Different Roles of C-terminal Cassettes in the Trafficking of Full-length NR1 Subunits to the Cell Surface

N-Methyl-d-aspartate (NMDA) receptors are glutamate-gated ion channels composed of NR1 and NR2 subunits. When expressed alone, the most prevalent NR1 splice variant and all NR2 subunits are retained in the endoplasmic reticulum (ER), whereas other NR1 splice variants reach the cell surface to varying degrees. Because similar trafficking patterns have been seen for single transmembrane domain chimeric proteins with appended C termini of NMDA receptor subunits, these chimeric proteins have been used as a model for studying the mechanisms underlying the ER retention and surface trafficking of NMDA receptors. Using this approach, an RRR motif in the C1 cassette has been identified as a major ER retention signal present in NR1 subunits, and the surface localization of other NR1 splice variants has been explained by the absence of the C1 cassette or by the presence of a PDZ/coatomer protein complex II-binding domain in the C2′ cassette. However, when we tested these conclusions using full-length NR1 constructs, a more complex role of the C-terminal cassettes in the trafficking of NR1 subunits emerged. Our experiments showed that two independent ER retention motifs in the C1 cassette, KKK and RRR, are the signals mediating ER retention of the full-length NR1 subunits and that the C2 cassette has an additional inhibitory effect on the forward trafficking of NR1 subunits. On the other hand, C0 and C2′ cassettes had an enhancing effect on the trafficking of NR1 subunits to the cell surface. Our observations identify the unique roles of C-terminal cassettes in the trafficking of full-length NR1 subunits. N-Methyl-d-aspartate (NMDA) receptors are glutamate-gated ion channels composed of NR1 and NR2 subunits. When expressed alone, the most prevalent NR1 splice variant and all NR2 subunits are retained in the endoplasmic reticulum (ER), whereas other NR1 splice variants reach the cell surface to varying degrees. Because similar trafficking patterns have been seen for single transmembrane domain chimeric proteins with appended C termini of NMDA receptor subunits, these chimeric proteins have been used as a model for studying the mechanisms underlying the ER retention and surface trafficking of NMDA receptors. Using this approach, an RRR motif in the C1 cassette has been identified as a major ER retention signal present in NR1 subunits, and the surface localization of other NR1 splice variants has been explained by the absence of the C1 cassette or by the presence of a PDZ/coatomer protein complex II-binding domain in the C2′ cassette. However, when we tested these conclusions using full-length NR1 constructs, a more complex role of the C-terminal cassettes in the trafficking of NR1 subunits emerged. Our experiments showed that two independent ER retention motifs in the C1 cassette, KKK and RRR, are the signals mediating ER retention of the full-length NR1 subunits and that the C2 cassette has an additional inhibitory effect on the forward trafficking of NR1 subunits. On the other hand, C0 and C2′ cassettes had an enhancing effect on the trafficking of NR1 subunits to the cell surface. Our observations identify the unique roles of C-terminal cassettes in the trafficking of full-length NR1 subunits. N-Methyl-d-aspartate (NMDA) 2The abbreviations used are: NMDA, N-methyl-d-aspartate; COPII, coatomer protein complex II; ER, endoplasmic reticulum; GFP, green fluorescent protein; M, membrane; PBS, phosphate-buffered saline; YFP, yellow fluorescent protein. receptors are glutamate-gated ion channels that play a central role in excitatory synaptic transmission as well as excitotoxicity (1Cull-Candy S. Brickley S. Farrant M. Curr. Opin. Neurobiol. 2001; 11: 327-335Crossref PubMed Scopus (1392) Google Scholar, 2Lau C.G. Zukin R.S. Nat. Rev. Neurosci. 2007; 8: 413-426Crossref PubMed Scopus (898) Google Scholar). The functional NMDA receptors are composed of NR1, NR2, and/or NR3 subunits. The NR1 subunit exists in eight different splice variants derived from a single gene, whereas NR2 subunits are products of four different genes, NR2A–D, and NR3 subunits are products of two different genes, NR3A and NR3B. All subunits have the same transmembrane topology with four membrane regions (M1–4), two extracellular regions, and an intracellular C-terminal region. Recent evidence suggests that abnormal surface expression and synaptic targeting of NMDA receptors play fundamental roles in the development of many disorders, including Alzheimer disease, Parkinson disease, and schizophrenia (1Cull-Candy S. Brickley S. Farrant M. Curr. Opin. Neurobiol. 2001; 11: 327-335Crossref PubMed Scopus (1392) Google Scholar, 2Lau C.G. Zukin R.S. Nat. Rev. Neurosci. 2007; 8: 413-426Crossref PubMed Scopus (898) Google Scholar). Although much is known about the biophysical properties of NMDA receptors, the mechanisms underlying their trafficking to the cell surface remain poorly understood. Previous studies have shown that full-length NR1-1 and NR2 subunits are retained intracellularly when expressed by themselves in heterologous cells, whereas other NR1 splice variants reach the cell surface (3McIlhinney R.A. Le Bourdelles B. Molnar E. Tricaud N. Streit P. Whiting P.J. Neuropharmacology. 1998; 37: 1355-1367Crossref PubMed Scopus (133) Google Scholar, 4Okabe S. Miwa A. Okado H. J. Neurosci. 1999; 19: 7781-7792Crossref PubMed Google Scholar, 5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Similar results have been obtained using single transmembrane domain chimeras with the appended C termini of NMDA receptor subunits, and thus, these chimeras have been used as a model for studying the mechanisms governing the trafficking of NMDA receptor subunits (5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 6Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar, 7Xia H. Hornby Z.D. Malenka R.C. Neuropharmacology. 2001; 41: 714-723Crossref PubMed Scopus (101) Google Scholar, 8Mu Y. Otsuka T. Horton A.C. Scott D.B. Ehlers M.D. Neuron. 2003; 40: 581-594Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 9Scott D.B. Blanpied T.A. Ehlers M.D. Neuropharmacology. 2003; 45: 755-767Crossref PubMed Scopus (157) Google Scholar). Hence, the RRR ER retention motif has been identified as a major ER retention signal in the NR1-1 subunit (5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 6Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar, 7Xia H. Hornby Z.D. Malenka R.C. Neuropharmacology. 2001; 41: 714-723Crossref PubMed Scopus (101) Google Scholar), and the reason why the NR1-3 splice variant reaches the cell surface has been explained by the presence of the PDZ- and/or coatomer protein complex II (COPII)-binding domain in the far C terminus of C2′ cassette, which can negate the RRR motif (5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 6Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar, 7Xia H. Hornby Z.D. Malenka R.C. Neuropharmacology. 2001; 41: 714-723Crossref PubMed Scopus (101) Google Scholar, 8Mu Y. Otsuka T. Horton A.C. Scott D.B. Ehlers M.D. Neuron. 2003; 40: 581-594Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). However, our recent study shows that the NR1 subunit truncated five residues after M4 traffics only slightly to the cell surface, likely due to the presence of an ER retention signal in the M3 domain (10Horak M. Chang K. Wenthold R.J. J. Neurosci. 2008; 28: 3500-3509Crossref PubMed Scopus (47) Google Scholar). This indicates that the role of the C terminus in the trafficking of full-length NR1 subunits is more complex than previous experiments suggested. In this study, we investigated the role of C-terminal cassettes in ER retention and forward trafficking of NR1 subunits. Using full-length and truncated yellow fluorescent protein (YFP)-NR1 constructs expressed in heterologous cells, we demonstrate that the C1 cassette contains two independent ER retention motifs, KKK and RRR, and that the C2 cassette has an additional inhibitory effect on the surface trafficking of NR1 subunits. We also found that both C0 and C2′ cassettes have an enhancing effect on surface targeting of the NR1 subunit and, unexpectedly, that the C2′ cassette can partially negate C1 cassette-mediated ER retention, even when the PDZ-binding domain is not functional. These that C-terminal cassette has a unique role in the trafficking of NR1 subunits to the cell surface. expression by a the and residues after the signal used K. J. Chang K. Wenthold R.J. S. J. Neurosci. PubMed Google Scholar). The by S. the expression (10Horak M. Chang K. Wenthold R.J. J. Neurosci. 2008; 28: 3500-3509Crossref PubMed Scopus (47) Google Scholar). All other constructs used in this study using the residues are as T. K. H. K. H. M. C. N. M. J. Full Text PDF PubMed Google Scholar). The and expression to and and are and The NR1 a of to the of and green in with with in The in in of their with of with of to the the with used for experiments after subunits in phosphate-buffered and with and on for The fluorescent protein in for in PBS, and the in for The in PBS, in in for and with from using a on a of and in and for in and when expression with in for The for in or with and for in the for surface expression and for in with in and with the for in and for in of in The with of and the using a The signal in with and from the obtained from are expressed as the is an of different in independent experiments the same using an C1 in the C terminus of the NR1 ER studies from that the full-length NR1-1 subunit not reach the surface membrane when expressed in the absence of NR2 subunits (3McIlhinney R.A. Le Bourdelles B. Molnar E. Tricaud N. Streit P. Whiting P.J. Neuropharmacology. 1998; 37: 1355-1367Crossref PubMed Scopus (133) Google Scholar, 4Okabe S. Miwa A. Okado H. J. Neurosci. 1999; 19: 7781-7792Crossref PubMed Google Scholar, 5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). identify the underlying ER retention of the NR1-1 single transmembrane domain chimeras the C termini of NR1 subunits in the of an RRR motif in the C1 cassette of the NR1-1 subunit as a major ER retention signal present on this subunit (5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 6Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar, 7Xia H. Hornby Z.D. Malenka R.C. Neuropharmacology. 2001; 41: 714-723Crossref PubMed Scopus (101) Google Scholar). However, in these the role of the RRR motif in ER retention of the full-length NR1-1 subunit not In this study, we investigated or not the C1 cassette contains functional ER retention when all of the of the NR1 subunit are we subunits truncated and after the C1 cassette, expressed in NMDA and the surface and expression of these constructs with the subunit using and of surface and expression with and the subunit not to the cell surface, similar to the the subunit surface This indicates that the C1 cassette contains ER retention we the RRR motif in the C1 cassette is a major ER retention signal in the full-length NR1-1 subunit of this motif for residues not in surface targeting of the that the RRR motif is not the ER retention motif or the only ER retention motif for the retention of the full-length NR1-1 subunit and Because our experiments with constructs that the C1 cassette contains an ER retention we the other ER retention present in the C1 cassette and surface targeting T. J. PubMed Scopus Google Scholar, T. M. Full Text PDF PubMed Scopus Google Scholar). Although this to to the surface more than the or constructs, we not a with the We both RRR and KKK motifs in the full-length subunit and expressed in and of surface expression surface expression of the with the the expression of all subunits not different with the our that both RRR and KKK motifs as independent ER retention motifs and are to the full-length NR1-1 subunit even when of is not functional. C1 ER C1 cassette contains four residues in to the RRR two of are known protein C and and of is a protein Ehlers M.D. K. C. J. Full Text Full Text PDF PubMed Scopus Google Scholar, Roche K.W. PubMed Scopus Google Scholar). previous study has shown that of with the protein C results in the of the chimeric protein the NR1-1 C terminus on the surface membrane and that of and to residues surface 6Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google also 5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google and 7Xia H. Hornby Z.D. Malenka R.C. Neuropharmacology. 2001; 41: 714-723Crossref PubMed Scopus (101) Google Scholar). study that the of a to protein results in surface expression of the chimeric proteins D.B. Blanpied T.A. Ehlers M.D. Neuropharmacology. 2003; 45: 755-767Crossref PubMed Scopus (157) Google Scholar). protein C and protein and negate the RRR ER retention motif in the chimeric protein the NR1-1 C However, these conclusions not using full-length NR1-1 subunits. Because we showed that in to the RRR is also a KKK motif for retention of the NR1-1 subunit we the of these residues can negate the ER retention of the full-length we single and in the full-length subunit by of and/or with residues and and experiments to their surface targeting and In all surface and expression of these constructs not different the we the subunit with C for and surface and receptor expression and These experiments showed with we expressed the subunit in and tested the effect of a of on the surface and expression of NR1 subunits. Our experiments showed and in the of both surface and expression not our results that protein C and/or protein is not to negate the ER retention of the full-length NR1-1 has been shown using single transmembrane domain chimeric proteins that the far C terminus of the C2′ cassette, which contains a PDZ-binding can negate the C1 cassette-mediated ER retention (5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 6Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar, 7Xia H. Hornby Z.D. Malenka R.C. Neuropharmacology. 2001; 41: 714-723Crossref PubMed Scopus (101) Google Scholar). this we the residues in the C terminus of the subunit with a PDZ-binding domain present in the C2′ cassette and expressed this subunit in and the PDZ-binding domain to the C terminus of the subunit surface not expression with the This suggests that the PDZ-binding domain can negate both the RRR and KKK ER retention motifs present in the C1 cassette. of the C0 in of the to the experiments showed that the subunit surface However, we that the NR1 subunit truncated five residues after M4 only surface trafficking (10Horak M. Chang K. Wenthold R.J. J. Neurosci. 2008; 28: 3500-3509Crossref PubMed Scopus (47) Google Scholar). This indicates that the C0 cassette surface targeting of the full-length NR1 we the surface trafficking of constructs truncated different in the C0 cassette These experiments showed that the subunit reaches the cell surface more than the even more surface seen for and subunits and our that the of the C0 cassette a role in the of the NR1 subunit to the cell surface. motif is for the C0 cassette-mediated we four different constructs, residues the residues in the C0 cassette surface expression for all of these constructs with the the constructs with and to the surface more than the other two constructs and The expression of all truncated and subunits not different with the In our that the presence of the C0 cassette can the surface trafficking of the NR1 subunit and that motif is likely in this of C2 and C2′ in of NR1 the full-length NR1-1 subunit is retained whereas has been that the other NR1 splice variants to the cell surface to varying S. Miwa A. Okado H. J. Neurosci. 1999; 19: 7781-7792Crossref PubMed Google Scholar, 5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). the roles of the C2 and C2′ cassettes in trafficking of the full-length subunits, we to the surface of all the NR1 splice variants with that of the and These experiments showed that the a C2 cassette, to the cell surface than the On the other hand, both and the C2′ cassette, to the cell surface more than the these results that the C2 cassette has an inhibitory effect on the trafficking of the full-length NR1 In the C2′ cassette can the surface trafficking of the full-length NR1 subunit in the absence or presence of the C1 cassette. The of C2′ has been Y. Otsuka T. Horton A.C. Scott D.B. Ehlers M.D. Neuron. 2003; 40: 581-594Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). This study showed that the two residues of the far C terminus in the C2′ cassette can and that this not a is for the of the C2′ proteins to the cell surface. We the effect of of and on the surface targeting of We the four residues in the subunit to both and we the in the PDZ-binding domain with that the with the C2′ cassette whereas the the expression of these constructs in the cells, we their surface expression with and subunits and the to the cell surface than the that of both and of the C2′ cassette. the surface expression with the similar to the These that the not the can the surface trafficking of the this we the with with that a PDZ-binding domain is a is surface and expression of this subunit not different with the subunit an not In with the surface of both and constructs that the C2′ cassette has an effect even when the PDZ-binding domain is not functional. that the C2′ cassette has an enhancing effect on the trafficking of the NR1 subunit even in the absence of the functional PDZ-binding we both the and and the constructs and their surface trafficking with and subunits surface trafficking of both and subunits with the the that an is for the surface of NR1 subunits. both and subunits to the cell surface more than the This shows that even when the PDZ-binding domain is not the C2′ cassette can negate the C1 cassette-mediated ER In our a model in which both C1 and C2 cassettes have an inhibitory whereas both C0 and C2′ cassettes have an enhancing effect on the surface trafficking of the full-length NR1 subunits. The and of NMDA receptors present surface are Although the biophysical properties of the NMDA receptors on the surface NMDA receptors in the ER, in the and in the However, these remain In this study, we have on the of the mechanisms governing the trafficking of the NR1 subunits to the cell surface. We found that two different ER retention motifs present in the C1 cassette, KKK and RRR, are for the ER retention of the full-length NR1 subunits. This ER retention can by the presence of the C2′ cassette even when the PDZ-binding domain in the far C terminus of this cassette is not functional. We that the C0 cassette has an enhancing effect and that the C2 cassette has an inhibitory effect on the surface targeting of the full-length NR1 subunits. ER of NR1 by the C1 studies identified an RRR motif as a major ER retention for the C1 cassette-mediated ER retention of the single transmembrane domain chimeric proteins with an appended C-terminal from the NR1-1 subunit (5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 6Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar, 7Xia H. Hornby Z.D. Malenka R.C. Neuropharmacology. 2001; 41: 714-723Crossref PubMed Scopus (101) Google Scholar). Our that are two independent ER retention motifs in the C1 cassette, KKK and RRR, shows that the trafficking of the full-length NR1 subunits is likely than the trafficking of the chimeric also suggests that a more complex than the ER retention of the NR1 subunits the M3 domain of the NR1 subunit contains an additional ER retention signal (10Horak M. Chang K. Wenthold R.J. J. Neurosci. 2008; 28: 3500-3509Crossref PubMed Scopus (47) Google Scholar). We the that the C2 cassette, which an inhibitory role in the trafficking of NR1 subunits, an additional role in the C1 cassette-mediated ER retention of NR1 subunits. recent has shown that the of the RRR ER retention motif with is for a of the full-length NR1-1 subunits to the cell surface of J. Full Text Full Text PDF PubMed Scopus Google Scholar), which is not in with our similar of ER retention ER retention motifs has been for the receptor subunit Y. S. Roche K.W. J. Neurosci. PubMed Scopus Google Scholar). The of residues to the RRR ER retention motif can ER retention of single transmembrane chimeric proteins the C terminus of the NR1-1 subunit D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar, 7Xia H. Hornby Z.D. Malenka R.C. Neuropharmacology. 2001; 41: 714-723Crossref PubMed Scopus (101) Google Scholar, 9Scott D.B. Blanpied T.A. Ehlers M.D. Neuropharmacology. 2003; 45: 755-767Crossref PubMed Scopus (157) Google Scholar). we have shown that protein C and of and/or with residues not negate the C1 cassette-mediated ER retention of the full-length This by our previous study (5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), is with our that two ER retention motifs, KKK and RRR, are present in the C1 cassette, and thus, both signals the subunit can the the presence of a PDZ-binding domain in the C terminus of the NR1-1 subunit to negate both ER retention motifs in the C1 cassette and the full-length subunit to the cell surface. this is in with the that the full-length NR1-3 subunit reaches the cell surface and indicates that other cassettes in the C terminus of the NR1 subunit play a role in the surface trafficking of the NR1 subunits to the cell surface S. Miwa A. Okado H. J. Neurosci. 1999; 19: 7781-7792Crossref PubMed Google Scholar, 5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). of C-terminal in of of NR1 that the NR1 subunit truncated five residues after M4 is slightly to the cell surface (10Horak M. Chang K. Wenthold R.J. J. Neurosci. 2008; 28: 3500-3509Crossref PubMed Scopus (47) Google Scholar). when we truncated the NR1 subunit after the C0 cassette, surface that the C0 cassette contains an The and of residues in the of the C0 cassette that motif is likely for the of the full-length subunit to the cell surface, in the surface of these constructs These the that the presence of the C0 cassette can negate the ER retention of the NR1 subunit the M3 is that C0 the of the M4 domain and other regions for the of the full-length NR1 subunit from the ER (10Horak M. Chang K. Wenthold R.J. J. Neurosci. 2008; 28: 3500-3509Crossref PubMed Scopus (47) Google Scholar). a M4 in the subunit and a M4 of the subunit have been shown to for the of receptors from the ER to the cell surface K. Chang K. C. Wenthold R.J. J. Full Text Full Text PDF PubMed Scopus Google Scholar, M. 2000; PubMed Scopus Google Scholar). We tested the effect of the C2 cassette on the trafficking of the NR1 subunits to the cell surface. Our experiments showed that the C2 cassette has an inhibitory effect on the surface targeting of the full-length which is with previous observations that the subunit traffics to the cell surface than the subunit and that the C2 cassette the ER of the chimeric protein this cassette S. Miwa A. Okado H. J. Neurosci. 1999; 19: 7781-7792Crossref PubMed Google Scholar, 8Mu Y. Otsuka T. Horton A.C. Scott D.B. Ehlers M.D. Neuron. 2003; 40: 581-594Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). we the role of the C2′ cassette in the surface targeting of NR1 subunits. Previous showed that the chimeric protein with an appended C terminus from the NR1-3 subunit reaches the cell surface that of the PDZ-binding domain from the C2′ cassette results in the ER retention of the chimeric protein (5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 6Scott D.B. Blanpied T.A. Swanson G.T. Zhang C. Ehlers M.D. J. Neurosci. 2001; 21: 3063-3072Crossref PubMed Google Scholar). the is that the PDZ-binding domain can negate the RRR ER retention motif in the C1 cassette. has been that an not is for the protein trafficking from the ER Y. Otsuka T. Horton A.C. Scott D.B. Ehlers M.D. Neuron. 2003; 40: 581-594Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Because these conclusions have not been tested using full-length subunits, we the and/or for of the surface trafficking of the subunit by the C2′ and/or for the C1 cassette-mediated ER retention in the NR1-3 Our experiments showed that the of and/or the surface trafficking of both the and the NR1-3 subunits and These results not the that a can forward trafficking of the NR1 subunits, obtained using chimeric proteins Y. Otsuka T. Horton A.C. Scott D.B. Ehlers M.D. Neuron. 2003; 40: 581-594Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). is likely by the which have been shown to to with the subunit in heterologous (5Standley S. Roche K.W. McCallum J. Sans N. Wenthold R.J. Neuron. 2000; 28: 887-898Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). of the of this is expressed in M. J. Sans N. J. PubMed Scopus Google Scholar). with the NR1 subunit truncated after the C0 cassette or with the NR1-1 the and the NR1-3 subunits the cell surface our that the not protein can the trafficking of the C2′ NR1 subunits to the cell surface and negate the ER retention by the C1 cassette. In our that the of the C2′ cassette the PDZ-binding domain contains domain that is to the surface trafficking of the NR1 subunit and negate the C1 cassette-mediated ER retention of NR1 subunits to a than the PDZ-binding results that cassette in the C terminus of NR1 subunits a unique role in the trafficking of the full-length subunit to the cell surface. are the of our that NR1 subunits are expressed in in the central and have in cells, the of ER retention and trafficking of NR1 subunits different by C-terminal cassettes can a in the of functional NMDA receptors Wenthold R.J. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). This is by the that the and subunits with NR1 splice variants in the central M. J. PubMed Scopus Google and that the C-terminal of NR1 subunits the of surface functional NMDA receptors S. Miwa A. Okado H. J. Neurosci. 1999; 19: 7781-7792Crossref PubMed Google Scholar). Our results that the of both KKK and RRR ER retention motifs in the C1 cassette not the surface targeting of receptors in heterologous This shows that the NR1 C-terminal mechanisms can after the of receptors. of different NR1 splice variants expressed in the central is that variant can with a different of and that these can intracellular trafficking of the NR1 subunits. the C1 cassette can with and M.D. R.J. J. Neurosci. 1998; PubMed Google Scholar, M.D. Zhang S. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. J. Neurosci. 1998; PubMed Google Scholar), and of the C1 cassette can with C. H. T. T. K. K. T. J. Full Text Full Text PDF PubMed Scopus Google Scholar). both ER retention motifs present in the C1 cassette, KKK and RRR, can likely with different that localization of the NR1 subunit can the of the mechanisms underlying the trafficking of the NR1 variants is by observations that the C1 cassette NMDA expression J. J. S. J. Neurosci. PubMed Scopus Google and that the presence of the C2 not the C2′ cassette is for the and of J. Neurosci. 2007; PubMed Scopus Google Scholar). We for and S. for on the