin-coated vesicles. AP-1 is found in the trans-Golgi network (TGN) and endosomes, whereas AP-2 localizes in the plasma membrane. AP-3 and AP-4 are localized mainly in the endosomes and TGN, respectively. AP complexes are heterotetrameric proteins formed by two large subunits (a, b1, b2, b3A, b3B, b4, d, e, and g), one medium subunit (m1A, m1B, m2, m3A, m3B, or m4), and one small subunit (s1, s2, s3, or s4). Subunits b3, m1, and m3 exist in two isoforms (A and B), which are expressed in a cell-specific manner. b3B and m3B are brain-specific, and m1B is an epithelial cell-specific protein. Among the AP complexes, epithelial cell-specific AP-1B and the more ubiquitous AP-4 mediate basolateral sorting in epithelial cells (Folsch et al., 1999; Simmen et al., 2002); when AP-1B or AP-4
is disrupted, basolaterally transported proteins are distributed nonselectively to both the basolateral and apical domains. In C. elegans olfactory neurons, odorant receptor ODR-10 is selectively transported to the dendrite in an AP-1-dependent manner (Dwyer et al., 2001). The protein that mediates dendrite-specific sorting in mammalian neurons, however, has not yet been identified. We recently examined the distribution of several somatodendritic proteins, including the AMPA receptor in AP-4 b (b subunit of AP-4)-deficient (AP-4b / ) mice (Matsuda et al., 2008). AMPA receptors were distributed nonselectively to both the axonal and somatodendritic domains of cultured hippocampal neurons from AP-4b / mice, whereas their distribution was restricted to the somatodendritic domain in wild-type neurons (Fig. 2). In contrast, the NR1 subunit of N-methyl-D-aspartate (NMDA) receptors and metabotropic glutamate receptor (mGluR) 1a proteins was selectively targeted to the somatodendritic domain, even in the AP-4b / hippocampal neurons. Thus, AP-4 mediates the somatodendritic transport of AMPA receptors, but the sorting of NMDA receptors and mGluR1a is not affected by AP-4. 4. Transmembrane AMPA receptor regulatory proteins (TARPs) link AMPA receptors to AP-4 Because AP-4 is necessary for somatodendritic targeting of the AMPA receptor, it may be that AP-4 binds directly to the AMPA receptor. The m subclass of AP complexes is generally responsible for recognizing cargo proteins (Boehm and Bonifacino, 2001). Indeed, the m2 subunit of AP-2 binds to the GluR2 subunit of the AMPA receptor (Lee et al., 2002). An interaction between m4 and AMPA receptors, however, is not observed in an immunoprecipitation assay (Matsuda et al., 2008). TARP family proteins g2, g3, g4, and g8 tightly associate with all AMPA receptor subunits (Chen et al., 2000; Tomita et al., 2005; Vandenberghe et al., 2005). Interestingly, TARP links the m4 subunit to the AMPA receptor, as indicated by an immunoprecipitation assay (Fig. 3A, left panel). Moreover, when TARPs are expressed in hippocampal neurons, they are excluded from axons in wild-type neurons, whereas they
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Fig. 2. Polarized sorting of the AMPA receptor is disrupted in AP-4b deficient neurons. (A) Hemagglutinin-tagged GluR1 subunits of the AMPA receptor were expressed in cultured hippocampal neurons from wild-type or AP-4b-deficient mice. Green fluorescent protein was also expressed to visualize the overall morphology. Upper panels show the entire neuron and lower panels show the enlarged images of the axonal region (white squared region in upper panel). In wild-type neurons, GluR1 proteins were selectively sorted to the somatodendritic domain. On the other hand, GluR1 proteins were sorted to both the somatodendritic and axonal domains in AP-4b-deficient neurons. (B) Schematic drawing of the distribution of the glutamate receptor in wild-type (left) and AP-4b-deficient neurons. AMPA receptors and TARPs were mislocalized to the axonal domains in AP-4b-deficient neurons, whereas the localization of NMDA receptors and mGluR1a was restricted to the somatodendritic domains, even in AP-4bdeficient neurons.
are missorted to the axons in AP-4b / hippocampal neurons (Fig. 2B). These findings suggest that AP-4 regulates the polarized sorting of TARPs, which in turn determines the somatodendritic distribution of the AMPA receptors in neurons. A deletion and point mutation analysis indicated that the three tyrosine and phenylalanine residues and the following five serine and threonine residues of the C-terminus of TARP are
required for the interaction with m4 (Matsuda et al., 2008). Mutant TARP, in which three tyrosine and phenylalanine and five serine and threonine residues are replaced with alanine (g38A), has very weak affinity to the m4 subunit of AP-4 (Fig. 3B, left panel). Immunocytochemical analysis indicated that g38A, but not g3wt, is missorted to the axons (Fig. 3B, right panel). Moreover, the expression of g38A, but not of g3wt, causes the
Fig. 3. An interaction between TARP and AP-4 is necessary for the polarized sorting of AMPA receptor. (A) Wild-type TARP (g3wt) binds to AP-4 at the TGN and links the AMPA receptor to AP-4 (left panel). This interaction induces AP-4-dependent dendrite-specific transport of the AMPA receptor–TARP complex (right panel). (B) The mutant TARP (g38A) whose tyrosine and phenylalanine and following five serine and threonine residues are mutated to alanine, does not bind to the AP-4 (left panel). When this mutant TARP is overexpressed in neurons, the AMPA receptor–mutant TARP complex is mislocalized to the axons (right panel).
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mislocalization of endogenous AMPA receptors in axons (Fig. 3A and B, right panels). These findings support the hypothesis that TARPs mediate the polarized sorting of AMPA receptors by interacting with AP-4. It has been reported that the certain kinds of intracellular trafficking of AMPA receptors depend on receptor subtypes (Malinow and Malenka, 2002). For example, GluR1 containing AMPA receptors are delivered to the spine surface in an activitydependent manner. On the other hand, AMPA receptors composed of GluR2 and GluR3 are continuously delivered to the spine surface. Does the somatodendritic sorting of AMPA receptor depend on receptor subtype? Since the TARP binds to the all subunits of the AMPA receptor (Chen et al., 2000), AP-4 seems to interact with all subtypes of the AMPA receptors. Moreover, both of GluR1 and GluR2 are selectively transported to the somatodendritic domain and excluded from axonal domain (Matsuda et al., 2008). Therefore, the polarized sorting of AMPA receptor does not seem to be subtype specific. 5. How does AP-4 mediate polarized sorting? Polarized sorting may be achieved via a biosynthetic or endocytic pathway. In the biosynthetic pathway, proteins are sorted at the TGN and directly transported to the target domain. On the other hand, in the endocytic pathway, proteins are transported to the plasma membrane nonselectively, and then sorted at the endosome. Because AP-4 is mainly localized in the TGN and the inhibition of endocytosis does not affect the polarized sorting of AMPA receptors (Matsuda et al., 2008), it is likely that AP-4 mediates biosynthetic sorting at the TGN. If AP-4 mediates the polarized sorting at the TGN, which is localized mainly at the soma, some kinds of motor proteins are necessary for transport from the TGN to the dendritic domain. Interestingly, KIF13, one of the kinesin super family proteins, binds to AP-1 (Nakagawa et al., 2000). A dendrite-specific motor protein, KIF17, associates with the NMDA receptor subunit NR2B indirectly (Setou et al., 2000). Moreover, Glutamate Receptor Interacting Protein (GRIP), which binds to the GluR2/3 subunit of the AMPA receptor, binds to KIF5 (Setou et al., 2002). Therefore, AP-4 may also associate with certain kinds of KIF family proteins to regulate the somatodendritic targeting of membrane proteins. 6. Missorted proteins are degraded by autophagy Although AMPA receptors are mislocalized to the axons in AP4b / neurons, they do not reach the cell surface. Electron microscopic analysis revealed the autophagosome-like structure in the axons of AP-4b / mice (Matsuda et al., 2008). Moreover, in the brains of AP-4b / mice, the amount of LC3-II, an autophagy marker, is increased, and the amount of p62/SQSTM1, which is degraded by autophagy, is reduced. Therefore, proteins that are missorted to axons are somehow captured and degraded by autophagy. 7. Remaining questions Several questions remain to be addressed. For example, although AMPA receptors and TARPs are missorted to axons, mGluR1a and NMDA receptors were normally transported to the somatodendritic domain in AP-4b / neurons. What kinds of proteins regulate the transport of these receptors? In C. elegans, AP1 excludes multiple postsynaptic proteins from axons (Margeta et al., 2009). Because AP-1B is an epithelial cell-specific protein, AP-1A may mediate the polarized sorting of NMDA receptors and mGluR1a.
In certain neurons and in neurons during development, AMPA receptors are localized at the presynaptic site (Fabian-Fine et al., 2000; Lu et al., 2002; Takago et al., 2005). Because AP-4 is expressed ubiquitously, it remains unclear how AMPA receptors escape from polarized sorting in these neurons. Finally, it is also unclear why mislocalized AMPA receptors do not reach the cell surface and instead accumulate in autophagosomes at the axons in AP-4b / mice. Increased autophagy is implicated in various neuronal disorders; therefore, further studies using AP-4b / mice are warranted to gain insight into the mechanisms that regulate autophagy in axons. The answers to these questions will shed additional light on the function of AP-4 and will contribute to a more complete understanding of the mechanism underlying polarized protein sorting in neurons. Acknowledgements This work was supported by a Grant-in-Aid for Young Scientists (to S.M.), and a Grant-in-Aid for Scientific Research on Priority Areas (to M.Y.). We are grateful to Drs. M. Watanabe, K. Kohda, K. Matsuda, W. Kakegawa, and E. Miura for useful discussions. We also thank J. Motohashi and S. Narumi for their technical assistance. References Anderson, E., Maday, S., Sfakianos, J., Hull, M., Winckler, B., Sheff, D., Folsch, H., Mellman, I., 2005. Transcytosis of NgCAM in epithelial cells reflects differential signal recognition on the endocytic and secretory pathways. J. Cell Biol. 170, 595–605. Arimura, N., Kaibuchi, K., 2005. Key regulators in neuronal polarity. Neuron 48, 881– 884. Bedoukian, M.A., Whitesell, J.D., Peterson, E.J., Clay, C.M., Partin, K.M., 2008. The stargazin C terminus encodes an intrinsic and transferable membrane sorting signal. J. Biol. Chem. 283, 1597–1600. Boehm, M., Bonifacino, J.S., 2001. Adaptins: the final recount. Mol. Biol. Cell 12, 2907–2920. Chen, L., Chetkovich, D.M., Petralia, R.S., Sweeney, N.T., Kawasaki, Y., Wenthold, R.J., Bredt, D.S., Nicoll, R.A., 2000. Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408, 936–943. Dotti, C.G., Simons, K., 1990. Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture. Cell 62, 63–72. Dwyer, N.D., Adler, C.E., Crump, J.G., L’Etoile, N.D., Bargmann, C.I., 2001. Polarized dendritic transport and the AP-1 mu1 clathrin adaptor UNC-101 localize odorant receptors to olfactory cilia. Neuron 31, 277–287. Fabian-Fine, R., Volknandt, W., Fine, A., Stewart, M.G., 2000. Age-dependent preand postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain. Eur. J. Neurosci. 12, 3687–3700. Folsch, H., Ohno, H., Bonifacino, J.S., Mellman, I., 1999. A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell 99, 189–198. Jareb, M., Banker, G., 1998. The polarized sorting of membrane proteins expressed in cultured hippocampal neurons using viral vectors. Neuron 20, 855–867. Lee, S.H., Liu, L., Wang, Y.T., Sheng, M., 2002. Clathrin adaptor AP2 and NSF interact with overlapping sites of GluR2 and play distinct roles in AMPA receptor trafficking and hippocampal LTD. Neuron 36, 661–674. Lu, C.R., Hwang, S.J., Phend, K.D., Rustioni, A., Valtschanoff, J.G., 2002. Primary afferent terminals in spinal cord express presynaptic AMPA receptors. J. Neurosci. 22, 9522–9529. Malinow, R., Malenka, R.C., 2002. AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 25, 103–126. Margeta, M.A., Wang, G.J., Shen, K., 2009. Clathrin adaptor AP-1 complex excludes multiple postsynaptic receptors from axons in C. elegans. Proc. Natl. Acad. Sci. U.S.A. 106, 1632–1637. Matsuda, S., Miura, E., Matsuda, K., Kakegawa, W., Kohda, K., Watanabe, M., Yuzaki, M., 2008. Accumulation of AMPA receptors in autophagosomes in neuronal axons lacking adaptor protein AP-4. Neuron 57, 730–745. Mitsui, S., Saito, M., Hayashi, K., Mori, K., Yoshihara, Y., 2005. A novel phenylalaninebased targeting signal directs telencephalin to neuronal dendrites. J. Neurosci. 25, 1122–1131. Nakagawa, T., Setou, M., Seog, D., Ogasawara, K., Dohmae, N., Takio, K., Hirokawa, N., 2000. A novel motor, KIF13A, transports mannose-6-phosphate receptor to plasma membrane through direct interaction with AP-1 complex. Cell 103, 569–581.
S. Matsuda, M. Yuzaki / Neuroscience Research 65 (2009) 1–5 Poyatos, I., Ruberti, F., Martinez-Maza, R., Gimenez, C., Dotti, C.G., Zafra, F., 2000. Polarized distribution of glycine transporter isoforms in epithelial and neuronal cells. Mol. Cell. Neurosci. 15, 99–111. Rivera, J.F., Ahmad, S., Quick, M.W., Liman, E.R., Arnold, D.B., 2003. An evolutionarily conserved dileucine motif in Shal K+ channels mediates dendritic targeting. Nat. Neurosci. 6, 243–250. Setou, M., Nakagawa, T., Seog, D.H., Hirokawa, N., 2000. Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport. Science 288, 1796–1802. Setou, M., Seog, D.H., Tanaka, Y., Kanai, Y., Takei, Y., Kawagishi, M., Hirokawa, N., 2002. Glutamate-receptor-interacting protein GRIP1 directly steers kinesin to dendrites. Nature 417, 83–87. Silverman, M.A., Peck, R., Glover, G., He, C., Carlin, C., Banker, G., 2005. Motifs that mediate dendritic targeting in hippocampal neurons: a comparison with basolateral targeting signals. Mol. Cell. Neurosci. 29, 173–180.
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Simmen, T., Honing, S., Icking, A., Tikkanen, R., Hunziker, W., 2002. AP-4 binds basolateral signals and participates in basolateral sorting in epithelial MDCK cells. Nat. Cell. Biol. 4, 154–159. Takago, H., Nakamura, Y., Takahashi, T., 2005. G protein-dependent presynaptic inhibition mediated by AMPA receptors at the calyx of Held. Proc. Natl. Acad. Sci. U.S.A. 102, 7368–7373. Tomita, S., Adesnik, H., Sekiguchi, M., Zhang, W., Wada, K., Howe, J.R., Nicoll, R.A., Bredt, D.S., 2005. Stargazin modulates AMPA receptor gating and trafficking by distinct domains. Nature 435, 1052–1058. Vandenberghe, W., Nicoll, R.A., Bredt, D.S., 2005. Stargazin is an AMPA receptor auxiliary subunit. Proc. Natl. Acad. Sci. U.S.A. 102, 485–490. Wisco, D., Anderson, E.D., Chang, M.C., Norden, C., Boiko, T., Folsch, H., Winckler, B., 2003. Uncovering multiple axonal targeting pathways in hippocampal neurons. J. Cell Biol. 162, 1317–1328.
Contents lists available at ScienceDirect
Neuroscience Research journal homepage: www.elsevier.com/locate/neures
Update article
Polarized sorting of AMPA receptors to the somatodendritic domain is regulated by adaptor protein AP-4 Shinji Matsuda *, Michisuke Yuzaki Department of Physiology, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 April 2009 Received in revised form 18 May 2009 Accepted 19 May 2009 Available online 27 May 2009
Neurons are highly polarized cells comprising somatodendritic and axonal domains. For proper neuronal function, such as neurotransmission and synaptic plasticity, membrane proteins must be transported to precise positions. a-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPA receptors) are membrane proteins localized in the somatodendritic domain. AMPA receptors mediate excitatory synaptic transmission; thus, regulation of the intracellular trafficking of AMPA receptors has a critical role in synaptic plasticity. An understanding of the molecular mechanisms that regulate AMPA receptor trafficking is essential for gaining further insight into neuronal function. Despite its importance, however, how neurons selectively transport AMPA receptors to the somatodendritic domain is largely unknown. In this Update Article, we discuss recent progress in studies of the mechanisms underlying the somatodendritic targeting of AMPA receptors in neurons. ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: AMPA receptor Glutamate receptor Stargazin Adaptor protein Membrane trafficking Polarized sorting Autophagy
1. Introduction Neurons are highly polarized cells comprising two morphologically and functionally distinct domains, the axonal and somatodendritic domains. The somatodendritic domain receives inputs from other neurons whereas the axonal domain transfers signals to other cells. The functional difference between the somatodendritic and axonal domains is determined by their resident proteins. For example, certain types of voltage-gated channels localize in the axonal domains to generate action potentials. On the other hand, certain neurotransmitter receptors localize in the somatodendritic domain to receive signals from other neurons. The a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor is an ionotropic glutamate receptor subtype that is selectively transported to the dendrites where it mediates the majority of excitatory synaptic transmission. Regulation of the number of AMPA receptors at the postsynaptic site plays a critical role in synaptic plasticity. Therefore, knowledge of the molecular mechanisms that regulate the intracellular trafficking of AMPA receptors is essential towards understanding neuronal function. How do neurons selectively transport certain kinds of membrane proteins, including AMPA receptors, to the somatoden-
dritic domain? During development, neurons extend several neurites, one of which becomes an axon whereas the others become dendrites. Once neuronal processes are destined to become axons or dendrites during development, neuronal polarity is maintained throughout adulthood by the selective sorting of membrane proteins such as ion channels, receptors, transporters, and adhesion molecules to their proper locations. Recent studies have begun to clarify the mechanisms by which neuronal polarity is established (Arimura and Kaibuchi, 2005), but the molecular mechanism that regulates polarized sorting in neurons is largely unknown. Polarized protein sorting has been studied mainly in epithelial cells. Epithelial cells have two distinct domains, the basolateral and apical domains. Interestingly, proteins sorted to the basolateral domain in epithelial cells are often (Dotti and Simons, 1990; Jareb and Banker, 1998), but not always (Silverman et al., 2005), selectively transported to the somatodendritic domain in neurons (Fig. 1). Therefore, neurons and epithelial cells may share some common polarized sorting machinery. We recently reported that an adaptor protein (AP) complex, AP-4, mediates dendritic sorting of the AMPA receptor (Matsuda et al., 2008). In this Update Article, we review AP-4-based dendriteselective sorting in neurons. 2. Tag sequences for dendrite-selective sorting
* Corresponding author. Tel.: +81 3 5363 3749; fax: +81 3 3359 0437. E-mail address: [email protected] (S. Matsuda).
Although a ‘tag’ sequence (an amino acid sequence that determines the subcellular localization of the protein) is required
0168-0102/$ – see front matter ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2009.05.007
2
S. Matsuda, M. Yuzaki / Neuroscience Research 65 (2009) 1–5
Fig. 1. Polarized sorting in neuronal cells and epithelial cells. Neurons and epithelial cells seem to have a common mechanism for the polarized sorting of membrane proteins. Vesicular stomatitis virus (VSV) G proteins are transported to the basolateral domains in epithelial cells and to the somatodendritic domains in neurons. Influenza hemagglutinin proteins are sorted to the apical membranes in epithelial cells and to the axonal domains in neurons (Dotti and Simons, 1990). A cell adhesion molecule, NgCAM, is transported to both the axonal (Wisco et al., 2003) and apical surfaces (Anderson et al., 2005). Epidermal growth factor (EGF) receptors (Silverman et al., 2005), transferrin (Tf) receptors, low-density lipoprotein (LDL) receptors (Jareb and Banker, 1998), and transmembrane AMPA receptor regulatory proteins (TARPs) (Bedoukian et al., 2008; Matsuda et al., 2008), are transported to the somatodendritic and basolateral domains.
for polarized sorting in neurons, the proteins that bind to these tag sequences and mediate polarized sorting are unclear. Dendriteselective sorting often requires a short stretch of amino acids, such as a dileucine, tyrosine-based motif, or phenylalanine motif within the cytosolic domain of the membrane protein that is to be sorted. For example, the dileucine motif has an essential role in the dendrite-specific sorting of Kv.4.2 K-channels and glycine transporters (GlyT1b) (Poyatos et al., 2000; Rivera et al., 2003). A tyrosine-based motif is required for dendrite targeting of the transferrin and low-density lipoprotein (LDL) receptors (Jareb and Banker, 1998). A phenylalanine-based motif transports telencephalin to the somatodendrite domain (Mitsui et al., 2005). Mutations of these motifs cause the mutant proteins to be transported to both the somatodendritic and axonal domains. Therefore, these motifs achieve dendrite-specific transport by inhibiting axonal transport rather than by actively facilitating dendritic transport. 3. AP-4-dependent polarized sorting of the AMPA receptor What kinds of proteins do the sorting motifs recognize? One candidate protein is the AP complex. Four AP complexes, AP-1, AP2, AP-3, and AP-4, control the sorting of membrane proteins in secretory and endocytic pathways (Boehm and Bonifacino, 2001). AP-1 and AP-2 are components of clathrin-coated vesicles. AP-1 is found in the trans-Golgi network (TGN) and endosomes, whereas AP-2 localizes in the plasma membrane. AP-3 and AP-4 are localized mainly in the endosomes and TGN, respectively. AP complexes are heterotetrameric proteins formed by two large subunits (a, b1, b2, b3A, b3B, b4, d, e, and g), one medium subunit (m1A, m1B, m2, m3A, m3B, or m4), and one small subunit (s1, s2, s3, or s4). Subunits b3, m1, and m3 exist in two isoforms (A and B), which are expressed in a cell-specific manner. b3B and m3B are brain-specific, and m1B is an epithelial cell-specific protein. Among the AP complexes, epithelial cell-specific AP-1B and the more ubiquitous AP-4 mediate basolateral sorting in epithelial cells (Folsch et al., 1999; Simmen et al., 2002); when AP-1B or AP-4
is disrupted, basolaterally transported proteins are distributed nonselectively to both the basolateral and apical domains. In C. elegans olfactory neurons, odorant receptor ODR-10 is selectively transported to the dendrite in an AP-1-dependent manner (Dwyer et al., 2001). The protein that mediates dendrite-specific sorting in mammalian neurons, however, has not yet been identified. We recently examined the distribution of several somatodendritic proteins, including the AMPA receptor in AP-4 b (b subunit of AP-4)-deficient (AP-4b / ) mice (Matsuda et al., 2008). AMPA receptors were distributed nonselectively to both the axonal and somatodendritic domains of cultured hippocampal neurons from AP-4b / mice, whereas their distribution was restricted to the somatodendritic domain in wild-type neurons (Fig. 2). In contrast, the NR1 subunit of N-methyl-D-aspartate (NMDA) receptors and metabotropic glutamate receptor (mGluR) 1a proteins was selectively targeted to the somatodendritic domain, even in the AP-4b / hippocampal neurons. Thus, AP-4 mediates the somatodendritic transport of AMPA receptors, but the sorting of NMDA receptors and mGluR1a is not affected by AP-4. 4. Transmembrane AMPA receptor regulatory proteins (TARPs) link AMPA receptors to AP-4 Because AP-4 is necessary for somatodendritic targeting of the AMPA receptor, it may be that AP-4 binds directly to the AMPA receptor. The m subclass of AP complexes is generally responsible for recognizing cargo proteins (Boehm and Bonifacino, 2001). Indeed, the m2 subunit of AP-2 binds to the GluR2 subunit of the AMPA receptor (Lee et al., 2002). An interaction between m4 and AMPA receptors, however, is not observed in an immunoprecipitation assay (Matsuda et al., 2008). TARP family proteins g2, g3, g4, and g8 tightly associate with all AMPA receptor subunits (Chen et al., 2000; Tomita et al., 2005; Vandenberghe et al., 2005). Interestingly, TARP links the m4 subunit to the AMPA receptor, as indicated by an immunoprecipitation assay (Fig. 3A, left panel). Moreover, when TARPs are expressed in hippocampal neurons, they are excluded from axons in wild-type neurons, whereas they
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Fig. 2. Polarized sorting of the AMPA receptor is disrupted in AP-4b deficient neurons. (A) Hemagglutinin-tagged GluR1 subunits of the AMPA receptor were expressed in cultured hippocampal neurons from wild-type or AP-4b-deficient mice. Green fluorescent protein was also expressed to visualize the overall morphology. Upper panels show the entire neuron and lower panels show the enlarged images of the axonal region (white squared region in upper panel). In wild-type neurons, GluR1 proteins were selectively sorted to the somatodendritic domain. On the other hand, GluR1 proteins were sorted to both the somatodendritic and axonal domains in AP-4b-deficient neurons. (B) Schematic drawing of the distribution of the glutamate receptor in wild-type (left) and AP-4b-deficient neurons. AMPA receptors and TARPs were mislocalized to the axonal domains in AP-4b-deficient neurons, whereas the localization of NMDA receptors and mGluR1a was restricted to the somatodendritic domains, even in AP-4bdeficient neurons.
are missorted to the axons in AP-4b / hippocampal neurons (Fig. 2B). These findings suggest that AP-4 regulates the polarized sorting of TARPs, which in turn determines the somatodendritic distribution of the AMPA receptors in neurons. A deletion and point mutation analysis indicated that the three tyrosine and phenylalanine residues and the following five serine and threonine residues of the C-terminus of TARP are
required for the interaction with m4 (Matsuda et al., 2008). Mutant TARP, in which three tyrosine and phenylalanine and five serine and threonine residues are replaced with alanine (g38A), has very weak affinity to the m4 subunit of AP-4 (Fig. 3B, left panel). Immunocytochemical analysis indicated that g38A, but not g3wt, is missorted to the axons (Fig. 3B, right panel). Moreover, the expression of g38A, but not of g3wt, causes the
Fig. 3. An interaction between TARP and AP-4 is necessary for the polarized sorting of AMPA receptor. (A) Wild-type TARP (g3wt) binds to AP-4 at the TGN and links the AMPA receptor to AP-4 (left panel). This interaction induces AP-4-dependent dendrite-specific transport of the AMPA receptor–TARP complex (right panel). (B) The mutant TARP (g38A) whose tyrosine and phenylalanine and following five serine and threonine residues are mutated to alanine, does not bind to the AP-4 (left panel). When this mutant TARP is overexpressed in neurons, the AMPA receptor–mutant TARP complex is mislocalized to the axons (right panel).
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mislocalization of endogenous AMPA receptors in axons (Fig. 3A and B, right panels). These findings support the hypothesis that TARPs mediate the polarized sorting of AMPA receptors by interacting with AP-4. It has been reported that the certain kinds of intracellular trafficking of AMPA receptors depend on receptor subtypes (Malinow and Malenka, 2002). For example, GluR1 containing AMPA receptors are delivered to the spine surface in an activitydependent manner. On the other hand, AMPA receptors composed of GluR2 and GluR3 are continuously delivered to the spine surface. Does the somatodendritic sorting of AMPA receptor depend on receptor subtype? Since the TARP binds to the all subunits of the AMPA receptor (Chen et al., 2000), AP-4 seems to interact with all subtypes of the AMPA receptors. Moreover, both of GluR1 and GluR2 are selectively transported to the somatodendritic domain and excluded from axonal domain (Matsuda et al., 2008). Therefore, the polarized sorting of AMPA receptor does not seem to be subtype specific. 5. How does AP-4 mediate polarized sorting? Polarized sorting may be achieved via a biosynthetic or endocytic pathway. In the biosynthetic pathway, proteins are sorted at the TGN and directly transported to the target domain. On the other hand, in the endocytic pathway, proteins are transported to the plasma membrane nonselectively, and then sorted at the endosome. Because AP-4 is mainly localized in the TGN and the inhibition of endocytosis does not affect the polarized sorting of AMPA receptors (Matsuda et al., 2008), it is likely that AP-4 mediates biosynthetic sorting at the TGN. If AP-4 mediates the polarized sorting at the TGN, which is localized mainly at the soma, some kinds of motor proteins are necessary for transport from the TGN to the dendritic domain. Interestingly, KIF13, one of the kinesin super family proteins, binds to AP-1 (Nakagawa et al., 2000). A dendrite-specific motor protein, KIF17, associates with the NMDA receptor subunit NR2B indirectly (Setou et al., 2000). Moreover, Glutamate Receptor Interacting Protein (GRIP), which binds to the GluR2/3 subunit of the AMPA receptor, binds to KIF5 (Setou et al., 2002). Therefore, AP-4 may also associate with certain kinds of KIF family proteins to regulate the somatodendritic targeting of membrane proteins. 6. Missorted proteins are degraded by autophagy Although AMPA receptors are mislocalized to the axons in AP4b / neurons, they do not reach the cell surface. Electron microscopic analysis revealed the autophagosome-like structure in the axons of AP-4b / mice (Matsuda et al., 2008). Moreover, in the brains of AP-4b / mice, the amount of LC3-II, an autophagy marker, is increased, and the amount of p62/SQSTM1, which is degraded by autophagy, is reduced. Therefore, proteins that are missorted to axons are somehow captured and degraded by autophagy. 7. Remaining questions Several questions remain to be addressed. For example, although AMPA receptors and TARPs are missorted to axons, mGluR1a and NMDA receptors were normally transported to the somatodendritic domain in AP-4b / neurons. What kinds of proteins regulate the transport of these receptors? In C. elegans, AP1 excludes multiple postsynaptic proteins from axons (Margeta et al., 2009). Because AP-1B is an epithelial cell-specific protein, AP-1A may mediate the polarized sorting of NMDA receptors and mGluR1a.
In certain neurons and in neurons during development, AMPA receptors are localized at the presynaptic site (Fabian-Fine et al., 2000; Lu et al., 2002; Takago et al., 2005). Because AP-4 is expressed ubiquitously, it remains unclear how AMPA receptors escape from polarized sorting in these neurons. Finally, it is also unclear why mislocalized AMPA receptors do not reach the cell surface and instead accumulate in autophagosomes at the axons in AP-4b / mice. Increased autophagy is implicated in various neuronal disorders; therefore, further studies using AP-4b / mice are warranted to gain insight into the mechanisms that regulate autophagy in axons. The answers to these questions will shed additional light on the function of AP-4 and will contribute to a more complete understanding of the mechanism underlying polarized protein sorting in neurons. Acknowledgements This work was supported by a Grant-in-Aid for Young Scientists (to S.M.), and a Grant-in-Aid for Scientific Research on Priority Areas (to M.Y.). We are grateful to Drs. M. Watanabe, K. Kohda, K. Matsuda, W. Kakegawa, and E. Miura for useful discussions. We also thank J. Motohashi and S. Narumi for their technical assistance. References Anderson, E., Maday, S., Sfakianos, J., Hull, M., Winckler, B., Sheff, D., Folsch, H., Mellman, I., 2005. Transcytosis of NgCAM in epithelial cells reflects differential signal recognition on the endocytic and secretory pathways. J. Cell Biol. 170, 595–605. Arimura, N., Kaibuchi, K., 2005. Key regulators in neuronal polarity. Neuron 48, 881– 884. Bedoukian, M.A., Whitesell, J.D., Peterson, E.J., Clay, C.M., Partin, K.M., 2008. The stargazin C terminus encodes an intrinsic and transferable membrane sorting signal. J. Biol. Chem. 283, 1597–1600. Boehm, M., Bonifacino, J.S., 2001. Adaptins: the final recount. Mol. Biol. Cell 12, 2907–2920. Chen, L., Chetkovich, D.M., Petralia, R.S., Sweeney, N.T., Kawasaki, Y., Wenthold, R.J., Bredt, D.S., Nicoll, R.A., 2000. Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408, 936–943. Dotti, C.G., Simons, K., 1990. Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture. Cell 62, 63–72. Dwyer, N.D., Adler, C.E., Crump, J.G., L’Etoile, N.D., Bargmann, C.I., 2001. Polarized dendritic transport and the AP-1 mu1 clathrin adaptor UNC-101 localize odorant receptors to olfactory cilia. Neuron 31, 277–287. Fabian-Fine, R., Volknandt, W., Fine, A., Stewart, M.G., 2000. Age-dependent preand postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain. Eur. J. Neurosci. 12, 3687–3700. Folsch, H., Ohno, H., Bonifacino, J.S., Mellman, I., 1999. A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell 99, 189–198. Jareb, M., Banker, G., 1998. The polarized sorting of membrane proteins expressed in cultured hippocampal neurons using viral vectors. Neuron 20, 855–867. Lee, S.H., Liu, L., Wang, Y.T., Sheng, M., 2002. Clathrin adaptor AP2 and NSF interact with overlapping sites of GluR2 and play distinct roles in AMPA receptor trafficking and hippocampal LTD. Neuron 36, 661–674. Lu, C.R., Hwang, S.J., Phend, K.D., Rustioni, A., Valtschanoff, J.G., 2002. Primary afferent terminals in spinal cord express presynaptic AMPA receptors. J. Neurosci. 22, 9522–9529. Malinow, R., Malenka, R.C., 2002. AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 25, 103–126. Margeta, M.A., Wang, G.J., Shen, K., 2009. Clathrin adaptor AP-1 complex excludes multiple postsynaptic receptors from axons in C. elegans. Proc. Natl. Acad. Sci. U.S.A. 106, 1632–1637. Matsuda, S., Miura, E., Matsuda, K., Kakegawa, W., Kohda, K., Watanabe, M., Yuzaki, M., 2008. Accumulation of AMPA receptors in autophagosomes in neuronal axons lacking adaptor protein AP-4. Neuron 57, 730–745. Mitsui, S., Saito, M., Hayashi, K., Mori, K., Yoshihara, Y., 2005. A novel phenylalaninebased targeting signal directs telencephalin to neuronal dendrites. J. Neurosci. 25, 1122–1131. Nakagawa, T., Setou, M., Seog, D., Ogasawara, K., Dohmae, N., Takio, K., Hirokawa, N., 2000. A novel motor, KIF13A, transports mannose-6-phosphate receptor to plasma membrane through direct interaction with AP-1 complex. Cell 103, 569–581.
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