Identification of Molecular Determinants That Are Important in the Assembly of N-Methyl-d-aspartate Receptors

Abstract

To determine which domains of theN-methyl-d-aspartate (NMDA) receptor are important for the assembly of functional receptors, a number of N- and C-terminal truncations of the NR1a subunit have been produced. Truncations containing a complete ligand binding domain bound glycine antagonist and gave binding constants similar to those of the native subunit, suggesting they were folding to form antagonist binding sites. Since NR2A is not transported to the cell surface unless it is associated with NR1 (McIlhinney, R. A. J., Le Bourdellès, B., Tricuad, N., Molnar, E., Streit, P., and Whiting, P. J. (1998) Neuropharmacology 37, 1355–1367), surface expression of NR2A can be used to monitor the association of the subunits. There was progressive loss of NR2A cell surface expression as the N terminus of NR1a was shortened, with complete loss when truncated beyond residue 380. Removal of the C terminus and/or the last transmembrane domain did not affect NR2A surface expression. Similar results were obtained in co-immunoprecipitation experiments. The oligomerization status of the co-expressed NR1a constructs and NR2A subunits was investigated using a non-denaturing gel electrophoresis system (blue native-polyacrylamide gel electrophoresis) and sucrose density gradient centrifugation. The blue native-polyacrylamide gel electrophoresis system also showed that the NR1a subunits could form a homodimer, which was confirmed using soluble constructs of the NR1a subunit. Together these results suggest the residues N-terminal of residue 380 are important for the association of NR2A with NR1a and that the complete N-terminal domain of the NR1a subunit is required for oligomerization with NR2A. To determine which domains of theN-methyl-d-aspartate (NMDA) receptor are important for the assembly of functional receptors, a number of N- and C-terminal truncations of the NR1a subunit have been produced. Truncations containing a complete ligand binding domain bound glycine antagonist and gave binding constants similar to those of the native subunit, suggesting they were folding to form antagonist binding sites. Since NR2A is not transported to the cell surface unless it is associated with NR1 (McIlhinney, R. A. J., Le Bourdellès, B., Tricuad, N., Molnar, E., Streit, P., and Whiting, P. J. (1998) Neuropharmacology 37, 1355–1367), surface expression of NR2A can be used to monitor the association of the subunits. There was progressive loss of NR2A cell surface expression as the N terminus of NR1a was shortened, with complete loss when truncated beyond residue 380. Removal of the C terminus and/or the last transmembrane domain did not affect NR2A surface expression. Similar results were obtained in co-immunoprecipitation experiments. The oligomerization status of the co-expressed NR1a constructs and NR2A subunits was investigated using a non-denaturing gel electrophoresis system (blue native-polyacrylamide gel electrophoresis) and sucrose density gradient centrifugation. The blue native-polyacrylamide gel electrophoresis system also showed that the NR1a subunits could form a homodimer, which was confirmed using soluble constructs of the NR1a subunit. Together these results suggest the residues N-terminal of residue 380 are important for the association of NR2A with NR1a and that the complete N-terminal domain of the NR1a subunit is required for oligomerization with NR2A. N-methyl-d-aspartate transmembrane domain nicotinic acetylcholine receptor human embryonic kidney polyacrylamide gel electrophoresis blue native α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane γ-aminobutyric acid subunit A The N-methyl-d-aspartate (NMDA)1 subtype of the glutamate receptor family is a hetero-oligomeric protein composed of two classes of NMDA receptor subunits: NR1 and NR2. The NR1 subunit is encoded by a single gene, which undergoes extensive splicing to generate eight different splice variants that differ in regional distribution and functional properties (2Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar). The NR2 subunit class consists of four different subunits, NR2A–NR2D, encoded by four separate but closely related genes (2Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar). A number of studies of mammalian cell lines either permanently or transiently transfected with NR1 alone have indicated that the NR1 subunit does not form glycine-glutamate-responsive channels and requires the presence of NR2 to do so (3Cik M. Chazot P.L. Stephenson F.A. Biochem. J. 1993; 296: 877-883Crossref PubMed Scopus (104) Google Scholar, 4Grimwood S. Le Bourdelles B. Whiting P.J. J. Neurochem. 1995; 64: 525-530Crossref PubMed Scopus (73) Google Scholar, 5Varney M.A. Jachec C. Deal C. J. Pharmacol. Exp. Ther. 1996; 279: 367-378PubMed Google Scholar). Other studies have shown that the NR1 and NR2 subunits contribute differently to the binding sites of a functional NMDA receptor. The NR1 subunit forms the glycine binding site (6Kuryatov A. Laube B. Betz H. Kuhse J. Neuron. 1994; 12: 1291-1300Abstract Full Text PDF PubMed Scopus (337) Google Scholar, 7Wafford K.A. Kathoria M. Bain C.J. Marshall G. Le Bourdelles B. Kemp J. Whiting P.J. Mol. Pharmacol. 1995; 47: 374-380PubMed Google Scholar, 8Hirai H. Kirsch J. Laube B. Betz H. Kuhse J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6031-6036Crossref PubMed Scopus (195) Google Scholar), and the NR2 subunit provides part of the glutamate binding site (9Laube B. Hirai H. Sturgess M. Betz H. Kushe J. Neuron. 1997; 18: 493-503Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 10Anson L.C. Chen P.E. Wyllie D.J.A. Colquhoun D. Schoepfer R. J. Neurosci. 1998; 18: 581-589Crossref PubMed Google Scholar). Thus, different combinations of both subunits co-assemble to form functionally distinct NMDA receptors. However, the biochemical and functional studies reported to date are ambiguous with regard to NMDA receptor subunit stoichiometry. Functional studies indicate that binding of at least two molecules of both glutamate and glycine is required for NMDA receptor activation, suggesting that at least four subunits must co-assemble (11Patneau D.K. Mayer M.L. J. Neurosci. 1990; 10: 2385-2399Crossref PubMed Google Scholar, 12Benveniste M. Mayer M.L. Br. J. Pharmacol. 1991; 104: 207-221Crossref PubMed Scopus (76) Google Scholar, 13Clements J.D. Westbrook G.L. Neuron. 1991; 7: 605-613Abstract Full Text PDF PubMed Scopus (273) Google Scholar). The molecular size of native NMDA receptors, as determined by both gel filtration and native polyacrylamide gel electrophoresis, is in the range of 605–850 kDa, which is consistent with the co-assembly of between four to five subunits (14Blahos J. Wenthold R.J. J. Biol. Chem. 1996; 271: 15669-15674Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 15Brose N. Gasic G.P. Vetter D.E. Sullivan J.M. Heinemann S.F. J. Biol. Chem. 1993; 268: 22663-22671Abstract Full Text PDF PubMed Google Scholar, 16Chazot P.L. Coleman S.K. Cik M. Stephenson F.A. J. Biol. Chem. 1994; 269: 24403-24409Abstract Full Text PDF PubMed Google Scholar). A recent biochemical study has suggested that there are three NR2 subunits per NMDA receptor complex, indicating that the NMDA receptor is at least a pentamer (17Hawkins L. Chazot P. Stephenson F.A. J. Biol. Chem. 1999; 274: 27211-27218Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Electrophysiological studies on the subunit stoichiometry using co-expressed wild-type and mutant forms of either the NR1 or NR2 subunits have also been inconclusive suggesting two or three NR1 subunits or two or three NR2 subunits per functional NMDA receptor complex (18Behe P. Stern P. Wylie D.J.A. M. Schoepfer R. Colquhoun D. Proc. R. Biol. Sci. 1995; PubMed Scopus Google Scholar, B. Kuhse J. Betz H. J. Neurosci. 1998; 18: PubMed Google Scholar, A. J. 1997; PubMed Scopus Google Scholar). The of NMDA receptor subunits the assembly of hetero-oligomeric NMDA to not been glutamate receptor subunits are to a transmembrane and domain with three transmembrane domains and a domain a that lines the N and C terminus R. Neuron. 1995; Full Text PDF PubMed Scopus Google Scholar, M. The Scholar, Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). The ligand binding domain is to be between part of the N terminus and the between and (6Kuryatov A. Laube B. Betz H. Kuhse J. Neuron. 1994; 12: 1291-1300Abstract Full Text PDF PubMed Scopus (337) Google Scholar, M. K. J. 1998; PubMed Scopus Google Scholar, B. M. P.J. Heinemann S.F. Neuron. 1994; Full Text PDF PubMed Scopus Google Scholar). the of the N terminus to the protein M. Neuron. Full Text PDF PubMed Scopus Google Scholar). The N-terminal domain has been suggested to be important in the assembly of receptor subunits in mammalian J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus (104) Google Scholar). study the of which domains of the NR1a NMDA receptor subunit are important for assembly receptor embryonic kidney were transfected with NR1a constructs containing to the N and C NR1a and soluble forms of the NR1a subunit and wild-type NR2A subunits. Since have shown that NR2A is not at the cell surface unless it is co-expressed with NR1 Le B. N. P. Whiting P.J. 1998; PubMed Scopus Google Scholar), cell surface expression of the NR2A subunit has been used to monitor subunit with co-immunoprecipitation of the different subunits. The of has been using a gel electrophoresis system and sucrose gradient the truncated NR1a subunits are a human NR1a and were by using in S. S. R.J. Whiting P.J. Le Bourdelles B. J. Neurochem. 1999; PubMed Scopus Google Scholar). was the N terminus between and C-terminal to the The constructs were by and by in binding using were as in S. Le Bourdelles B. Whiting P.J. J. Neurochem. 1995; 64: 525-530Crossref PubMed Scopus (73) Google Scholar, S. S. R.J. Whiting P.J. Le Bourdelles B. J. Neurochem. 1999; PubMed Scopus Google Scholar). were and transfected using the The NMDA receptor NR1a and NR2A subunits in the expression and were transfected at a of the were in the presence of and were the using and as A. J. Neurochem. 1999; PubMed Scopus Google that was to the to cell were by in a of acid with the with to that of the in which the was for and and were to for the were with of at containing different combinations of human NMDA subunit the expression or NR1a truncations and NR2A were at a of were at in of at with and for to were in a and at with were with two containing and at were in the the of the was transfected were with containing of the at in was by the for with The were with on in a and The was at and a of the for cell The surface in the were with of for at The were with and with and the by of The were using and the The that the the the was for by the cell and for the protein those in which was were used for transfected were on with containing and The was at and was for cell A of the was to with and at with A of was to the and by at for The were by and the was in and with these of the subunits was The were the by with and the the and cell were by and The used for were a Le B. N. P. Whiting P.J. 1998; PubMed Scopus Google and the The was using A. P. J. Neurochem. 1994; PubMed Scopus Google Scholar). were by using polyacrylamide to using a cell the were in in for The to the at were a and the which the N terminus of NR2A Le B. N. P. Whiting P.J. 1998; PubMed Scopus Google Scholar). The NR1 subunit was using to residues or to residues of the subunit or the C-terminal The were using the or to in with the of the of native protein was by using a of the of as H. G. Biochem. 1991; Scopus Google Scholar, H. G. Biochem. 1994; PubMed Scopus Google Scholar). were by with and at for of was to and on for The were at and with blue The and were with blue and with the on a gel containing The and were at and at a The gel was to a the was with in The were with and to the protein The of the containing the was with and in the as the to the of and were The used for were for the and for the which on the subunit. and were by and of cell were by in in containing and density of cell was using cell on sucrose in a for at were gradient and by for protein were transfected with of containing and with and was and the were for of The with was at for the and using The was and by on a using a The were by using and and both using the The of the different NR1a constructs used in study are in of the NR1a truncated subunits, with the of the the NR1a N-terminal and the binding sites for the glycine antagonist when in S. S. R.J. Whiting P.J. Le Bourdelles B. J. Neurochem. 1999; PubMed Scopus Google Scholar). Since the NR1 truncations that did not the glycine antagonist that of the domain important for the of the glycine binding these results are in with of the of glutamate receptor subunits. the NR1a constructs containing to residue 380 in the N terminus and NR2A were for the expression of functional channels NR1a and the and constructs functional channels with NR2A at the cell to residue 380 the N-terminal domain of NR1a NMDA Similar were also obtained in not The in the and constructs were in with those with the wild-type NR1a and NR2A subunits, suggesting that these truncated subunits form of functional channels at the cell of the C terminus and also functional channels with NR2A. Since forms a functional glycine binding these results that the acid residue 380 is important for the of functional NMDA and the NR1a truncations with using glutamate glycine were in with the indicated NR1a constructs and wild-type NR2A. in a Electrophysiological using glutamate glycine were in with the indicated NR1a constructs and wild-type NR2A. surface expression of the NR2A subunit was determined by cell surface with the NR1a reported Le B. N. P. Whiting P.J. 1998; PubMed Scopus Google Scholar), of NR1a and NR2A in a which was when NR2A was alone to the N-terminal domain of NR1a to a progressive in the cell surface expression of NR2A with there was surface NR2A of cell surface NR2A were when the subunit was co-expressed with and not the presence of a complete N terminus but C terminus and of NR1a and did not affect cell surface expression of NR2A be that the of the NR1a and NR2A were in the cell is that the in the surface expression of the constructs of expression of the the of the cell surface expression are in with the functional which that the subunits can to form functional channels the in the N terminus of NR1a residue 380. The of expression of with NR2A must of the loss of the NR1a subunit, does to surface expression of NR2A. Since cell surface expression of NR2A both subunit association and co-immunoprecipitation studies were to determine the of association of NR2A with the different NR1a The results showed that of NR1a at the C terminus on the association of the subunits as for However, of the N terminus in a progressive loss of which was with or Thus, there to be association of with subunit does not to either functional channels or surface expression of NR2A when the subunits are The results could be as suggesting that the N-terminal of NR1a residues are for subunit To they are for have NR2A with NR1a truncated the of the domain and a of NR1 the N terminus of NR1a that of of the NR1 and with NR2A in did not to cell surface expression did they with NR2A that when and NR2A are co-expressed is to be and could suggest that the NR1a N-terminal alone not be for association with NR2A. The by H. G. Biochem. 1991; Scopus Google Scholar, H. G. Biochem. 1994; PubMed Scopus Google was to a for the of NMDA receptor subunit the of these the system has also been used to oligomerization and the domains that are important in glycine receptor assembly A. J. G. J. 1999; PubMed Scopus Google Scholar, N. C. A. Kuhse J. G. Betz H. J. 1999; 18: PubMed Scopus (105) Google Scholar). is used to the NR1a subunit with molecular of and can be The of the was and could the different expression of the subunits in the different NR2A was co-expressed with the molecular of the NR1a to a with a molecular of kDa, with two at and NR2A could also be in the but was not in the NR1a However, NR2A could also be in with a molecular of when the is used for the of with the The of that of the NR1a could be The expression of the NR2A subunit alone in in of the suggesting that the NR2A subunit not be folding in the of NR1a the molecular of the receptor by have been reported to be the gel system used for the NMDA was used to and of stoichiometry H. G. Biochem. 1994; PubMed Scopus Google Scholar, A. J. A. G. G. J. 1998; PubMed Scopus Google Scholar). the with molecular of kDa, that subunit The complex in these molecular of kDa, which is the pentamer was also with molecular of kDa, which a of the complex a between the subunits. of the complex using and as A. J. A. G. G. J. 1998; PubMed Scopus Google Scholar), to the of suggesting the stoichiometry of the complex by was not of the oligomerization of the NR1a N-terminal constructs co-expressed with NR2A in of which for the NR1a constructs and NR2A could be when the were for either NR1a and NR2A that in the N terminus of the NR1a subunit or of the subunits, association of the subunits is in with the surface expression and functional of the C-terminal truncations of NR1a and with NR2A did not affect the of a molecular containing both NR1a and of the by the complex was in size to the and gave to at on the to the for when for a at is also in the which that is a containing the two subunits. and were they on similar to those with the NR1a subunit and with the in molecular not results that the oligomerization of the subunits is not by the C-terminal and they to surface expression of NR2A. to the system was a of NR1a and NR2A the assembly of the subunits was also by sucrose gradient centrifugation. of the gradient cell either the NR1a or NR2A subunit alone that NR1a has a NR2A The NR1a subunit to the which suggest that the NR1a subunit is a as in the The NR2A subunit has a with a of NR2A at the of the gradient The of NR2A the gradient and the at the of the gradient that the NR2A subunit not when it is the obtained the cell NR1a and NR2A subunits were on sucrose two of for NR1a were was in the as the and the was the gradient in the the also the of the NR2A which was in a of the the suggest that the NR1a and NR2A subunits are to form a complex that is not when either NR1a or NR2A subunits are Thus, the of receptor oligomerization was by the sucrose gradient The of the NR1a subunit transmembrane domains for subunit association with NR2A was using soluble constructs of the NR1a subunit. The consists of the and domains of NR1a by a The has the N-terminal domain of NR1a to the domain in the as of these constructs in results in a soluble form of the in the the of the transfected by size and two of the and a which the The protein the to a of the protein A similar of showed that also as two of the and the the of the form of the has molecular of kDa, the suggest that the containing a complete N-terminal domain can to the of the NR1a subunit using and were co-expressed in with NR2A and cell surface used to monitor NR2A cell surface expression. NR2A could be at the cell surface when co-expressed with the that a complete N-terminal domain but not could also be in the study have used the surface expression of NR2A when co-expressed with different NR1a truncations to determine the of NR1a that are important for subunit association and the C terminus of NR1a to residue on the surface expression of between residues at the N terminus of NR1a cell surface expression of NR2A. The progressive loss of surface expression of NR2A with of NR1a N terminus a progressive on the association of the two subunits Electrophysiological studies the on cell surface expression of as the NR1a N-terminal truncations with to residue showed with the subunit. between residues and 380 of the NR1a subunit in functional channels The the of the NR1a N-terminal truncations suggest they are or folding that alone the of subunit of the NR1a N-terminal truncations to glycine antagonist binding sites with the of functional However, it is that the of the domain of NR1a contribute to the folding of the NR1a subunit. that it has been that of the subunit at the N terminus of subunit folding when these are in mammalian S.K. K. Neurosci. Proc. 12: Scholar). the N-terminal domain of NR1a residues to be for the association with NR2A. However, the obtained the NR1a truncated N terminus and to suggest that the N-terminal alone of NR1a alone not be to cell surface expression of NR2A and not be for subunit association of cell surface expression of NR2A when co-expressed with the NR1a with cell surface could be in could a of the subunits to a of or of and/or between the NR1a and NR2A subunits. the association of NR1a with NR2A using co-immunoprecipitation was The results showed that progressive to the N-terminal domain of NR1a a in the of NR2A that could be residue 380 to the association of NR2A with suggesting that residues are important for Since co-immunoprecipitation of NR2A when co-expressed with but not when co-expressed with the NR1 N-terminal or the that residues 380 in to subunit association as the However, it is that residues are for NMDA receptor subunit association and similar to the in the receptor by J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Since cell surface expression also subunit the assembly status of the subunits was using the of the oligomerization of the NMDA receptor a of kDa, which was for both NR1a and when using with both NR1a and NR2A subunits. The size of complex is similar to that by of the NMDA receptor complex in (14Blahos J. Wenthold R.J. J. Biol. Chem. 1996; 271: 15669-15674Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 15Brose N. Gasic G.P. Vetter D.E. Sullivan J.M. Heinemann S.F. J. Biol. Chem. 1993; 268: 22663-22671Abstract Full Text PDF PubMed Google Scholar, 16Chazot P.L. Coleman S.K. Cik M. Stephenson F.A. J. Biol. Chem. 1994; 269: 24403-24409Abstract Full Text PDF PubMed Google Scholar). To the of receptor stoichiometry using the used to the stoichiometry of the and the that the system molecular for the has been which is consistent with A. J. A. G. G. J. 1998; PubMed Scopus Google Scholar). The molecular of these that and/or binding the of receptor the gel it to determine receptor subunit stoichiometry these However, by the molecular of the receptor and the with those obtained by the system used it that there is consistent of receptor by between and a similar was on the NMDA receptor subunits, the molecular for the NR1a when the subunit is be in the range of and kDa, which suggest a and of the subunit. the when NR1a and NR2A are co-expressed be and kDa, consistent with a of two NR1 and two NR2A subunits and a of NR1a and However, the of the and the of the molecular on these at the molecular a of stoichiometry for the receptor. the that the NR1a and NR2A undergoes a when the subunits are that these do the oligomerization of the NMDA receptor to be that the NR1a when the subunit is alone do the and of with the molecular but it is consistent with the sucrose gradient and which also suggest that NR1a can form Together these suggest that the NR1a subunit can to form as suggested by studies of soluble NR1 and receptor subunits A. M. K. J. 1995; PubMed Scopus Google Scholar, A. H. Laube B. Kuhse J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the system also showed that NR2A not be folding when suggesting that subunit not form the NR1a subunit is also consistent with the sucrose gradient of cell NR2A alone The NR1a truncations with C terminus and and were to co-assemble with NR2A and a complex at the cell surface similar to that with the wild-type NR1a and NR2A subunits and However, of the C terminus and of NR1a to of a complex with NR2A in studies Thus, of the NR1a subunit is for of functional NMDA receptor but of the C-terminal and of NR1a does not affect NMDA receptor subunit association or it has been suggested that of subunits is the but with the and domains to form a functional J. 1996; Full Text PDF PubMed Scopus Google Scholar). The of the transmembrane domains in the assembly of the NMDA receptor was using soluble NR1a either with or the similar to those in studies on both and NR1 subunits A. H. Laube B. Kuhse J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google A. R. K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. H. Biochem. J. 1999; PubMed Google Scholar, N. Chen 1998; PubMed Scopus Google Scholar). The constructs were in and the cell and for the presence of the of the constructs with molecular of and in the and cell and molecular are consistent with the molecular size of the the The of the oligomerization of receptor subunits of containing the constructs not so size was used as to the soluble NR1a constructs The of in the with with molecular of that the which is with a of the protein as a with the of Other studies the oligomerization of soluble domains of glutamate receptor subunits have also that the soluble forms of the domains as in A. R. K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, R. D. K. 1999; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). However, using a soluble domain of the NMDA receptor similar to the A. H. Laube B. Kuhse J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google that the protein as as to be a The in the properties of protein and that the that have protein in mammalian in The a protein that with a molecular of kDa, which the of a of the using glutamate receptor subunit constructs with a complete N-terminal domain have also with molecular similar to that for a A. R. K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). the obtained the NR1 and constructs are consistent with these and that the NR1a subunit can form a homodimer, which is on the presence of the the that could with as in cell surface studies the of of the NR1a subunit, as as the presence of a complete N-terminal for assembly with NR2A. The that can surface expression of NR2A that the transmembrane domains of NR1a are not for subunit do not that they be important for either oligomerization of the subunits or for subunit the that the domain of NR1a a the of the subunit as as with NR2A. However, the N-terminal domain of NR1a alone not be for association with NR2A. the NR1a transmembrane domains are not for association with are the NR1a C terminus or study also that in the N terminus of NR1a affect and that NR2A not in the of which has for the of studies at closely the of these subunits that are for subunit