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Early events in glutamate receptor trafficking
Early events in glutamate receptor trafficking Wim Vandenberghe1 and David S Bredt2 Glutamate receptors are the primary mediators of excitatory synaptic transmission in the mammalian central nervous system. Activity-dependent changes in the number of postsynaptic glutamate receptors underlie aspects of synaptic plasticity and provide a mechanism for information storage in the brain. Recent work shows that receptor exit from the endoplasmic reticulum represents a critical regulatory step in glutamate receptor trafficking to the neuronal cell surface. Addresses 1 Department of Physiology, University of California at San Francisco, Genentech Hall N274, 600 16th Street, San Francisco, CA 94143, USA e-mail: [email protected] 2 Department of Physiology, University of California at San Francisco, Genentech Hall N272F, 600 16th Street, San Francisco, CA 94143, USA
Current Opinion in Cell Biology 2004, 16:134–139 This review comes from a themed issue on Cell regulation Edited by Craig Montell and Peter Devreotes 0955-0674/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved.
that contain both NR1 and NR2 (NR2A–D) subunits, and in some cases NR3 subunits. Among the mGluR subunits, mGluR1 and mGluR5 are predominant at postsynaptic sites, and these receptors probably exist on the cell surface as homodimers. Metabotropic GluR subunits are seventransmembrane proteins, whereas iGluR subunits contain three transmembrane domains and a pore-lining, reentrant membrane loop (Figure 1). All GluR subunits contain an extracellular N-terminal domain and a cytoplasmic C terminus. GluRs are highly concentrated at postsynaptic sites, where they interact with a host of structural and signaling proteins collectively referred to as the postsynaptic density (PSD). Tremendous progress has been made in dissecting the molecular anatomy of the PSD [5]. In addition, it has become clear that changing the numbers of postsynaptic glutamate receptors, especially AMPA receptors, is a key mechanism of activity-dependent modulation of synaptic strength, a phenomenon thought to underlie aspects of learning and memory [6–8]. Accordingly, GluR trafficking at the postsynaptic membrane has been intensively studied.
DOI 10.1016/j.ceb.2004.01.003
Abbreviations AMPA a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid COPII coat protein complex II ER endoplasmic reticulum ERAD ER-associated degradation ERGIC ER–Golgi intermediate compartment GABA g-amino butyric acid GluR glutamate receptor iGluR ionotropic glutamate receptor mGluR metabotropic glutamate receptor NMDA N-methyl-D-aspartic acid PDZ PSD-95/Dlg/ZO-1 domain PKC protein kinase C PSD postsynaptic density QC quality control
Introduction Glutamate, the principal excitatory neurotransmitter in the mammalian central nervous system, activates two types of postsynaptic receptors: ionotropic receptors (iGluRs), which are glutamate-gated cation channels, and metabotropic receptors (mGluRs), which are linked through Gproteins to second messenger systems [1–4]. Ionotropic GluRs can be subdivided into three families on the basis of pharmacology, electrophysiology and sequence homology: AMPA receptors, which are homo- or hetero-tetramers composed of the subunits GluR1–4; kainate receptors, homo- or hetero-tetramers of the subunits GluR5–7, KA1 and KA2; and NMDA receptors, hetero-tetramers Current Opinion in Cell Biology 2004, 16:134–139
Classical studies in non-neuronal cells have shown that exit from the endoplasmic reticulum (ER) is often the most stringently controlled step in integral membrane protein transport to the cell surface [9]. Similarly, recent work reveals that GluR trafficking through this early compartment of the secretory pathway is tightly regulated and that mechanisms controlling ER exit have a major impact on synaptic GluR abundance. This review highlights these recent discoveries of rules governing ER exit of GluRs (Figure 2).
ER quality control of GluRs Like other multimeric cell membrane proteins, GluRs are synthesized, folded and assembled in the ER. For large multi-subunit membrane proteins such as ion channels, folding of individual subunits often continues throughout formation of the multimeric protein complex [10]. Nglycosylation and disulphide bond formation, which often promote proper protein folding [11,12], also occur in the ER. All GluR subunits possess N-glycosylation sites [2,4], and iGluR [13] and mGluR [4] subunits contain intraand intermolecular disulphide bridges, respectively, but whether these modifications play central roles in GluR folding and trafficking to the cell surface is not fully understood [4,14,15]. The ER uses a rigorous quality control (QC) system to ensure that only correctly folded and assembled proteins www.sciencedirect.com
Early events in glutamate receptor trafficking Vandenberghe and Bredt 135
Figure 1
iGluR subunit (a)
evidence suggests that these signals mediate cargo retrieval from the ER–Golgi intermediate compartment (ERGIC) or from the Golgi to the ER by direct or indirect interactions with coat protein complex I (COPI), a set of cytosolic proteins involved in retrograde vesicle transport through the secretory pathway.
mGluR subunit
(b) (c) (d) (e) Current Opinion in Cell Biology
Membrane topology and positions of ER export and retention signals in GluR subunits (see also Table 1). Green and red stars represent ER export and retention/retrieval signals, respectively, as found in (a) GluR1, (b) GluR2, (c) KA2, (d) NR1 and (e) mGluR1.
exit to the Golgi [9]. Endoplasmic reticulum QC operates both at a general level (primary QC) and at a substrateselective level (secondary QC) [9]. Primary QC mechanisms accurately discriminate between native and nonnative structures in a wide variety of unrelated nascent polypeptides. Prominent components of primary QC include the ER chaperones BiP and calnexin, which both assist the folding process and serve as retention anchors for unfolded proteins. The secondary QC machinery comprises a wide array of specialized factors required for efficient ER export of selected protein species. An example of this rapidly expanding group is Boca/MESD, a chaperone specific for certain members of the lowdensity-lipoprotein receptor family [16,17]. Persistently misfolded proteins are retrotranslocated into the cytosol, polyubiquitinated and degraded by proteasomes in a process called ER-associated degredation (ERAD) [18]. Very few specific data are available about QC of GluRs. AMPA receptors in brain are substrates of primary QC, as demonstrated by their co-immunoprecipitation with BiP and calnexin [19]; however, no GluR-specific chaperones have been identified. Serrando and colleagues serendipitously found cytosolic inclusion bodies containing GluR1, ubiquitin and proteasomes in a subset of spinal interneurons during a specific developmental time window [20]. These inclusions may represent misfolded, retrotranslocated GluR1. This raises the possibility of a developmental mismatch between GluR1 synthesis and the capacity for folding support and/or proteasomal degradation. Ubiquitination of GLR-1, an AMPA receptor homologue of C. elegans, occurs in vivo and apparently regulates receptor endocytosis [21]. Ubiquitination of mammalian GluRs has not been reported.
ER retention/retrieval signals in GluRs ER retrieval signals, such as the KDEL and KKXX motifs, help anchor ER resident proteins [9,22]. Current www.sciencedirect.com
ER retention/retrieval signals are also present in some proteins destined for transport to the cell surface. The best known of these signals is the RXR motif (where X is any amino acid except an acidic one) first described in the cytosolic domains of subunits of ATP-sensitive potassium channels (KATP channels) and GABAB receptors [22]. This arginine-rich motif helps retain unassembled subunits and is fully masked only after complete heteromeric assembly. This provides a QC mechanism for ensuring correct subunit association and stoichiometry. The anchor that binds to the exposed RXR signal has not been identified. Arginine-based ER retention/retrieval signals have been identified in members of each GluR subfamily (Figure 1, Table 1). To form a functional NMDA receptor, the NR1 subunit assembles with one of the four NR2 subunits [23]. NR2 subunits expressed alone are retained in the ER and fail to reach the cell surface [24,25]. Co-expression of NR1 results in robust surface expression of the heteromeric complex [24]. Still unknown is whether an ER retention signal prevents export of unassembled NR2 subunits. For NR1 subunits expressed alone, the extent of surface expression strongly depends on the splice variant [26–28]. There are eight NR1 splice variants, including four variants of the C-terminal cytoplasmic tail (NR1-1, NR1-2, NR1-3 and NR1-4). When expressed without NR2, all isoforms except NR1-1 are able to reach the cell surface [26–28]. This is explained by the presence of an ER retention/retrieval signal (RRR) in the cytoplasmic tail of NR1-1 [27–29]. The same signal also occurs in the cytoplasmic tail of NR1-3 splice variants, but ER retention of NR1-3 is overcome by the binding of PDZ proteins to a PDZ-binding site present in NR1-3 but absent in NR1-1 [27–29]. In a manner analogous to the shielding of the RXR motif of KATP channels and GABAB receptors by heteromeric assembly [22], association with NR2 apparently releases ER retention of NR1-1 by masking the RRR motif, but the molecular details of this masking process are not understood [23]. A related ER retention/retrieval motif was recently reported in the kainate receptor subunit KA2 [30]. KA2 forms functional receptors only when co-expressed with GluR5, GluR6 or GluR7, thereby producing channels with properties different from GluR5-7 homomers. Homomeric KA2 complexes fail to reach the plasma membrane owing to an ER retention/retrieval motif that Current Opinion in Cell Biology 2004, 16:134–139
136 Cell regulation
Figure 2
Synapse
Golgi
?
COPII vesicle
ER export of correctly folded and assembled GluRs ERAD
ER lumen
Elimination
ER retention of unfolded and unassembled GluRs Cytosol
GluR subunit Current Opinion in Cell Biology
ER exit is a critical, highly regulated step in the trafficking of GluRs to the synapse. Correctly folded and assembled GluRs are exported to the Golgi, whereas the QC machinery retains misfolded or misassembled GluRs. Persistently misfolded or misassembled GluRs are probably eliminated by ERAD. Whether GluRs can also be retrieved to the ER from post-ER compartments is not known. For simplicity, fully assembled GluRs are depicted here as tetramers.
consists of a string of five arginine residues in its cytoplasmic tail [30]. Although the precise molecular mechanism remains unclear, heteromeric assembly with GluR6 silences this signal.
The surface expression of the mGluR1b splice variant is also limited by an ER retention/retrieval signal (RRKK) in its C-terminal cytoplasmic domain [31]. The same motif is present in mGluR1a, but another C-terminal
Table 1 Published ER retention/retrieval and export signals in GluR subunits. GluR subfamily
Subunit
Sequence
Orientation
Effect
References
NMDA receptor AMPA receptor
NR1 (isoforms NR1-1 and 1-3) GluR1 GluR2 KA2 mGluR1
RRR893–895 IQI25–27 R607 RRRRR826–866 RRKK877–880
Cytosolic Luminal Membrane Cytosolic Cytosolic
Retention/retrieval Export Retention Retention/retrieval Retention/retrieval
[27–29] [37] [32] [30] [31]
Kainate receptor mGluR
The numbers next to the sequences refer to the amino acid positions in rat, with number 1 corresponding to the first residue of the signal peptide. For NR1, numbering is based on NR1-1a.
Current Opinion in Cell Biology 2004, 16:134–139
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Early events in glutamate receptor trafficking Vandenberghe and Bredt 137
sequence specific to mGluR1a somehow overrides this effect [31]. Finally, the AMPA receptor subunit GluR2 contains a distinct arginine-based ER retention motif that consists of just a single arginine residue (R607) in a transmembrane domain [32]. A positively charged residue in a membrane segment of T-cell receptor subunits also mediates ER retention [33]. In the case of AMPA receptors, the critical arginine (R607 in GluR2) is generated by mRNA editing [34], and resides in a re-entrant membrane loop that lines the pore of fully assembled AMPA receptor channels. This arginine critically controls calcium permeability and other channel properties [2,34]. The additional role for R607 in ER retention presumably results from its interaction with an unidentified ER anchor. As GluR2 is a component of functional AMPA receptors in many neuronal cell types [35], a highly efficient, as yet unknown mechanism must exist to selectively suppress the ER retention effect of R607 while preserving its influence on channel properties such as calcium permeability. It is possible that assembly of a tetrameric AMPA receptor channel shields R607 from an ER anchor outside the receptor complex.
ER export signals in GluRs Vesicles export cargo from the ER to the Golgi. Budding of export vesicles is driven by interactions of ER membranes with coat protein complex II (COPII), a polymer formed by ordered assembly of cytosolic subunits [36]. According to the bulk-flow model, a default pathway packages properly folded and assembled membrane proteins into COPII-coated vesicles at their prevailing concentration in the ER. On the other hand, some membrane proteins contain specific export signals that interact with COPII subunits, and these proteins become highly enriched in COPII vesicles [22,36]. The two major classes of such cytoplasmic COPIIbinding ER export signals are diacidic and dihydrophobic motifs. A recently reported ER export signal in GluR1 (Figure 1, Table 1) is distinct from the canonical motifs in two respects [37]. First, the sequence of this motif in GluR1 (IQI) is unlike that of any known ER export signal. Second, the IQI motif is part of the extracellular N-terminal domain of GluR1, whereas all other known ER export signals occur in cytosolic regions. Either this GluR1 motif may interact with a transmembrane protein capable of binding COPII subunits, or its forward trafficking effect may be completely unrelated to the COPII coat. An identical IQI motif is present in GluR2, and similar ISI and VQI signals are found in GluR3 and GluR4, respectively. However, deletion of the entire N-terminal domain of GluR4, including the VQI sequence, does not have any significant impact on GluR4 trafficking [38], suggesting that the role of this motif differs among AMPA receptor subunits. www.sciencedirect.com
Regulation of ER exit of GluRs by phosphorylation The RRR retention motif in NR1-1 and NR1-3 is adjacent to two serine residues that are phosphorylated by protein kinase A and protein kinase C (PKC). Mutation of these serines to negatively charged residues to mimic phosphorylation suppresses ER retention [28,29]. In addition, pharmacological activation of PKC increases NR1 surface expression [27–29]. The effect of phosphorylation on NR1 export from the ER adds an activity-dependent dimension to the control of this trafficking step.
Regulation of ER exit of GluRs by GluRbinding proteins In the past eight years a large number of GluR-binding proteins have been identified. Most of these interacting proteins localize to the PSD [5]; however, some interacting proteins associate with GluRs in the proximal secretory pathway and affect ER export. As mentioned earlier, binding of PDZ proteins suppresses the NR1-3 ER retention signal [27–29]. Synapse-associated protein 97, a PDZ domain-containing binding partner of GluR1, preferentially binds immature GluR1 in the ER and cis-Golgi [39], but the effect of this association on AMPA receptor trafficking has not yet been clarified. And the PDZ protein SAP102 links NR2 to sec8, a component of the exocyst [40], a cytosolic complex that directs secretory vesicles to their fusion sites in the plasma membrane. Sec8, SAP102 and NR2 form a complex in brain and this mediates surface delivery of NMDA receptors. Furthermore, findings in heterologous cells argue that these complexes form before NMDA receptors exit the ER [40], but whether this facilitates ER export is not clear. GluR-interacting proteins can also inhibit ER export. Homer 1b, a binding partner of mGluRs, causes ER retention of mGluR5 [41]. This may reflect the ability of Homer 1b homomultimers to cross-link mGluR5 to the IP3 receptor, an ER resident protein.
ER in dendrites and spines Most GluRs are synthesized and processed in somatic ER and then trafficked to dendrites and spines. This is illustrated by the subcellular redistribution of NMDA receptor subunits in mice specifically lacking NR1 in hippocampal CA1 pyramidal neurons [25]. As discussed above, NR2 subunits cannot exit the ER without NR1. In CA1 pyramidal cells of CA1–NR1 knockout mice, NR2 subunits are nearly absent from dendrites and accumulate in somatic ER [25], implying that somatic ER represents the primary site for receptor synthesis. However, a particularly exciting discovery is the identification of ribosomes, rough ER (containing translocons, chaperones and exit sites), ERGIC, Golgi complex and trans-Golgi network in proximal and distal dendrites and even in synaptic spines [42–45]. Furthermore, Current Opinion in Cell Biology 2004, 16:134–139
138 Cell regulation
hippocampal dendrites contain low levels of iGluR mRNAs [46–48]. Thus, some dendrites may be equipped with all the necessary ingredients for local iGluR synthesis and processing. This provides an attractive mechanism for rapidly adjusting iGluR content of individual synapses. Brefeldin A, a blocker of ER-to-Golgi transport, inhibits a chemically induced form of hippocampal synaptic potentiation and the accompanying increase in AMPA receptor content of synaptic membranes, without affecting basal synaptic transmission [49]. This suggests that regulated exit of AMPA receptors from dendritic ER may contribute to the expression of some forms of synaptic plasticity.
Conclusions The complexity of ER export of GluRs is only beginning to be understood. GluR exit from ER is regulated by diverse mechanisms including mRNA splicing and editing, as well as receptor phosphorylation, association with specific binding partners, and interaction with the QC machinery. By altering the efficiency of GluR surface expression, these events influence neuronal responsiveness to glutamate and vulnerability to excitotoxicity. In addition, the presence of ER in dendrites and spines allows synapse-specific regulation of receptor delivery. It will now be interesting to see whether the altered postsynaptic AMPA receptor insertion observed during activity-dependent synaptic plasticity [6–8] involves changes in receptor exit from local ER.
Update Mu and colleagues recently reported that neuronal activity regulates ER export of NMDA receptors by controlling alternative splicing of the C-terminal tail of the NR1 subunit [50]. Chronically decreasing neuronal activity favors production of splice variants that efficiently exit the ER, whereas chronically increasing activity has the opposite effect. Consistent with previous studies [27–29], accelerated ER export of NR1 depends on a C-terminal – TVV sequence present in NR1-3 and NR1-4 but not in other splice variants. However, in contrast to the previously proposed model [27–29], this –TVV sequence does not merely function by interacting with PDZ proteins but rather promotes ER export by acting as a dihydrophobic binding partner for COPII subunits [50].
Acknowledgements WV is supported by a postdoctoral fellowship of the Fund for Scientific Research-Flanders. DSB is supported by grants from the National Institutes of Health and is an established investigator of the American Heart Association.
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26. Okabe S, Miwa A, Okado H: Alternative splicing of the C-terminal domain regulates cell surface expression of the NMDA receptor NR1 subunit. J Neurosci 1999, 19:7781-7792. 27. Standley S, Roche KW, McCallum J, Sans N, Wenthold RJ: PDZ domain suppression of an ER retention signal in NMDA receptor NR1 splice variants. Neuron 2000, 28:887-898. 28. Scott DB, Blanpied TA, Swanson GT, Zhang C, Ehlers MD: An NMDA receptor ER retention signal regulated by phosphorylation and alternative splicing. J Neurosci 2001, 21:3063-3072. 29. Xia H, Hornby ZD, Malenka RC: An ER retention signal explains differences in surface expression of NMDA and AMPA receptor subunits. Neuropharmacology 2001, 41:714-723. 30. Ren Z, Riley NJ, Garcia EP, Sanders JM, Swanson GT, Marshall J: Multiple trafficking signals regulate kainate receptor KA2 subunit surface expression. J Neurosci 2003, 23:6608-6616. This study identifies an arginine-rich ER retention signal in the cytosolic C-terminal tail of KA2, explaining the inability of this subunit to reach the cell surface when expressed alone. 31. Chan WY, Soloviev MM, Ciruela F, McIlhinney RAJ: Molecular determinants of metabotropic glutamate receptor 1b trafficking. Mol Cell Neurosci 2001, 17:577-588. 32. Greger IH, Khatri L, Ziff EB: RNA editing at Arg607 controls AMPA receptor exit from the endoplasmic reticulum. Neuron 2002, 34:759-772. The authors identify R607 as an ER retention signal for GluR2 in hippocampal neurons. Mutation of R607 to glutamine increases both the rate of GluR2 ER export and the fraction of GluR2 present on the cell surface. Conversely, mutation of the corresponding glutamine residue in GluR1 to arginine reduces surface expression of GluR1.
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Current Opinion in Cell Biology 2004, 16:134–139
Current Opinion in Cell Biology 2004, 16:134–139 This review comes from a themed issue on Cell regulation Edited by Craig Montell and Peter Devreotes 0955-0674/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved.
that contain both NR1 and NR2 (NR2A–D) subunits, and in some cases NR3 subunits. Among the mGluR subunits, mGluR1 and mGluR5 are predominant at postsynaptic sites, and these receptors probably exist on the cell surface as homodimers. Metabotropic GluR subunits are seventransmembrane proteins, whereas iGluR subunits contain three transmembrane domains and a pore-lining, reentrant membrane loop (Figure 1). All GluR subunits contain an extracellular N-terminal domain and a cytoplasmic C terminus. GluRs are highly concentrated at postsynaptic sites, where they interact with a host of structural and signaling proteins collectively referred to as the postsynaptic density (PSD). Tremendous progress has been made in dissecting the molecular anatomy of the PSD [5]. In addition, it has become clear that changing the numbers of postsynaptic glutamate receptors, especially AMPA receptors, is a key mechanism of activity-dependent modulation of synaptic strength, a phenomenon thought to underlie aspects of learning and memory [6–8]. Accordingly, GluR trafficking at the postsynaptic membrane has been intensively studied.
DOI 10.1016/j.ceb.2004.01.003
Abbreviations AMPA a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid COPII coat protein complex II ER endoplasmic reticulum ERAD ER-associated degradation ERGIC ER–Golgi intermediate compartment GABA g-amino butyric acid GluR glutamate receptor iGluR ionotropic glutamate receptor mGluR metabotropic glutamate receptor NMDA N-methyl-D-aspartic acid PDZ PSD-95/Dlg/ZO-1 domain PKC protein kinase C PSD postsynaptic density QC quality control
Introduction Glutamate, the principal excitatory neurotransmitter in the mammalian central nervous system, activates two types of postsynaptic receptors: ionotropic receptors (iGluRs), which are glutamate-gated cation channels, and metabotropic receptors (mGluRs), which are linked through Gproteins to second messenger systems [1–4]. Ionotropic GluRs can be subdivided into three families on the basis of pharmacology, electrophysiology and sequence homology: AMPA receptors, which are homo- or hetero-tetramers composed of the subunits GluR1–4; kainate receptors, homo- or hetero-tetramers of the subunits GluR5–7, KA1 and KA2; and NMDA receptors, hetero-tetramers Current Opinion in Cell Biology 2004, 16:134–139
Classical studies in non-neuronal cells have shown that exit from the endoplasmic reticulum (ER) is often the most stringently controlled step in integral membrane protein transport to the cell surface [9]. Similarly, recent work reveals that GluR trafficking through this early compartment of the secretory pathway is tightly regulated and that mechanisms controlling ER exit have a major impact on synaptic GluR abundance. This review highlights these recent discoveries of rules governing ER exit of GluRs (Figure 2).
ER quality control of GluRs Like other multimeric cell membrane proteins, GluRs are synthesized, folded and assembled in the ER. For large multi-subunit membrane proteins such as ion channels, folding of individual subunits often continues throughout formation of the multimeric protein complex [10]. Nglycosylation and disulphide bond formation, which often promote proper protein folding [11,12], also occur in the ER. All GluR subunits possess N-glycosylation sites [2,4], and iGluR [13] and mGluR [4] subunits contain intraand intermolecular disulphide bridges, respectively, but whether these modifications play central roles in GluR folding and trafficking to the cell surface is not fully understood [4,14,15]. The ER uses a rigorous quality control (QC) system to ensure that only correctly folded and assembled proteins www.sciencedirect.com
Early events in glutamate receptor trafficking Vandenberghe and Bredt 135
Figure 1
iGluR subunit (a)
evidence suggests that these signals mediate cargo retrieval from the ER–Golgi intermediate compartment (ERGIC) or from the Golgi to the ER by direct or indirect interactions with coat protein complex I (COPI), a set of cytosolic proteins involved in retrograde vesicle transport through the secretory pathway.
mGluR subunit
(b) (c) (d) (e) Current Opinion in Cell Biology
Membrane topology and positions of ER export and retention signals in GluR subunits (see also Table 1). Green and red stars represent ER export and retention/retrieval signals, respectively, as found in (a) GluR1, (b) GluR2, (c) KA2, (d) NR1 and (e) mGluR1.
exit to the Golgi [9]. Endoplasmic reticulum QC operates both at a general level (primary QC) and at a substrateselective level (secondary QC) [9]. Primary QC mechanisms accurately discriminate between native and nonnative structures in a wide variety of unrelated nascent polypeptides. Prominent components of primary QC include the ER chaperones BiP and calnexin, which both assist the folding process and serve as retention anchors for unfolded proteins. The secondary QC machinery comprises a wide array of specialized factors required for efficient ER export of selected protein species. An example of this rapidly expanding group is Boca/MESD, a chaperone specific for certain members of the lowdensity-lipoprotein receptor family [16,17]. Persistently misfolded proteins are retrotranslocated into the cytosol, polyubiquitinated and degraded by proteasomes in a process called ER-associated degredation (ERAD) [18]. Very few specific data are available about QC of GluRs. AMPA receptors in brain are substrates of primary QC, as demonstrated by their co-immunoprecipitation with BiP and calnexin [19]; however, no GluR-specific chaperones have been identified. Serrando and colleagues serendipitously found cytosolic inclusion bodies containing GluR1, ubiquitin and proteasomes in a subset of spinal interneurons during a specific developmental time window [20]. These inclusions may represent misfolded, retrotranslocated GluR1. This raises the possibility of a developmental mismatch between GluR1 synthesis and the capacity for folding support and/or proteasomal degradation. Ubiquitination of GLR-1, an AMPA receptor homologue of C. elegans, occurs in vivo and apparently regulates receptor endocytosis [21]. Ubiquitination of mammalian GluRs has not been reported.
ER retention/retrieval signals in GluRs ER retrieval signals, such as the KDEL and KKXX motifs, help anchor ER resident proteins [9,22]. Current www.sciencedirect.com
ER retention/retrieval signals are also present in some proteins destined for transport to the cell surface. The best known of these signals is the RXR motif (where X is any amino acid except an acidic one) first described in the cytosolic domains of subunits of ATP-sensitive potassium channels (KATP channels) and GABAB receptors [22]. This arginine-rich motif helps retain unassembled subunits and is fully masked only after complete heteromeric assembly. This provides a QC mechanism for ensuring correct subunit association and stoichiometry. The anchor that binds to the exposed RXR signal has not been identified. Arginine-based ER retention/retrieval signals have been identified in members of each GluR subfamily (Figure 1, Table 1). To form a functional NMDA receptor, the NR1 subunit assembles with one of the four NR2 subunits [23]. NR2 subunits expressed alone are retained in the ER and fail to reach the cell surface [24,25]. Co-expression of NR1 results in robust surface expression of the heteromeric complex [24]. Still unknown is whether an ER retention signal prevents export of unassembled NR2 subunits. For NR1 subunits expressed alone, the extent of surface expression strongly depends on the splice variant [26–28]. There are eight NR1 splice variants, including four variants of the C-terminal cytoplasmic tail (NR1-1, NR1-2, NR1-3 and NR1-4). When expressed without NR2, all isoforms except NR1-1 are able to reach the cell surface [26–28]. This is explained by the presence of an ER retention/retrieval signal (RRR) in the cytoplasmic tail of NR1-1 [27–29]. The same signal also occurs in the cytoplasmic tail of NR1-3 splice variants, but ER retention of NR1-3 is overcome by the binding of PDZ proteins to a PDZ-binding site present in NR1-3 but absent in NR1-1 [27–29]. In a manner analogous to the shielding of the RXR motif of KATP channels and GABAB receptors by heteromeric assembly [22], association with NR2 apparently releases ER retention of NR1-1 by masking the RRR motif, but the molecular details of this masking process are not understood [23]. A related ER retention/retrieval motif was recently reported in the kainate receptor subunit KA2 [30]. KA2 forms functional receptors only when co-expressed with GluR5, GluR6 or GluR7, thereby producing channels with properties different from GluR5-7 homomers. Homomeric KA2 complexes fail to reach the plasma membrane owing to an ER retention/retrieval motif that Current Opinion in Cell Biology 2004, 16:134–139
136 Cell regulation
Figure 2
Synapse
Golgi
?
COPII vesicle
ER export of correctly folded and assembled GluRs ERAD
ER lumen
Elimination
ER retention of unfolded and unassembled GluRs Cytosol
GluR subunit Current Opinion in Cell Biology
ER exit is a critical, highly regulated step in the trafficking of GluRs to the synapse. Correctly folded and assembled GluRs are exported to the Golgi, whereas the QC machinery retains misfolded or misassembled GluRs. Persistently misfolded or misassembled GluRs are probably eliminated by ERAD. Whether GluRs can also be retrieved to the ER from post-ER compartments is not known. For simplicity, fully assembled GluRs are depicted here as tetramers.
consists of a string of five arginine residues in its cytoplasmic tail [30]. Although the precise molecular mechanism remains unclear, heteromeric assembly with GluR6 silences this signal.
The surface expression of the mGluR1b splice variant is also limited by an ER retention/retrieval signal (RRKK) in its C-terminal cytoplasmic domain [31]. The same motif is present in mGluR1a, but another C-terminal
Table 1 Published ER retention/retrieval and export signals in GluR subunits. GluR subfamily
Subunit
Sequence
Orientation
Effect
References
NMDA receptor AMPA receptor
NR1 (isoforms NR1-1 and 1-3) GluR1 GluR2 KA2 mGluR1
RRR893–895 IQI25–27 R607 RRRRR826–866 RRKK877–880
Cytosolic Luminal Membrane Cytosolic Cytosolic
Retention/retrieval Export Retention Retention/retrieval Retention/retrieval
[27–29] [37] [32] [30] [31]
Kainate receptor mGluR
The numbers next to the sequences refer to the amino acid positions in rat, with number 1 corresponding to the first residue of the signal peptide. For NR1, numbering is based on NR1-1a.
Current Opinion in Cell Biology 2004, 16:134–139
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Early events in glutamate receptor trafficking Vandenberghe and Bredt 137
sequence specific to mGluR1a somehow overrides this effect [31]. Finally, the AMPA receptor subunit GluR2 contains a distinct arginine-based ER retention motif that consists of just a single arginine residue (R607) in a transmembrane domain [32]. A positively charged residue in a membrane segment of T-cell receptor subunits also mediates ER retention [33]. In the case of AMPA receptors, the critical arginine (R607 in GluR2) is generated by mRNA editing [34], and resides in a re-entrant membrane loop that lines the pore of fully assembled AMPA receptor channels. This arginine critically controls calcium permeability and other channel properties [2,34]. The additional role for R607 in ER retention presumably results from its interaction with an unidentified ER anchor. As GluR2 is a component of functional AMPA receptors in many neuronal cell types [35], a highly efficient, as yet unknown mechanism must exist to selectively suppress the ER retention effect of R607 while preserving its influence on channel properties such as calcium permeability. It is possible that assembly of a tetrameric AMPA receptor channel shields R607 from an ER anchor outside the receptor complex.
ER export signals in GluRs Vesicles export cargo from the ER to the Golgi. Budding of export vesicles is driven by interactions of ER membranes with coat protein complex II (COPII), a polymer formed by ordered assembly of cytosolic subunits [36]. According to the bulk-flow model, a default pathway packages properly folded and assembled membrane proteins into COPII-coated vesicles at their prevailing concentration in the ER. On the other hand, some membrane proteins contain specific export signals that interact with COPII subunits, and these proteins become highly enriched in COPII vesicles [22,36]. The two major classes of such cytoplasmic COPIIbinding ER export signals are diacidic and dihydrophobic motifs. A recently reported ER export signal in GluR1 (Figure 1, Table 1) is distinct from the canonical motifs in two respects [37]. First, the sequence of this motif in GluR1 (IQI) is unlike that of any known ER export signal. Second, the IQI motif is part of the extracellular N-terminal domain of GluR1, whereas all other known ER export signals occur in cytosolic regions. Either this GluR1 motif may interact with a transmembrane protein capable of binding COPII subunits, or its forward trafficking effect may be completely unrelated to the COPII coat. An identical IQI motif is present in GluR2, and similar ISI and VQI signals are found in GluR3 and GluR4, respectively. However, deletion of the entire N-terminal domain of GluR4, including the VQI sequence, does not have any significant impact on GluR4 trafficking [38], suggesting that the role of this motif differs among AMPA receptor subunits. www.sciencedirect.com
Regulation of ER exit of GluRs by phosphorylation The RRR retention motif in NR1-1 and NR1-3 is adjacent to two serine residues that are phosphorylated by protein kinase A and protein kinase C (PKC). Mutation of these serines to negatively charged residues to mimic phosphorylation suppresses ER retention [28,29]. In addition, pharmacological activation of PKC increases NR1 surface expression [27–29]. The effect of phosphorylation on NR1 export from the ER adds an activity-dependent dimension to the control of this trafficking step.
Regulation of ER exit of GluRs by GluRbinding proteins In the past eight years a large number of GluR-binding proteins have been identified. Most of these interacting proteins localize to the PSD [5]; however, some interacting proteins associate with GluRs in the proximal secretory pathway and affect ER export. As mentioned earlier, binding of PDZ proteins suppresses the NR1-3 ER retention signal [27–29]. Synapse-associated protein 97, a PDZ domain-containing binding partner of GluR1, preferentially binds immature GluR1 in the ER and cis-Golgi [39], but the effect of this association on AMPA receptor trafficking has not yet been clarified. And the PDZ protein SAP102 links NR2 to sec8, a component of the exocyst [40], a cytosolic complex that directs secretory vesicles to their fusion sites in the plasma membrane. Sec8, SAP102 and NR2 form a complex in brain and this mediates surface delivery of NMDA receptors. Furthermore, findings in heterologous cells argue that these complexes form before NMDA receptors exit the ER [40], but whether this facilitates ER export is not clear. GluR-interacting proteins can also inhibit ER export. Homer 1b, a binding partner of mGluRs, causes ER retention of mGluR5 [41]. This may reflect the ability of Homer 1b homomultimers to cross-link mGluR5 to the IP3 receptor, an ER resident protein.
ER in dendrites and spines Most GluRs are synthesized and processed in somatic ER and then trafficked to dendrites and spines. This is illustrated by the subcellular redistribution of NMDA receptor subunits in mice specifically lacking NR1 in hippocampal CA1 pyramidal neurons [25]. As discussed above, NR2 subunits cannot exit the ER without NR1. In CA1 pyramidal cells of CA1–NR1 knockout mice, NR2 subunits are nearly absent from dendrites and accumulate in somatic ER [25], implying that somatic ER represents the primary site for receptor synthesis. However, a particularly exciting discovery is the identification of ribosomes, rough ER (containing translocons, chaperones and exit sites), ERGIC, Golgi complex and trans-Golgi network in proximal and distal dendrites and even in synaptic spines [42–45]. Furthermore, Current Opinion in Cell Biology 2004, 16:134–139
138 Cell regulation
hippocampal dendrites contain low levels of iGluR mRNAs [46–48]. Thus, some dendrites may be equipped with all the necessary ingredients for local iGluR synthesis and processing. This provides an attractive mechanism for rapidly adjusting iGluR content of individual synapses. Brefeldin A, a blocker of ER-to-Golgi transport, inhibits a chemically induced form of hippocampal synaptic potentiation and the accompanying increase in AMPA receptor content of synaptic membranes, without affecting basal synaptic transmission [49]. This suggests that regulated exit of AMPA receptors from dendritic ER may contribute to the expression of some forms of synaptic plasticity.
Conclusions The complexity of ER export of GluRs is only beginning to be understood. GluR exit from ER is regulated by diverse mechanisms including mRNA splicing and editing, as well as receptor phosphorylation, association with specific binding partners, and interaction with the QC machinery. By altering the efficiency of GluR surface expression, these events influence neuronal responsiveness to glutamate and vulnerability to excitotoxicity. In addition, the presence of ER in dendrites and spines allows synapse-specific regulation of receptor delivery. It will now be interesting to see whether the altered postsynaptic AMPA receptor insertion observed during activity-dependent synaptic plasticity [6–8] involves changes in receptor exit from local ER.
Update Mu and colleagues recently reported that neuronal activity regulates ER export of NMDA receptors by controlling alternative splicing of the C-terminal tail of the NR1 subunit [50]. Chronically decreasing neuronal activity favors production of splice variants that efficiently exit the ER, whereas chronically increasing activity has the opposite effect. Consistent with previous studies [27–29], accelerated ER export of NR1 depends on a C-terminal – TVV sequence present in NR1-3 and NR1-4 but not in other splice variants. However, in contrast to the previously proposed model [27–29], this –TVV sequence does not merely function by interacting with PDZ proteins but rather promotes ER export by acting as a dihydrophobic binding partner for COPII subunits [50].
Acknowledgements WV is supported by a postdoctoral fellowship of the Fund for Scientific Research-Flanders. DSB is supported by grants from the National Institutes of Health and is an established investigator of the American Heart Association.
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