The Stokes radius of the pro- teins in the fractions that had the ..... al., 1993) and 42 and 100 kDa (Henley et al., 1992; Kerry et al.,. 1993) add credence to the ...
THE JOWAL OF BIOL~CICAL CHEMISTRY
Vol. 269,No. 44, Issue of November 4, pp. 27384-27393, 1994 Printed in U S A .
Purification and Pharmacological and Immunochemical Characterization of Synaptic MembraneProteins with Ligand-binding Properties of N-Methyl-D-aspartate Receptors* (Received for publication, April 20, 1994, and in revised form, August 29, 1994)
Keshava N. KumarS, Kent K. BabcockS, Peter S. JohnsonOn, Xingyu Cheni, Katy T.Eggemanll, and Elias K. Michaelis$** From the Department of Pharmacology and Toxicology and the Center for Neurobiology and Immunology Research, University of Kansas, Lawrence, Kansas 66045, the §National Institute of Drug Abuse, Laboratory of Molecular Neurobiolo.gy, Baltimore, Maryland 21224, and the IDepartment of Pharmacology, University of Colorado Health Sciences Center, De&er, Colorado 80262
A method was developedfor the solubilization of approximately 50% of proteins in synaptic membranes that have ligand-binding characteristics of N-methyl+aspartate (NMDA) receptors. Affinity chromatographic separation of the solubilized proteins through L-glutamate-derivatized matrices and subsequent elution by NMDA-containing buffers led to the purification of four predominant proteins with estimated sizes of 67-70,5362, 41-43, and 28-36 kDa. The co-purification of NMDAsensitive L-glutamate binding, dizoeilpine-sensitive thienylcyclohexylpiperidine (TCP)-binding,and strychnine-insensitive glycine-binding proteins was achieved by this affinity chromatographic procedure. Glutamate, glycine, and the polyamine spermidine increased both the “on” rate and the equilibrium level of [%I]TCP binding to the isolated proteins. The group of proteins eluted by NMDA from the glutamate-derivatized matrices could be further purified through size exclusion chromatography without loss of ligand binding activity or separation of the NMDA-sensitive glutamate-binding from the dizocilpine-sensitiveTCP-binding proteins. Polyclonal and monoclonal antibodies to the cloned NMDA receptor protein NMDARl did not react with any proteins in the solubilized membrane proteins or the purified fractions. However, immunoreaction of antibodies raised against a glutamate-bindingprotein and a phosphonoaminocarboxylic acid-binding protein indicated that these are two of the major proteins in the purified fractions. Thesestudies indicate that these two proteins might be components of a complex that has some of the characteristics of NMDA receptors and that neuronal membranes may contain varieties of NMDAlike receptors composed of protein subunits that differ from the NMDARl and NMDAR.2 receptor proteins.
to their sensitivity to the specific agonists, kainate, a-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA),’ and N-methyl-D-aspartate (NMDA) (Watkins et al., 1990). Great success has beenachieved in understanding the molecular structure of these receptors through the cloning of the cDNAs for the proteins that represent kainate, AMPA, and NMDA receptor ion channels (e.g. Hollman et al. (1989), Bettler et al. (19901, Boulter et al. (1990), Keinanen et al. (1990), Moriyoshi et al. (19911, Monyer et al. (19921, Kutsuwada et al. (19921, and Hollmann and Heinemann(1994)). The cDNAs for these receptor proteins were identified by functional expression cloning. An r3H1AMPA-bindingprotein was also recently purified and identified by immunochemical procedures as being related to a cloned kainate/AMPA receptor species (Hunter andWenthold, 1992). A group of proteins previously purified from rat brain has recognition sites for NMDA receptor ligands such as L-glutamate, NMDA, glycine, 2-amino-5-phosphonopentanoate (2-AP5), thienylcyclohexyl piperidine (TCP), phencyclidine (PCP), and dizocilpine (MK-801) (Ikin et al., 1990). Synaptic membrane proteins,some of which had very similar molecular sizes to those isolated by Ikin et al. (19901, were isolated and reconstituted into liposomes, and the proteoliposomes formed with these proteins exhibited both L-glutamate and NMDAactivated cation fluxes (Ly and Michaelis, 1991). The glutamate-activated cation fluxes into liposomes were blocked by 2-AP5. The molecular sizes of the proteins reported in these two studies are not those expected of subunits of the cloned NMDA receptor proteins, i.e. the NMDARl(105 kDa)and NMDAR2 (130-165 kDa) (Moriyoshi et al., 1991; Monyer et al., 1992; Kutsuwada et al., 1992). It is possible, though, that the proteins of smaller molecular size (Ikin et al., 1990; Ly and Michaelis, 1991) are breakdown products of the NMDARl or NMDAFt2 families of proteins. The present study was focused on obtaining information L-Glutamic acid is theprincipal excitatory neurotransmitter about the ligand binding characteristicsof the 63-67- 53-57in the mammalian central nervous system (Fonnum, 1984; 41-46-, and 31-36-kDa proteins purified from rat brain synapCotman and Monaghan, 1987). The glutamate receptors that tic membranes, especially those characteristicsthat may idenform ion channels areclassified into threecategories according tify theseproteinsas components of potentially functional NMDA receptors. These studieswere also designed totrace by * This work was supported in part by Grant AA04732 from the National Institute on Alcohol Abuse and Alcoholism andGrant DAAL03The abbreviations used are: AMPA, a-amino-3-hydroxy-5-methyl-491-G-00167 fromthe Army Research Office. The costsof publication of this article were defrayedin part by the payment of page charges. This isoxazolepropionic acid; 2-AP5, 2-amino-5-phosphonopentanoicacid; acid; CGP article must thereforebe hereby marked “advertisement”in accordance CPP, 3-~~~~-2-carboxypiperazine-4-yl)-propyl-l-phosphonic with 18 U.S.C. Section 1734 solely to indicate this fact. 39653, (-c)-(E)-2-amino-4-propyl-5-phosphonopentenoic acid; CHAPS, 1 Supported by Training GrantDA 05472 from the National Institute 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate; MK-801, ~+)-5-methyl-l0,ll-dihydro-5H-dibenzo[a,d]cyclohepten-5,10on Drug Abuse. ** To whom correspondence should be addressed: Dept. of Pharma- imine; NMDA, N-methybaspartate; NMDAR1, clonedN-methybascology and Toxicology, University of Kansas, Lawrence, KS 66045. ”el.: partate receptor protein;PCP, phencyclidine; PAGE, polyacrylamide gel electrophoresis; TCP,N-(l-(2-thienyl)cyclohexyl)piperidine. 913-864-4001;Fax: 913-864-5219.
27384
NMDA Ligand-binding
Proteins Synaptic from Membranes
27385
sence of L-glutamate and glycine, it was determined that the binding reactions were a t equilibrium after 50 min of incubation at 23 "C. Complete saturation binding isotherms were obtained for NMDA-sensitive ~-[~H]glutamate and MK-801-sensitive r3H1TCPbinding to the isolated proteins by measuring ligand binding to the proteins in triplicate samples for each concentration of the ligand. The respective nonspecific ligand binding at each concentration was determined also in triplicate samples in the presence of an excess competing ligand, i.e. NMDA or MK-801. All ligand binding data were analyzed according to Munson (1983) with the LIGAND program. Immunochemical Characterization of Synaptic Membrane Proteins EXPERIMENTALPROCEDURES and Isolated Protein Fractions-The synaptic membrane proteins, soluPurification of Glutamate and NMDA-binding Proteins by Affznity bilized membrane proteins, and all protein fractions isolated through Chromatography-The isolation of synaptic membranes from 2 whole chromatographic procedures weresubjected to SDS-PAGE according to the procedures of Park and Labbe (1989). All protein samples were rat brain homogenates was performed as described previouslyWhen et mM dithioal., 1988). The purified synaptic membranes were solubilizedin 10 m~ dissolved in sample buffer that contained 2% (w/v) SDS, 250 potassium phosphate buffer, pH 7.4, that contained four protease in- threitol, and 500 m~ iodoacetamide. These high concentrations of dihibitors (0.3 mM phenylmethanesulfonyl fluoride, 10 mM €-amino ca- thiothreitol and iodoacetamide were foundto inhibit the appearance of proic acid, 0.1 mM EGTA, 0.1 mM benzamide), 1.5% (w/v) CHAPS,0.5% band artifacts in silver- and gold-stained gels and membranes, respectively (see also Park and Labb6 (1989)).Western blot analyses of the (w/v) n-octylglucoside, and 10% (v/v) glycerol. The solubilized membrane proteins were loaded onto a ReactiGel column that had been proteins isolated at various steps of the purification scheme used were (Chen et al., reacted with L-glutamate (Wanget al., 1992),and fractions were eluted performed accordingto the procedures described previously by the successive introduction of 30 m~ KC1,5 m~ NMDA, and 1M KC1 1988; Eaton et al., 1990). The antiserum to the NMDARl receptor subunit (obtained from R. Wenthold) was raised in rabbits against as described previously(Kumar et al., 1991b).All fractions representing peptide (LQNQKDTVLPRRAIEREEBpeaks of protein were pooled and dialyzed at 4 "C against 40 m~ Trid a synthetic, 30-aminoacid SO, buffer, pH 7.2. The fraction eluted by the introduction of 5 m~ QLQLCSRHRES) at thecarboxyl-terminalof NMDARl (Petralia et al., 1994).A monoclonal antibody (MAb54.1) raised against residues 660NMDA was purified further by gel filtration chromatography on a 1.5 x 44-cm column of Sephacryl S-400 HR according to the procedures de- 811 of a bacterial fusion protein (Brose et al., 1993) was also used in scribed previously (Kumar et al., 1991a). The protein species in each immunoblot studies with synaptic membranes and solubilized fracfraction were analyzed by sodium dodecyl sulfate-polyacrylamide gel tions. The other antisera used in theWestern blots were the antiglutamate-binding protein (Eaton et al., 1990) and the anti-CPP-binding electrophoresis (SDS-PAGE). Radioligand Binding Assays-Ligand binding to the various subcel- protein (Eggemanet al., 1993)antisera from rabbits. The dilution of all antisera used in Western blots was 1:1000, and the same dilution was lular and protein fractions was assayed by a polyethyleneglycol-yusedfrom the secondary anti-rabbit antibody-alkaline phosphatase described previously globulin precipitationkentrifugation method (Chen et al., 1988). Aliquotsof synaptic membranes or solubilized mem- conjugate. brane proteins (400-500 pg) or purified proteins (400-600 ng) were RESULTS incubated in Microfuge tubes with 40 mM Tris/SO, buffer, pH 7.2, that contained 0.5% (w/v) y-globulinand varying concentrations of radioacLigand Binding Characteristics of Proteins Eluted from binding tive ligands or displacing agents. Nonspecific ~-[~Hlglutamate Glutamate AftinityMatrices-Treatment of synaptic memwas defined as radioactive ligand binding measured in the presence of branes with the combination of detergents employed in this 100 p~ NMDA or 100 p~ L-glutamate, as indicated under "Results."The study (1.5% CHAPS and 0.5% n-octylglucoside)brought about nonspecific binding defined by incubating in the presence of 100 p~ NMDA averaged 72% of the total binding in isolated protein fractions the solubilization of 44% of synaptic membrane proteins. and 80% in membranes and solubilized membrane fractions. The aver- NMDA-sensitive ~-[~Hlglutamate binding and MK-801-sensiage NMDA-sensitive specific binding at 150 nM ~-[~Hlglutamate was tive [3H]TCP binding to the solubilized synaptic membrane 3,140 cpm (+320 S.E., n = 8) for the isolated protein fractions, 1,714 proteins was measured after 2-3 days of dialysis of the ex(+400 S.E., n = 5 ) for synaptic membranes, and 2,320 k175 S.E., n = 3) for solubilized synaptic membranes. Nonspecific L3HlTCP binding was tracted proteins against several changes of buffer that did not defined as ligand bound to various preparations in the presence of 100 contain any detergents. Ligand binding to the solubilized memp~ MK-801, nonspecific L3H1CPP binding was defined as that deter- brane proteins was markedly activated as compared with the mined in the presence of 100 p~ 2-AP5, and nonspecific L3H1glycine interaction of the same ligands with synaptic membranes prior binding was defined as ligand binding measured in the presence of to solubilization. For example, NMDA-sensitive ~-[~H]glutaeither 100 p~ glycine or D-serine. All incubation mixtures used to determine L3Hlglycine binding also contained 100 1.1~strychnine in order mate binding to the solubilized proteins was 14.1 pmol/mg proand that for MK-801-sensitive to prevent glycine binding to strychnine-sensitive receptors. The non- tein (150 nM ~-[~Hlglutamate) specific binding of L3HlTCP to isolated proteins averaged 49% of total L3H1TCPbinding was 16.7 pmol/mgprotein (100 nM [3H]TCP,n TCP binding while that to synaptic membranes and solubilized mem- = three determinations). The specific activity of ligand-binding brane proteins averaged 73% of total TCP binding. Specific MK-801- sites associated with the proteins in the membrane pellet that ) to the isolated proteins was 2,957 remained after the solubilization step was enhanced by the sensitive PHITCP (96 n ~ binding cpm k286 S.E., n = 4), to synaptic membranes, 2,061 cpm (+303 S.E., n detergent extraction procedure to a level identical t o that = 4), and to solubilized membrane proteins, 4,800 cpm(400S.E., n = 3). The nonspecificbinding of L3H1glycineto isolated proteins averaged 76% observed for the detergent-solubilized proteins. The values for of total glycine binding while that to synaptic membranes and solubi- ligand binding to the two fractions following treatment with lized membrane proteins was 83%. Specific strychnine-insensitive the detergents and glycerol were approximately 10 times the L3HlgIycine (100 nM) binding to isolated proteins averaged 2,410 cpm specific activity of ligand-binding proteins in intact synaptic (+175 S.E., n = 4); to synaptic membranes averaged 2,328 cpm (+175 membranes (Table I). S.E., n = 3); andto solubilized membrane proteins averaged 3,823 cpm Because of the apparently equal activation by CHAPS and (2823 S.E., n = 6). Binding of ['HIAMPA to kainate and quisqualate-sensitive sites was n-octylglucoside of ligand binding to both the solubilized and determined as described previously(Keinanen et al., 1990)and nonspe- nonsolubilized synaptic membrane proteins, the relative bindcific binding was defined as L3H1AMPAbindingin thepresence of 100p~ ing activities associated with these two fractions following dekainate or 100 PM quisqualate. All incubations of the brain subcellular tergent extraction of membrane proteins could be used to estifractions and the isolated proteins with the various ligands were con- mate the solubilization efficiency of the procedures employed. ducted at 23 "C for either 30 min (with the ligands ~-[~H]glutamate, L3H3glycine,L3HICPP, and I3H1AMPA) or 60 min (with the ligand Based on the estimatesof MK-801-sensitiveL3H1TCPbinding to [3HlTCP).The 60-min incubation period with [3H]TCPwas chosen be- the solubilized and nonsolubilized synaptic membrane procause in preliminary studies of the kinetics of C3H]TCPbinding to either teins, the solubilization efficiency with respect to these binding cell subfractions or isolated proteins, performed in the presence or ab- entities was 51%. Similar calculations established a solubiliza-
immunochemical procedures the presence in the purified protein fractions of the key subunit of cloned NMDA receptor proteins, the NMDARl (Moriyoshiet al., 1991), and of two other proteins with glutamate andNMDA receptor-like binding characteristics, the 63-71-kDa glutamate-binding protein (Chen et al., 1988; Kumar et al., 1991b) and the 53-58-kDa carboxypiperazinyl-propylphosphonic acid (CPP)-binding protein (Cunningham and Michaelis, 1990; Eggeman et al., 1993).
NMDA Ligand-binding Proteins from Synaptic Membranes
27386
TABLEI Purification of NMDA-sensitiue ~-['H]glutamate, MK-801-sensitiuer3H]TG'e and strychnine-insensitiue[3H]glycine-binding proteins Specific binding activity" Fraction
Total protein
~-['H]Glutamate
['HITCP pmol lmg protein
0.13* 0.05 (6) 0.24f 0.04 (6) 1.45 * 0.24
Homogenate p2
Synaptic membranes
(6)
30 m KC1 eluate
114 -c 40 (9)
[3HlGlycine
0.20* 0.06 (6) 0.27-c 0.03 (6) 1.31_+ 0.18 (6) ND
5 mM NMDA eluate
0.017f 0.002 (9)
270 f 28 (9)
218 f 94 (5)
1 M KC1 eluate
0.021-c 0.003 (9)
60 k 23 (9)
ND
L-[3HlGlutamatebinding was measured in thepresence of 150 nM ~-[~H]glutamate. Nonspecific binding was determined in thepresence of 100 I3H1TCPbinding was measured in the presence of 100 rm L3H1TCPand non-specificbinding was estimated as the binding in the presence of 100 I.~M MK-801. Strychnine-insensitive [3H]glycinebinding was measured in the presence o f 9 5 nM [3H]glycine. Nonspecific binding was measured in the presence of 100 I.~Mglycine. All values are the mean (* S.E.) of the number of determinations shown in parentheses. ND, not determined. L-glutamate These values of ~-[~Hlglutamate binding were determined under identical conditions as those described above except that 100 was used as the displacing agent to define nonspecificbinding to the proteins. a
~ uNMDA. l
4
tion eficiency of 55% for the strychnine-insensitive, D-serine0.05 sensitive [3H]glycine-binding proteins. The solubilized fraction of synaptic membrane proteins was subjected to affinity chromatography on L-glutamate-derivatized ReactiGel matrices, and the proteins that were retained on the matrixwere eluted by the sequentialintroduction into the elution buffer of 30 lll~KCl, followed by 5 mM NMDA, and 1 M KC1 (Fig. 1).The pooled fractions eluted ineach of these steps represented a n enrichment of both NMDA-sensitive ~-[~H]glutamate-binding proteins and MK-801-sensitive L3H]TCP-binding entities (Table I). Both the NMDA-sensitive L-glutamateI I I I I I I I I I I binding entities and MK-801-sensitive TCP recognition 10 20 30 40 proteins were most enriched in theNMDA-eluted protein fraction. The proteins eluted by the introduction of 5 m NMDA FRACTION NUMBER exhibitedapproximately equalenrichmentin L-glutamateFIG.1. Affinity chromatographyof solubilized synaptic membinding entities andTCP-binding proteins, as well as glycine- brane proteins on an L-glutamate-derived ReactiGel matrix.A binding proteins (Table I). The NMDA-eluted fractions con- typical chromatogram from the affinity chromatography step isshown. tained also 2-AP-5-sensitive L3H]CPP-binding proteins (100 e It includes the three steps in the elution of bound proteins from the 25 pmoVmg protein, 100 nM CPP, n = six determinations), but column (marked by arrows) following loading of the proteins and washing of the matrix with buffer as described under "Experimental their enrichment in this fraction was less than that for the Procedures." glutamate, TCP, and glycine-binding entities. Some of the CPPbinding proteins remained tightlybound to the ReactiGel ma- chromatographic separation on glutamate-ReactiGel matrices trix and were recovered in the fractions collected following were NMDA-sensitive sites, as shown by the fact that displaceproteins by 100 VM elution of the column with 1 M KC1 (data not shown). Never- ment of ~-[~Hlglutamate binding to these theless, four ligand-binding entities thatdefine the pharmaco- glutamate gave values for the specific glutamate binding to the logical features of NMDA receptors in neuronal membranes, isolated protein fractions essentially identical to those for displacement by 100 1.1~NMDA (Table I). Furthermore, there was were co-purified in the NMDA-eluted fraction. The fractions eluted by 30 II~MKC1 exhibited a greater en- no trace of either [3H]kainate or [3H]AMPAbinding to thefracrichment of TCP-binding entities compared t o glutamate-bind- tion that was isolated from the glutamate-ReactiGel columns by the NMDA-containing buffers. The lack of kainate and ing proteins, whereas those eluted by 1 M KC1 were highly AMPA-binding proteins in theisolated fraction was consistent enriched in NMDA-sensitive ~-[~H]glutamate-bindng sites but exhibited a considerably lower enrichment in PHITCP-binding with the observations that nearly all glutamate-binding entientities (Table I). This mightindicate that either partialdisso- ties in thisfraction were sensitive to NMDA (Table I). The values for specific ligand bindingto theisolated fractions ciation of ligand-binding proteins or loss of activity of some of the proteins hasoccurred during elution with theKCl-contain- shown in Table I were obtainedfollowing 2 days of dialysis of all ing buffers. Approximately 80-100% of the glutamate-binding samples. However, the extent to which detergent molecules sites associated with the proteins that were isolated through remaining in the isolated fractions affected ligand binding to
NMDA Ligand-binding Proteins Synaptic from Membranes 1.5
27387 TABLE I1
I
Kinetic constants for PHITCP binding to the fraction eluted by 5 mM NMDA measured in the presence or absence of glutamate and glycine or spermidine Kinetic constants' Condition
Control 5 p~ glutamate plus 5 p~ glycine 1 mM spermidine 5 p~ glutamate plus 5 PM glycine plus 1 mM
k,,, ( m i d )
B,(pmol/mg)
0.040f 0.004 0.069 2 0.006
326 f 15 447 f 13
0.060 f 0.009
434 2 24 471 f 26
0.074f 0.012
spermidine L3H1TCPbinding was measured as describedin Table I. The values represent the mean (2 S.E.) of six determinations from two separate purification experiments. The estimates of k, and Be, are based on computer-assistednonlinear least squares fitting of the data t o a pseudo-first order rate equation. a
0
100
200
[ 3H]TCP
300
400
(nM)
FIG.2. Isotherm of MK-801-sensitive ['HITCP binding to the proteins elutedfrom the ReactiGel columnby buffers that contain 5 m~ NMDA. Binding of [3HlTCP to the isolated proteins was measured at concentrations of the ligand ranging between 5 and 400 nM. Each value is the mean of triplicate determinations from two protein the nonspecific preparations of the total [3H]TCPbindingminus was measured at each [3H]TCP binding. The nonspecific ligand binding TCP concentration and inthe presence of 100 p~ MK-801. The nonspecific binding was linear across the ligand concentrations used in these assays and was subtracted from the total L3HITCP boundto the protein. All incubations with the radioactive ligand were for 60 min. The curve drawn represents the calculated values for ligand bound obtained by computer-assisted fitting of the data as described under "Experimental Procedures." Optimal fitting of the data to the generalized Scatchard equation was achieved when the constants KD = 73 n~ and B,, = 1.5 nmoVmg protein were used. [3HITCP binding to the isolated proteins was measured in the presence of 5 p~ L-glutamate, 5 p~ glycine, and 1
fraction. The KO and B,, for [3HlTCP binding to the purified proteins were estimated by conducting the ligand binding studies in assay media thatcontained 5 p~ L-glutamate, 5 1.1~glycine, and 1mM spermidine. TheKD for L3H1TCPbinding to the purified proteins measured in the absence of L-glutamate, glycine, or spermidine was 53 nM and the B,, was 560 pmoVmg protein. Interaction of the agonists NMDA or glutamate and of the allosteric co-activatorglycine with NMDAreceptors in synaptic membranes produces an increase in both the observed association rate constant (kob8)and the maximal amount of c3H1TCP bound to these receptors at equilibrium (Eleq) (Bonhaus and mM spermidine. McNamara, 1988; Javitt and Zukin, 1989). The occupation of the proteins in those fractions wasdifficult to assess. No esti- the polyamine sites on the NMDA-receptor complex by spermof mation of the purification factor for the various ligand-binding ine or spermidinealsoenhancesthemaximumamount proteins isolated by chromatographic separation on L-gluta- 13H]TCPbound to membrane receptors at equilibrium (Ransom mate-derivatized columns was attemptedbecause of the uncer- andStec, 1988). Inthepresentstudies, MK-801-sensitive tainty about the potential activation of the ligand-binding pro- L3H1TCP(100 n ~binding ) to the NMDA-eluted protein fraction teins by any remaining detergents in these preparations. The exhibited kinetics that were best described by a single compoNMDA-eluted fractions from a few preparations weresubjected nent, first-order rate equation. The kobsand Be, shown inTable t o extensive dialysis forup to 12 days post-purification in order I1 represent the best estimatesof these constants obtained by to remove contaminants of small molecular size that might be computer-assisted, nonlinear least-squares fitting of the data present in these fractions. Such extensive dialysis of the puri- to such a rate equation. L-Glutamate and glycine added to the fied fractions increased the ligand binding activities of the iso- assay medium at a concentration of 5 PM and spermidine at 1 kobsand theB , for the binding lated proteins. For example, NMDA-sensitive ~-[~H]glutamate mM increased both the estimated binding was increasedfrom 195 2 15 pmoVmg protein at day 2 of L3H1TCP t o the isolated protein fraction (Table 11).The estiof dialysis to 434 2 34 pmoVmg protein ( n = four determina- mated k,,, and Be, for TCP binding to theisolated proteins in tions) at day 12following the initiationof dialysis. This maybe the presence of glutamate and glycine or in the presence of an indication that some substance present in thepurified frac- spermidine alonewere approximately 50-73 and 33-37% tion, possibly remaining NMDA, interfered with ligand binding greater than the k,, and Beq, respectively, in the absence of to the isolated proteins. these receptor activators. The concentrations of glutamate, glyBecause of the relative enrichment of ligand-binding entities cine, and spermidine usedt o determine thepossible activation in the NMDA-eluted fraction in comparison with the other of L3H1TCPbinding to the isolated proteins were in the range of this fraction was concentrations that were previously shown to bring about neareluates from the glutamate-ReactiGel matrix, used for the further characterization of the ligand binding prop- maximal activation of [3HlMK-801 binding to deoxycholateerties of the isolated proteins. The estimated equilibrium dis- solubilized synapticmembraneproteins(McKernan et al., binding to thepro- 1989; Bakker et al., 1991). sociation constant (K,) for ~-[~Hlglutamate teinsinthefractioneluted from theglutamate-ReactiGel Protein Composition of the Fractions Isolated by Glutamate capac- Affznity Chromatography-The molecular sizes of the proteins matrix by NMDA was 99 n M and the maximum binding for these sites was1.22 nmoVmg protein (valuesfrom in theNMDA-eluted fraction were estimatedby SDS-PAGE to ity (El,,) three purification experiments). The proteins used in the con- be 70,62,43 and 41 (doublet), and 36 and (doublet) 31 kDa (see duct of these assays were dialyzed for only 2 days following Fig. 1in Kumaret al. (1991b)). The proteins around 41-43 and their isolation by chromatographic procedures. The inhibitory 31-36 kDa frequently migratedon SDS-PAGE either asdiffuse constant (K,)for NMDA displacement of ~-[~H]glutamate bound bands or as doublets. Concentrations of NMDA as low as 1m~ to these proteins was 490 I". The estimated KD for [3H]TCP wereused successfully t o elute the same proteins as those of proteins was 73nM and theB,, binding to the same fraction eluted from the glutamate-derivatized matrix by 5 mM NMDA. was 1.5 nmol/mg protein (Fig. 2),a value that is very similar to Two protein groups, those of 41-43 and 31-36 kDa size, were t o this protein the most highly enriched proteins in the fraction eluted from the estimated B,, for ~-[~H]glutamate binding
27388
NMDA Ligand-binding
Proteins Synaptic fromMembranes
the column following the introduction of 30 mM KC1, but this fraction also containedother proteins ranging molecular in size from 26 to 100 kDa (data not shown). The protein fraction eluted by the introduction of 1 M KC1 contained many more protein bands inaddition to thefour majorbands present in the fraction eluted by NMDA-eluted fraction or those present in the 30 mM KC1. If ReactiGel matrices that were derivatized with ethanolamine rather thanL-glutamate were used in the chromatographic separationof solubilized synaptic membranes, the fractions eluted by KCl, glutamate, or NMDA introduced into the elution buffers were heavily contaminated by many proteins anddid not exhibit any apparent enrichment of the group .” 0 5 10 20 15 25 35 30 of proteins described above (data not shown). Molecular size-exclusion chromatography on Sephacryl S-400 HR was used to purify further the proteins in fraction the eluted from the glutamate-ReactiGel columnsby NMDA. A major protein peak that eluted between fractions 21 and 24 contained the highestactivity of NMDA-sensitive ~-[~H]glutamate and MK-801-sensitive [3HlTCP binding entities (Fig. 3, A and B ) . There was good reproducibility across a series of purification experiments in the elution pattern of the ligand-binding proteins from the Sephacryl column. In different chromatographicanalyses,thepeak of ligand-binding proteinswas eluted at fractions 21, 22, or 23. The Stokes radius of the proteins in the fractions that had the highestligand binding activities was estimatedaccording to procedures described previ0 5 10 15 20 25 30 35 ously (Kumar et al., 1991a) and had an average value of 5.1 nm. FRACTION Y For the experiments shown in Fig. 3, the NMDA-sensitive ~-[~H]glutamate binding activity associated with the proteins infractionnumber23 was 490 pmol/mg protein(150 nM ~-[~H]glutamate) and the MK-801 sensitive [3HlTCP binding activity was 408 pmol/mg protein (100 nM L3H1TCP) (averages from three purification experiments). The values of glutamate and TCP binding to purified the proteins indicated only a 2-fold purification of the binding proteins when comparedwith their levels in thefraction eluted by NMDA-containing buffers from the glutamate-ReactiGel column. This relatively small level of enrichment of ligand-binding proteins in the peak fractions following chromatographic separation on Sephacryl S-400 HR might have been due primarily to the removal of substances that inhibitedligand binding to these proteins rather than an 21 2 2 2 3 2 4 2528 enrichment of the binding proteins. Fractkn Number The protein fractions isolated by Sephacryl S-400 HR that binding activities FIG. 3. Elution profile of proteins and ligand had the highest ligand binding activity contained four major SephacrylS-400 HR chromatography of the proteins that protein species and a few minor ones (Fig. 3C). Some of the from were eluted from a glutamate ReactiGel column by 5 mM NMDA. proteins formed doublet, triplet, or diffuse bands under the A and B, pattern of elution of proteins and the NMDA-sensitive ( A ) and of MK-801-sensitive [’HITCPconditions of SDS-PAGE. The average molecular sizes for the ~-[~Hlglutamate-binding entities four major protein species were obtained from analyses of R, binding proteins (B).Conditions for application, elution, and detection proteins during the conduct ofsize exclusion chromatography were as values determined by SDS-PAGE and were 61, 51, 41, and 28 of described under “ExperimentalProcedures.”The results shown are the kDa ( n = five purification experiments). The sameprotein frac- average absorbance values obtained from four chromatographic sepations also contained two or three protein bands withmolecular rations of one protein sample. The ligand binding data are the average of triplicate determinations from three of these chromatographic sepasizes ranging between 75 and 90 kDa(Fig. 3C). rations. C, electrophoretic analysis of the proteins eluted from the Immunochemical Characterization of the Isolated ProteinsSephacryl column. SDS-PAGE was performed on all fractions that repNMDARl is the subunitof NMDA receptors that is thought to resented peaks in the absorbance monitored at 280 nm from one chroexpress all ligandrecognition sites associated with NMDA re- matographic elution. The fractions shown (numbers 21-26) are those ceptors (Moriyoshi et al., 1991). The possible presence of that correspond to the peak of protein which had the ligand binding NMDARl in theprotein fraction obtained following treatment characteristics shown in A and B. The arrows on the left mark the positions of the major protein species detected that have the following of synaptic membranes with CHAPS and n-octylglucoside or estimated sizes: 70, 50, 46, 35, and 25 kDa. in the fractionsisolated by chromatography on glutamateReactiGel columns was assessedby the useof specific antibodies raised against a 30-amino acid long peptide synthesized to membrane pellet obtained after detergent treatment of the represent thecarboxyl-terminal sequence of the NMDARl (see membranes, but this protein was almost completely absent “Experimental Procedures”). As shown in Fig. 4 A , the antifrom the solubilized extract of the synaptic membranes (Fig. NMDARl antibodies labeled only a single protein in synaptic 4A). The fractions isolated through chromatographic separamembranes, a protein of estimated size equal to 107 kDa. A tion on L-glutamate-derivatized ReactiGel matrices did not conprotein band with an identical size was also labeled in the tain any traceof the 107-kDa protein that was labeled by the
C
1
NMDA Ligand-bindingProteins from Synaptic Membranes
27389
0kDa
11697 66 -
200
45
-
31
-
21
a a
b
c
kDa
97 66
--
b
0
f'i
kDa
97 66
--
4545-
31 31
21
21
-
-
C
I '
:.
I
a
b
c
FIG. 5. Immune labeling with an antiserum to the glutamatebinding protein of synaptic membrane and solubilized synaptic membrane proteins, and of protein fractions isolated through FIG.4. Immune labeling with an antiserum to the NMDARl of glutamate-ReactiGel columns.A, Western blots of synaptic memsynaptic membrane and solubilized synaptic membrane probrane proteins(lane a,27 pg), solubilized synaptic membrane proteins teins, and of protein fractions isolated through glutamate-&- (lane b, 24 pg), andnonsolubilized membrane pellet proteins(lane c, 37 actiGel columns. A, Western blots of synaptic membrane proteins pg). B , Western blotsof synaptic membrane proteins (lane a, 28 pg) and (lane a , 27 pg), solubilized synaptic membrane proteins(lane b, 24 pg), proteins eluted from glutamate-derivatized columns by either 30 mM and nonsolubilized membrane pellet proteins (lane c, 37 pg). B, Western KC1 (lane b, 4 pg) or 5 mM NMDA (lane c, 2.3 pg).All procedures in the blots of synaptic membrane proteins(lane a,28 pg) and proteins elutedconduct of the studiesshown in this figure were performed a s described from glutamate-derivatized columnsby either 30 mM KC1 (lane b, 4 pg) in the legend to Fig. 4. The estimated molecular sizes of the proteins or 5 mM NMDA (lane c, 2.3 pg). The isolationof membranes, solubili- labeled are presented in the text. zation of membrane proteins, isolationof various protein fractions, and conduct of SDS-PAGE and Western blot analyses were performed as bands of 45-50 and approximately 30 kDa were labeled by this described under "Experimental Procedures." The estimated molecular antibody. None of these bands appeared in soluble the extract of size of the protein labeled is presented in the text.
a
b
c
the synaptic membrane. The isolated fractions were examined for the presence of anti-NMDAR1 antiserum in synaptic membranes (Fig. 4B). Since the anti-NMDAR1 antiserum used in these studies was other proteins that are known to contain ligand-binding sites raised against a region of the protein that may not be present for L-glutamate, CPP, (+)-(E)-2-amino-4-propyl-5-phosphonoin some of the variantforms of NMDARl (Sugihara et al., 1992; pentenoic acid (CGP 39653), 2-AP5, and NMDA. Antibodies Hollmann et al., 1993) or may have been proteolytically cleaved previously raised against the purified 63-71-kDa glutamateduring solubilization of the membrane proteins, a monoclonal binding protein (Chenet al., 1988; Eaton et al., 1990) were used antibody raised against a 151-amino acid region between the t o examine thepresence of this protein in theisolated fractions. putative transmembrane domains I11 and IV (Brose et al., The antiserum labeled primarily a 65-67-kDa protein band in 1993) was also used in Western blot analyses. This antibody the synaptic membranes, the solubilized synaptic membrane labeled a major band of 112 kDa in synaptic membranes and inprotein fraction, and the pelleted membrane fraction following the pellet of nonsolubilized proteins, butdid not labelany pro- extraction with the detergents(Fig. 5A). Bands with estimated size equal to 61-63 kDa were also labeled weakly by these tein bands in the soluble extract from synaptic membranes (data not shown). This monoclonal antibody appeared to react antibodies in a pattern that wasvery similar to thatdescribed purified glutaalso with probable degradation products of the NMDARl pro- previously for the synaptic membranes and the tein in the pellet of nonsolubilized proteins as 2-3 protein mate-binding protein (Chen et al., 1988; Eaton et al., 1990). A
NMDA Ligand-binding Proteins from Synaptic Membranes
27390
0
kDa
66
-
45
-
200 11697
31
21
a
b
J
0
kDa 97
-
66
-
45
-
31
-
a
b
C
8
kDa
97
-
66
-
45
-
31
-
21
-
-
a b FIG. 6. Immune labeling with antisera to the CPP-binding and
doublet of protein bands with estimated sizes of 69 (most heavily labeled) and 61kDa was labeled in anotherblot of synaptic membrane proteins and of the 30 mM KC1 and 5 mM NMDAeluted fractions from the glutamate-ReactiGel columns (Fig. 5B). Another protein that has ligand-binding sites that may be associated with NMDA receptors is a 54-58-kDa CPP-binding and CGP 39653-binding protein (Cunningham and Michaelis, were 1990; Eggeman et al., 1993). In synaptic membranes that isolated in the presence of protease inhibitors, treated with SDS immediately following their isolation, and stored at -70 "C, the antibodies labeled mostheavily two protein bands, an 83- and 179-197-kDa protein (Fig. 6A). Several less welllabeled bands were also detected, the most prominent being a 140- and 39-kDa band (Fig. 6A). If the SDS-PAGE was performed on membrane proteins that were solubilized in SDS but were not treated with dithiothreitol and iodoacetamide, then the major band detected was that which migrated at a molecular size between 186 and 197 kDa (Fig. 6A). Storage of synaptic membranesfor 2 to 4 days at 4 "C prior to theaddition of SDS led to the appearanceof a broad band between 55 and 60 kDa, a more prominent band at 39 kDa, a complete disappearance of the 179-197-kDa species, and a partial disappearance of the 83-kDa band recognized by the anti-CPP-binding protein antiserum (data not shown). When the membrane proteins were solubilized in the buffer that contained the detergents CHAPS and n-octylglucoside in preparation for the conduct of affinitychromatography, the anti-CPP-binding-protein antiserum labeled a broad protein band of 53-57 kDa size, as well as a n 81-kDa band (Fig. 6B). The higher molecular mass protein(s) of 179-197 kDa labeled by the antiserum in synaptic membranes was absent from the solubilized synaptic membrane protein fraction. The same antiserum labeled a 53-kDa protein in the fractions eluted from the glutamate-ReactiGel column by either 30 m~ KC1- or 5 mM NMDA-containing buffers (Fig. 6B). In thesefractions, however, both the 81-83- and the 179-197-kDa proteins were either absentor not recognized by the antibodies. In protein fractions that represented a component of the peak of glutamate andTCP-binding proteins beingeluted from the Sephacryl S-400 HR column, each of the antiglutamatebinding protein and anti-CPP-binding protein antibodies recognized one major protein species. The antiglutamate-binding protein antiserum labeled most heavily a 68-kDa protein and less strongly proteins of estimated size between48 and 63kDa (Fig. 6C). In the same fraction, the anti-CPP-binding protein antiserum labeled a broad band of proteins between 53 and62 kDa (Fig. 6C). The proteins labeled by the two antisera corresponded to two of the four predominant protein bands in this fraction purified through size exclusion chromatography (data not shown). DISCUSSION
A complex of proteins with molecular sizes of 61-70, 51-62, glutamate-binding proteinsof synaptic membrane and solubi- 4 1 4 6 , and 28-36 has been isolated in two laboratories using lized synapticmembrane proteins, andof protein fractionsiso- three differentaffinitychromatographic procedures: affinity lated through glutamate-ReactiGel and Sephacryl S-400HR colchromatography of cholate-solubilized synaptic membraneproumns. A, analysis of proteins in synaptic membranes labeled by a n antiserum to the CPP-binding protein. Lune a represents a Western blot teinsthrough amino-PCPderivatized matrices(Ikin et al., of synaptic membrane proteins (34 pg) subjected t o dithiothreitol plus 1990), affinity chromatography of n-octylglucoside-solubilized iodoacetamide treatment (see "Experimental Procedures") prior to SDSsynaptic membrane proteins through a glutamate-derivatized PAGE and Western blot analysis. Lune b represents synaptic membrane proteins (12 pg) prepared under identical conditions a s for lane a but glass fiber matrix (Ly and Michaelis, 1991), and CHAPS plus not treated with dithiothreitol and iodoacetamide. B , Western blot of solubilized synaptic membrane proteins(lane a, 23 pg), and of proteins eluted from glutamate-derivatized columns by either 30 mM KC1 (lane b, proteins chromatographed on Sephacryl S-400HR were those isolated by elution with5 mM NMDA. All 4 pg) or 5 mM NMDA (lane c, 2.3 pg). C , immune labeling of proteins through glutamate ReactiGel columns isolatedthroughSephacryl S-400 HR (fraction number 21, 2 pg of procedures in the conduct of the studies shown in this figure were protein per lane) and reacted with antiglutamate-binding protein antiperformed as described in the legend Fig. to 4. The estimated molecular serum (lane a ) and anti-CPP-binding protein antiserum (lane b ) . The sizes of the proteins labeled are presented in the text.
ins Membranes from NMDA Ligand-bindingProteiSynaptic
27391
mately equal t o the “fast” rate of L3H1MK-801 binding to membrane receptors (Javitt and Zukin, 1989). However, the increase in kabs for [3H]TCP binding t o the isolated proteins produced by glutamate, glycine, and spermidine was greater than that detected for the activation by glutamate and glycine References of [3H]MK-801binding to synaptic membranes. Despite such an increase in the kobsof TCP binding to the isolated proteins in nM the presence of glutamate and glycine, the KD for13HlTCP 48 McVittie and Sibly (1989) TCP binding to these proteins was not affected substantially by the 35 Ambar et al. (1988) Ikin et al. (1990) 60 presence of the two agonists. This might have been due t o a Present study 73 concomitant increase induced by glutamate andglycine in both the “on” and “off rates of I3H]TCP binding to the isolated proMcKernan Glutamate 126‘ et al. (1989) teins. NMDA and glutamate have been shown t o have such an 150” Ikin al. et (1990) effect on the on and off rates of TCP binding to synaptic mem99 Present study branes (Bonhaus and McNamara, 1988).The major effectof the NMDA McKernan 874‘ et al. (1989) agonists on I3H]TCPbinding to the isolated proteins appeared 500” Ikin al. et (1990) to be on the Bmm.This type of increase in B,, for l3H1TCP 490 Present study binding to the proteins may be due to the opening by glutamate, glycine, and spermidine of a path for the ligand t o gain a The values from published studies for glutamate and NMDA interaction with solubilized membrane proteins or purified proteins repre- access t o an occluded binding site as was previously suggested sent estimates of& or the effective concentrationfor 50%activation of by Javitt et al. (1990) to explain the increases in Be, for the TCP or MK-801 binding to the proteins. binding of [3HlMK-801to synaptic membranes. An increase in Be, was observed in the present study as well as in studies of n-octylglucoside-solubilized synaptic membrane proteins L3H]TCP binding to synaptic membranes when an agonist, through glutamate-derivatized ReactiGel matrices (present NMDA, was added to the incubation medium (Bonhaus and study). The protein fractions purified byLy and Michaelis (1991) contained also a broad band with an estimated molecu- McNamara, 1988). If the fractions purified in this study through chromatolar size of 25-28 kDa. All three approaches of solubilization and purification mentioned above ledto the isolation of entities that graphic separation on L-glutamate-derivatized matrices conhave ligand binding (Ikin et al. (1990) and present study) or tained intact NMDA-receptor complexes,then itseemed likely ion-channel-like characteristics (Ly and Michaelis, 1991) of an that these fractions would also contain the proteins that are known to be associated with NMDA receptors in rat brain, NMDA subtype of glutamate receptor. A comparison of ligand binding constants for the proteins especially the NMDAR1. However, there was no apparent enisolated by Ikin et al. (1990) with those isolated in the present richment of proteins with molecular sizes equal to those of the study is shown in Table 111. In the same Table are also pre- NMDARl or NMDAR2 subunits in the protein fractions isosentedthe binding constants reported in other studies for lated and analyzed by SDS-PAGE and silver staining. Of the [3HlTCP binding t o solubilized synaptic membrane protein subunits of the NMDA receptor identified by functional expresNMDARl subunit that appears to have all preparations, as well as the constants for activation by gluta- sion cloning,it is the mate and NMDAof the [3HlMK-801 or l3H1TCP binding to the ligand recognition sites present in intact brain receptors. solubilized proteins. In the study by Ikin et al. (19901, only Therefore, the presence of either the intact NMDARl or proI3H1TCPbinding to the various protein fractions was measured teolytic fragments of the NMDARl might have accounted for and the constants reported for L-glutamate and NMDA repre- the ligand binding activities associated with the isolated prosent the estimated concentrations of these agonists that pro- tein fractions. Immunochemical analyses of the isolated protein duced half-maximal activation of l3H1TCP binding to the iso- fractions for the possible presence of the NMDARl protein, lated proteins. The ligand binding constants estimated in the however, revealed a complete absence of any protein bands in present study are very similar to those reported by other in- the isolated fractions that reacted either with a polyclonal anvestigators for the interaction of the same ligands with either tiserum (Fig. 4) or with a monoclonal antibody against solubilized synaptic membrane proteins or purified proteins NMDARl. The 107-112-kDa protein band that was labeled by (Table 111).An important characteristic of the proteins isolated the anti-NMDAR1 antibodies was essentially absent from the by the methods described in this paper (Table 11) as well as solubilized synaptic membrane proteins, as well as from the those reported by Ikin et al. (19901, is the activation by L- purified protein fractions. This would indicate that either the glutamate, glycine, and spermidine of I3H]TCP binding t o the NMDARl protein is not solubilized by the procedures used in protein fractions isolated from solubilized synaptic membrane this study or that following solubilization all peptides related to proteins. Based on these observations, it would appear that the NMDARl are no longer recognized by two different antibodies glutamate, glycine, and polyamine-recognizingproteins are not that react with two different regions of the protein, one near onlyco-purified through the various purification stepsbut the carboxyl-terminal, the other between two putative transmust also be interacting in a mannersimilar to that observed membrane domains of the NMDAR1. It was recently reported for theintact NMDA-receptorcomplexes in synaptic mem- also by Brose et al. (1993) that NMDARl was not efficiently branes (e.g. Bonhaus and McNamara (1988),Javitt and Zukin extracted from synaptic membranes by treatment of the mem(19891, and Ransom and Stec (1988)) or for the complexes in branes with CHAPS or cholate, an observation that isconsistsolubilized preparations of synaptic membrane proteins (Am- ent with the observations described in the present studies. bar et al., 1988; McKernanet al., 1989; Bakker et al., 1991). Other investigators have reported limited success in solubiThe activation by glutamate and glycine of I3H]TCP binding lizing ligand-binding proteins related to NMDA receptors in to the NMDA-eluted protein fractions from the glutamate af- synaptic membranes through the use of cholate-containing finity chromatography was not a purely kinetic activation of buffers. For example, solubilization of only 1.8%of the [3H]MKligand binding. L3H1TCPbinding t o the isolated proteins exhib- 801-binding proteins (McKernan et al., 1989) or of 20-30% of ited only a single kinetic component and the k,, was approxi- 13HlTCP-bindingentities insynaptic membranes was achieved TABLE I11 Comparative values of the KB or KWtfor TCR glutamate, and NMDA binding to solubilized synaptic membrane proteinsor isolated protein fractions
27392
NMDA
Ligand-binding
Proteins from Synaptic
by treatment with buffers containing 1% cholate (Ambar et al., 1988). In previous studies where either CHAPS alone (0.50.6%) or n-octylglucoside alone (O&0.9%) were used in the solubilization of synaptic membrane proteins, the solubilization efficiency for 13H]TCP-binding proteins (Ambar et al., 1988) or for 13H]MK-801-binding entities (McKernan et al., 1989) in the presence of each detergent varied from 0.4 to 14%. There is no previously reported attempt to use both detergents simultaneously at the concentrations and under the conditions employed in the present study in order to achieve solubilization of TCP/MK-801and glutamate/NMDA-binding proteins. The results presented in this study indicate that approximately 50% of glutamate, TCP, and glycine sites are solubilized by the procedures used. Two proteins were identified in the fractions that were enriched in NMDA-sensitive glutamate and MK-801-sensitive TCP-binding proteins. These were a glutamate-binding protein and a CPP-binding protein. The 61-69-kDa protein recognized by the antiglutamate-binding protein antibodies in the synaptic membranes and in the isolated protein fractions probably corresponds to the protein previously purified from synaptic membranes by glutamate affinity chromatography (Chen et al., 1988; Eaton et al., 1990; Wang et al., 1992). On the other hand, the antiserum raised against the CPP and CGP 39653-binding protein that was previously purified from synaptic membranes (Cunningham and Michaelis, 1991; Eggeman et al., 1993) labeled proteins in intact synaptic membranes that were considerably larger (83 and 179-197 kDa) than the isolated CPPbinding protein (53-58 kDa). The larger of these two protein species (179-197 kDa) probably represents a dimer of the 83kDa protein, as it was the major protein labeled by the antiserum in Western blots of synaptic membrane proteins when nonreducing and nonalkylating conditions were used (Fig. 6A). Although it was described previously that these antibodies recognize a protein in synaptic membranes with an estimated molecular size equal to 54 kDa, it was also noted in that study that the antiserum reacts in some membrane preparations with protein bands that have sizes of -80 and 30 kDa (Eggeman et al., 1993). This was interpreted as an indication that the primary antigen is a protein of larger molecular size, possibly an -80-kDa protein, and that the -54- and -30~kDa bands are the degradation products of the larger protein (Eggeman et al., 1993). This interpretation appears to be correct, as in the present study it was shown that in membranes isolated with extreme care so that proteolytic degradation was minimized, the anti-CPP-binding protein antibodies labeled a predominant protein band of 81-83 kDa. It was also noted in the present studies that the 81-83-kDa protein species was very susceptible to degradation upon solubilization or upon prolonged storage of the synaptic membranes at 4 “C. Therefore, it seems likely that the 54-kDa protein identified by the antibodies is a breakdown product of a larger protein, probably the 81-83-kDa species. The size of a CPP-binding protein in rat brain was estimated previously on the basis of radiation inactivation studies to be that of an 83-kDa protein (Honor6 et al., 1989), i.e. a molecular size that is fairly close to the estimated molecular size of the CPP-binding proteins identified by the antiserum used in the present study. However, when the photoaffinity ligand 13Hlazidophencyclidine (azido-PCP) was used to label neuronal proteins in a crude particulate fraction, five bands of proteins with estimated molecular sizes of 90, 62, 49, 40, and 33 kDa were labeled specifically by this ligand (Haring et al., 1986). Care was taken in that study to prevent proteolysis by including multiple protease inhibitors at all stages of the isolation of the membranes. Since some of the bands labeled by the photoaf-
Membranes
tinity reagent were broad, it is not possible to compare directly the estimates of molecular sizes of the labeled proteins with those of protein bands in synaptic membranes or in the isolated protein fractions labeled by the antisera used in the present study. Nevertheless, some of the sizes of the proteins labeled by azido-PCP are fairly similar to the molecular sizes of the proteins identified in the present study. The lower molecular sizes of the major protein bands in the fraction isolated through the step of Sephacryl S-400 HR chromatography (see Fig. 2C) as compared with the size of the proteins in intact synaptic membranes or in the fractions isolated through glutamate affinity chromatography was probably due to the continuous proteolysis of the isolated proteins. A mixture of additional or alternate protease inhibitors needs to be tested in future protein purification efforts in order to achieve optimal inhibition of the protein degradation that seems to occur during the purification procedure. Because of the apparent degradation that occurred during the purification of these proteins, the composition of a potential “receptor complex” formed by these proteins is not certain. Therefore, no effort was made in the present study to obtain an estimate of the molecular size of such a putative complex. It was reported previously by Ikin et al. (1990) that the size of the complex of proteins isolated through the amino-PCP affinity chromatography was 209 kDa. However, since there was no effort made in that study to determine whether the isolated proteins had undergone proteolytic degradation during their purification, nor was an attempt made to correct for the presence of attached detergent molecules, the true molecular size of any complex formed by the purified proteins remains unknown. Honor6 et al. (1989) in their studies with radiation inactivation reported that the NMDA-receptor complex in rat brain that was labeled by 13H1CPP is 203 kDa. If the estimates by Ikin et al. (1990) and Honor-6 et al. (1989) of the molecular size of a protein complex are assumed to be correct, then the B max for NMDA-sensitive L-glutamate binding (1.22 nmol/mg) and MK-801-sensitive TCP binding (1.5 nmoYmg protein) to the NMDA-eluted proteins would indicate that the purity of the ligand-binding entities in this fraction is equal to 2530% of the expected stoichiometry of one ligand binding site per 203-209kDa complex. These calculations of the stoichiometry of ligandbinding sites per molecule of the protein complex may be wrong, as the true molecular size of a complex of these proteins may be larger than the assumed size of 203-209 kDa. It is, of course, possible that some of the proteins co-purifying with the glutamate-binding and CPP-binding proteins are contaminants that represent at least 70-75% of the proteins in these fractions. Alternatively, all proteins in the isolated fractions may contribute to the formation of a receptor complex, but because of the progressive degradation of the proteins during their purification, there is a loss of ligand binding activity and a lower than expected stoichiometry. It would appear that synaptic membrane proteins such as the 63-71-kDa glutamate-binding protein, the 81-83~kDa CPP-binding protein, and possibly others may function as components of a different type of NMDA-like receptors in neuronal membranes. The results from studies which have reported on the presence of functional NMDA receptors that are formed by proteins with estimated molecular sizes of 33 kDa (Smirnova et al., 1993) and 42 and 100 kDa (Henley et al., 1992; Kerry et al., 1993) add credence to the idea that there may be proteins other than the NMDARl and NMDARP that can form NMDA-receptor-like complexes. Furthermore, the evidence gathered from studies with primary neuronal cultures indicates that the antibodies to the glutamate-binding protein protect neurons from NMDA-induced cell toxicity (Mattson et al., 1991), as does
NMDA Ligand-binding Proteins from Synaptic Membranes treatment of these neurons with antisense oligonucleotides that arebased on the sequence of the cDNA for the glutamatebinding protein (Mattson et al., 1993).These observations suggest that the glutamate-binding protein plays an important role in NMDA-induced neurotoxicityand may be a component of some type of neuronal NMDA receptors. Acknowledgments-We thank Dr. Robert Wenthold for his generous gift of the anti-NMDAR1 antiserum and Drs. JamesBoulter and Doug Vetter for their gift of MAb54.1 anti-NMDAR1 antibody used in this study. The support of the Center for Neurobiology and Immunology Research at the University of Kansas, is acknowledged. REFERENCES Ambar, I., Kloog, Y., and Sokolovsky, M. (1988)J. Neurochem. 51, 133-140 Bakker, M. H. M., McKernan, R. M., Wong, E. H. F., and Foster, A. C. (1991)J. Neurochem. 57,39.45 Bettler, B., Boulter, J., Hermans-Borgmeyer, I., @Shea-Greenfield, A., Deneris, E. S . , Moll, C., Borgmeyer, U., Hollmann, M., and Heinemann,S . (1990)Neuron 5, 583-595 Bonhaus, D. W., and McNamara, J. 0. (1988)Mol. Pharmacol. 34, 250-255 Boulter, J., Hollmann, M., @Shea-Greenfield, Hartley, M., Deneris, E.,Maron, C., and Heinemann, S., (1990)Science 249, 1033-1037 Brose, N., Gasic, G. P., Vetter, B. E., Sullivan, J. M., and Heinemann, S . F. (1993) J. Biol. Chem. 288,22663-22671 Chen, J.-W., Cunningham, M. D., Galton, N., andMichaelis, E. K. (1988)J . Biol. Chem. 283,417427 Cotman, C. W., and Monaghan, D. T. (1987)in Psychopharmacology: The Third Generation of Progress (Metzler, H. Y., Coyle, J. T.,Bunney, W. E., Kopin, I. J., and Davis, K. L., eds)pp. 197-210, Raven Press, New York Cunningham, M. D., and Michaelis, E. K. (1990)J . Biol. Chem. 265,7768-7778 Eaton, M. J., Chen, J.-W., Kumar, K N., Gong, Y., and Michaelis, E. K. (1990)J . Biol. Chem. 285, 16195-16204 Eggeman, K. T., Pal, R., Walsh, J., Kumar, K. N., and Michaelis, E. K (1993) Neurosci. Lett. 158, 173-176 Fonnum, F. (1984)J . Neurochem. 42, 1-11 Haring, R., Kloog, Y., and Sokolovsky, M. (1986)Biochemistry 25,612-620 Henley, J. M., Ambrosini, A,, Rodriguez-Ithurralde, D., Sudan, H., Brackley, P., Kerry, C., Mellor, I., Abutidze, K., Usherwood, P. N.R., and Barnard, E. A.
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