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• W2163518851 abstract "Article17 June 2002free access Gain control of N-methyl-D-aspartate receptor activity by receptor-like protein tyrosine phosphatase α Gang Lei Gang Lei Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Sheng Xue Sheng Xue Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Nadège Chéry Nadège Chéry Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Qiang Liu Qiang Liu Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Jindong Xu Jindong Xu Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Chun L. Kwan Chun L. Kwan Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Yang-Ping Fu Yang-Ping Fu Neuroscience Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, T2N 4N1 Canada Search for more papers by this author You-Ming Lu You-Ming Lu Neuroscience Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, T2N 4N1 Canada Search for more papers by this author Mingyao Liu Mingyao Liu Division of Cellular and Molecular Biology, University Health Network, Toronto General Hospital, Toronto, Ontario, M5G 2C4 Canada Search for more papers by this author Kenneth W. Harder Kenneth W. Harder Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, 3050 Australia Search for more papers by this author Xian-Min Yu Corresponding Author Xian-Min Yu Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Gang Lei Gang Lei Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Sheng Xue Sheng Xue Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Nadège Chéry Nadège Chéry Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Qiang Liu Qiang Liu Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Jindong Xu Jindong Xu Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Chun L. Kwan Chun L. Kwan Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Yang-Ping Fu Yang-Ping Fu Neuroscience Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, T2N 4N1 Canada Search for more papers by this author You-Ming Lu You-Ming Lu Neuroscience Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, T2N 4N1 Canada Search for more papers by this author Mingyao Liu Mingyao Liu Division of Cellular and Molecular Biology, University Health Network, Toronto General Hospital, Toronto, Ontario, M5G 2C4 Canada Search for more papers by this author Kenneth W. Harder Kenneth W. Harder Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, 3050 Australia Search for more papers by this author Xian-Min Yu Corresponding Author Xian-Min Yu Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada Search for more papers by this author Author Information Gang Lei1, Sheng Xue1, Nadège Chéry1, Qiang Liu1, Jindong Xu1, Chun L. Kwan1, Yang-Ping Fu2, You-Ming Lu2, Mingyao Liu3, Kenneth W. Harder4 and Xian-Min Yu 1 1Center for Addiction and Mental Health, Faculty of Dentistry and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 Canada 2Neuroscience Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, T2N 4N1 Canada 3Division of Cellular and Molecular Biology, University Health Network, Toronto General Hospital, Toronto, Ontario, M5G 2C4 Canada 4Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, 3050 Australia *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:2977-2989https://doi.org/10.1093/emboj/cdf292 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Src kinase regulation of N-methyl-D-aspartate (NMDA) subtype glutamate receptors in the central nervous system (CNS) has been found to play an important role in processes related to learning and memory, ethanol sensitivity and epilepsy. However, little is known regarding the mechanisms underlying the regulation of Src family kinase activity in the control of NMDA receptors. Here we report that the distal phosphatase domain (D2) of protein tyrosine phosphatase α (PTPα) binds to the PDZ2 domain of post-synaptic density 95 (PSD95). Thus, Src kinase, its activator (PTPα) and substrate (NMDA receptors) are linked by the same scaffold protein, PSD95. Removal of PTPα does not affect the association of Src with NMDA receptors, but turns off the constitutive regulation of NMDA receptors by the kinase. Further more, we found that application of the PTPα catalytic domains (D1 + D2) into neurones enhances NMDA receptor-mediated synaptic responses. Conversely, the blockade of endogenous PTPα inhibits NMDA receptor activity and the induction of long-term potentiation in hippocampal neurones. Thus, PTPα is a novel up-regulator of synaptic strength in the CNS. Introduction The reversible phosphorylation of proteins on serine, threonine and tyrosine residues represents a primary post-translational mechanism underlying the regulation of cell functions, including DNA replication, energy metabolism, cell growth and signalling (Thomas and Brugge, 1997). An abundance of data has demonstrated that Src family protein tyrosine kinases (PTKs) may play a central role in the regulation of N-methyl-D-aspartate (NMDA) receptors by multiple signalling pathways (Yu and Salter, 1998; Lu et al., 1999; Huang et al., 2001; Manzerra et al., 2001; Vissel et al., 2001). Recent studies have documented that Src family PTKs may potentiate NMDA receptor functions via both the enhancement of the channel gating and an increase in the number of NMDA receptors on the cell surface (Yu et al., 1997; Grosshans et al., 2001). However, it remains unknown how the activity of Src family PTKs is controlled in the regulation of NMDA receptors. The Src kinase family is comprised of a total of nine members, at least five of which—Src, Fyn, Lyn, Lck and Yes—are known to be expressed in the central nervous system (CNS) (Thomas and Brugge, 1997; Ali and Salter, 2001). All members of the Src family contain highly homologous regions, including an inhibitory C-terminal sequence, as well as catalytic, SH2 and SH3 domains (Brown and Cooper, 1996). The activity of these PTKs is tightly regulated by the reversible phosphorylation of a C-terminal tyrosine residue (Tyr527 in chicken c-Src) (Cooper et al., 1986; Nada et al., 1991; Brown and Cooper, 1996). Although phosphatases have long been proposed to be responsible for the dephosphorylation of phosphorylated Tyr527, and consequently increase Src family PTK activity (Brown and Cooper, 1996), it has been demonstrated recently via inactivation of the protein tyrosine phosphatase α (PTPα) gene by homologous recombination (Ponniah et al., 1999; Su et al., 1999) that PTPα may act as an endogenous activator of Src family PTKs by dephosphorylating phosphorylated Tyr527 (Zheng et al., 1992; den Hertog et al., 1993). PTPα belongs to the family of receptor-like PTPs (Neel and Tonks, 1997; den Hertog, 1999), which at present includes seven members discovered in mammalian cells (den Hertog, 1999). Most receptor-like PTPs contain two phosphatase domains, a single transmembrane region and a variable extracellular N-terminus (Neel and Tonks, 1997; den Hertog, 1999). PTPα is highly expressed in the CNS (Sap et al., 1990; Van Vactor, 1998), but the function of this enzyme in the CNS is still poorly understood. To clarify functions of phosphatases in the CNS, we examined the role of PTPα in the regulation of NMDA receptor activity. Results PTPα complexes with NMDA receptors by directly binding to the PDZ2 domain of PSD95 In order to determine whether PTPα is a critical factor in the regulation of NMDA receptors, we first set out to determine whether PTPα is an integral component of the NMDA receptor-associated signalling complex in the CNS. We performed co-immunoprecipitation experiments using antibodies recognizing either the NMDA receptor NR1 subunit (which is present in all functional NMDA receptors; Dingledine et al., 1999; Cull-Candy et al., 2001), PTPα or Src from membrane protein extracts of adult rat brain solubilized under non-denaturing conditions. We found that antibodies recognizing either the NMDA receptor NR1 subunit, PTPα or Src could co-precipitate the other two remaining proteins (Figure 1A–C). In contrast, none of these proteins were detected in immunoprecipitates using non-specific IgG (Figure 1A–C). These data indicate that PTPα, Src and the NMDA receptor may associate in the CNS. Figure 1.PTPα, Src and the NMDA receptor form a complex in the CNS. The lane immediately left of (A) was loaded with solubilized membrane protein (20 μg, SM) which was prepared from rat brain tissues and used for immunoprecipitation shown in (A–C). (A) Immuno precipitates with non-specific IgG (mouse) and an antibody against NMDA NR1 subunit (mouse IgG) were loaded in the lanes (left to right) of the gel. Approximately 88 ± 4% (mean ± SE, n = 4) of the NR1 subunit protein was precipitated from SM with the anti-NR1 antibody. (B) Immunoprecipitates with non-specific IgG (mouse) and an antibody against Src (mouse IgG) were loaded in the lanes (left to right) of the gel. (C) Immunoprecipitates with non-specific IgG (rabbit) and an antibody against PTPα (rabbit IgG) (Harder et al., 1998) were loaded in the lanes (left to right) of the gel. The filters shown in (A–C) were stripped sequentially and immunoblotted with the antibodies against PTPα, NR1 and Src as indicated next to the arrows. (D) Immunoprecipitates from three consecutive immunoprecipitations with anti-PSD95 antibody (mouse IgG) were loaded in the lanes (left to right) of the gel. (E) Immunodepletion of PSD95 significantly reduced the amount of PTPα co-precipitated with NMDA receptors. The left blots in (E) show the effect of PSD95 immunodepletion on PTPα association with NMDA receptors. NR1 immunoprecipitates from brain lysates without PSD95 immunodepletion were loaded in the left lane of the gel. The middle lane was loaded with NR1 immunoprecipitates from the supernatant after immunoprecipitation was performed twice with PSD95 antibodies. PSD95 immunoprecipitates were loaded in the right lane. The right blots in (E) show the effect of immunoprecipitation with non-specific IgG (mouse) on PTPα association with NMDA receptors. NR1 immunoprecipitates from brain lysates without IgG immunoprecipitation were loaded in the left lane of the gel. The middle lane of the gel was loaded with NR1 immunoprecipitates from the supernatant after two consecutive immunoprecipitations with non- specific IgG. IgG immunoprecipitates were loaded in the right lane. The filters were stripped sequentially and immunoblotted with antibodies against proteins as indicated next to the arrows. (F) The bar graph shows the mean ratios (±SE, four experiments) of band intensity of co-precipitated PTPα versus that of NR1 subunit protein detected in NR1 immunoprecipitates. P < 0.05 (t-test). All experiments shown in this figure were repeated >3 times. Numbers to the left of the blots indicate the position and size (kDa) of molecular mass markers. IP, immunoprecipitation. Download figure Download PowerPoint Recently, mechanisms underlying the association of Src family PTKs, Src and Fyn, with NMDA receptors have been investigated in depth by other groups (Tezuka et al., 1999; Hajdur et al., 2001). It is found that Src and Fyn may bind directly to the PDZ domain-containing protein, post-synaptic density 95 (PSD95), and thereby associate with NMDA receptors (Tezuka et al., 1999; Hajdur et al., 2001). In order to get an indication as to how the Src family PTK activator PTPα associates with NMDA receptors, we examined whether PSD95 may also be involved in the association of PTPα with NMDA receptors. For this purpose, we first investigated whether the association of PTPα with NMDA receptors was affected by a PSD95 depletion. PSD95 immunoprecipitation was conducted twice to deplete PSD95 from the rat brain lysates, after which very little (<3%) PSD95 was found remaining in the supernatant (Figure 1D). The left blots of Figure 1E show an example of PSD95 depletion experiments. PSD95 antibodies co-precipitated both the NMDA NR1 subunit and PTPα (the right lane of the left blots). After PSD95 immunoprecipitation, the supernatant was subjected to immunoprecipitation with anti-NR1 antibodies. The resultant precipitates were loaded in the middle lane of the gel. Compared with the amount of PTPα co-precipitated with NR1 antibodies from rat brain lysates without the initial PSD95 immunodepletion (the left lane of the left blots in Figure 1E), there was much less PTPα detected in NR1 immunoprecipitates after the PSD95 depletion (the middle lane of the left blots in Figure 1E). As a control, immunoprecipitation performed twice with non-specific IgG produced no significant change in PTPα association with NMDA receptors (the right blots of Figure 1E). The fact that the ratio of band intensity of co-precipitated PTPα versus that of NR1 subunit protein detected in NR1 immunoprecipitates was significantly reduced by PSD95 depletion (Figure 1F) strongly suggests that PTPα may associate mainly with those NMDA receptors that are also associated with PSD95. It is known that the PDZ1 and PDZ2 domains of PSD95 may bind to the C-terminus of NMDA NR2 subunits (Kornau et al., 1995; Sheng and Sala, 2001). To identify whether PSD95 may act as a linker to recruit PTPα into the NMDA receptor complex, we examined whether PSD95 expression is sufficient to enable PTPα–NMDA receptor association in non-neuronal cells that normally express neither NMDA receptors nor PSD95. HEK293 and COS7 cells were transfected with cDNAs encoding NMDA NR1-1a, NR2A subunits (which are found in most NMDA receptors of adult animals; Dingledine et al., 1999; Cull-Candy et al., 2001) and/or PSD95. Co-immunoprecipitation experiments were performed on cell lysates using an antibody against PTPα. We found that expressed PSD95 was co-precipitated with PTPα from the lysate of cells lacking NMDA receptor subunit co-expression (Figure 2A). In contrast, PTPα antibodies could not precipitate overexpressed NMDA receptor subunit proteins without PSD95 co-expression (Figure 2A). Moreover, without NMDA NR2A subunit co-expression, PTPα failed to be co-precipitated with NMDA NR1-1a subunit, even if PSD95 was co-expressed (Figure 2A). Thus, PTPα may associate with a functional NMDA receptor by way of PSD95 association with the NR2A subunit. Figure 2.PTPα associates with NMDA receptors through the PDZ domain-containing protein, PSD95. (A) The lanes of the gel (left to right) were loaded with cell lysates (40 μg) and non-specific IgG or PTPα immunoprecipitates obtained from lysates of HEK293 cells transfected with cDNAs encoding NMDA receptor subunits and/or PSD95 as indicated under the blots by − (not transfected) or + (transfected). The filters were stripped sequentially and immunoblotted with antibodies against these proteins as indicated next to the arrows. (B) The lanes (left to right) in the left blot show PSD95 deletion mutants lacking the guanylate kinase (GK) domain (ΔGK), both the SH3 and GK domains [Δ(SH3 + GK)], and the PDZ3 (ΔPDZ3), PDZ2 (ΔPDZ2) and PDZ1 (ΔPDZ1) domains (Tezuka et al., 1999) expressed in HEK293 cells, and detected with an antibody against amino acids 1–54 of PSD95 (anti-PSD951–54). The right blot shows the PSD95 mutants, as indicated with arrows, detected in PTPα immunoprecipitates from these cells. C, cell lysates; IP, immunoprecipitation; IB, immunoblotting. Download figure Download PowerPoint The data above led us to infer that PTPα may bind to PSD95, leading to its association with NMDA receptors. To verify this hypothesis, we directed our investigation towards determining how PTPα interacts with PSD95. cDNAs encoding PSD95 mutants, in which a segment corresponding to either PDZ1, 2, 3 or the SH3 and/or GK domain of PSD95 was deleted (Tezuka et al., 1999), were transfected into HEK293 or COS7 cells. The left blot in Figure 2B shows the expression of these mutants in HEK293 cells by immunoblotting with an antibody against PSD95 residues 1–54 (anti-PSD951–54). We found that all of the expressed PSD95 mutants except for one lacking residues 157–256 (corresponding to the PDZ2 domain of PSD95) could be co-immunoprecipitated by anti-PTPα antibodies (Figure 2B). These data suggest that PTPα association with PSD95 may be due to the binding of PTPα to the PDZ2 domain of PSD95. We substantiated this finding by performing in vitro GST fusion protein precipitation assays. Figure 3A shows the constructs of PTPα peptides used in these assays. Figure 3B shows that 35S-labelled peptides produced by in vitro transcription/translation corresponding to the entire cytoplasmic portion (D1 + D2) and the membrane-distal phosphatase domain including the C-terminal (D2), but not the membrane-proximal phosphatase domain (D1) of PTPα, could be precipitated by glutathione–agarose beads bound to GST fusion proteins containing the PSD95 PDZ2 domain. In contrast, neither GST alone nor the GST fusion proteins containing PSD95 PDZ1 or the PDZ3 domain could precipitate any of these 35S-labelled peptides (Figure 3B). Thus, the PTPα D2 domain appears to be involved in the interaction between PTPα and PSD95. Figure 3.The membrane-distal phosphatase domain (D2) of PTPα binds to the PSD95 PDZ2 domain in vitro. (A) Schematics of constructs encoding the entire intracellular portion (D1 + D2), D1 and D2 domains of PTPα, and a segment (amino acids 539–711) in the D2 domain. (B) Autoradiograph showing 35S-labelled PTPα peptides (Input) as indicated in (A), and PTPα peptides precipitated by GST fusion proteins conjugated with the PDZ1 (GST–PDZ1), PDZ2 (GST–PDZ2) or PDZ3 (GST–PDZ3) domain of PSD95. Numbers to the right of the blots indicate the position and size (kDa) of molecular mass markers. Download figure Download PowerPoint Since there is no typical E(S/T)XV motif at the C-terminus of PTPα available for PSD95 PDZ2 domain binding, and recent studies have documented that the PDZ domain of PSD95 may also bind to the internal peptides of proteins lacking the typical E(S/T)XV terminal motif (Hillier et al., 1999; Tezuka et al., 1999; Sheng and Sala, 2001), we sought to identify further the sequence which may underlie the binding between the PTPα D2 and PSD95 PDZ2 domains. By using a D2 domain that was truncated from its N- and/or C-terminus, we found that a GST fusion protein containing the amino acids 539–711 of PTPα could still bind to the PDZ2 domain of PSD95 in vitro (Figure 3B). Thus, we conclude that PTPα binds to PSD95 via the D2–PDZ2 domain interaction, and thereby complexes with NMDA receptors. In non-neuronal cells, it has been found that Src (Harder et al., 1998; Zheng et al., 2000) and Fyn (Bhandari et al., 1998) may complex with PTPα. To identify the role of PTPα in the regulation of NMDA receptor activity, we then investigated whether PTPα expression changes Src expression or the physical association of this kinase with NMDA receptors. cDNAs encoding NMDA receptor subunits and wild-type PSD95 were transfected into PTPα-deficient (PTPα−/−; Su et al., 1999) fibroblasts. Figure 4A shows the results of co-immunoprecipitation experiments using antibodies against NR2A/B subunits on lysates of these cells with or without the re-introduction of PTPα by transfection of PTPα cDNA into PTPα−/− fibroblasts. Compared with that observed in the fibroblasts without the re-introduction of PTPα, it was found that PTPα expression did not alter the ability of PSD95 or Src to associate with co-expressed NMDA receptors (Figure 4A). Western blot analysis using antibodies against Src (clone327), or C-terminally tyrosine-phosphorylated Src (Src-pTyr529), showed no significant difference in the total amount of Src in cells with PTPα expression versus those cells lacking PTPα, while a marked reduction in anti-Src-pTyr529 immunoreactivity, indicative of increased Src activity (Zheng et al., 1992; den Hertog et al., 1993; Ponniah et al., 1999; Su et al., 1999; Zheng et al., 2000), was found in the cells into which PTPα was re-introduced (Figure 4B and C). Figure 4.Removal of PTPα does not affect the association of Src with NMDA receptors, but reduces Src activity. (A) The left blots show immunoprecipitates obtained using an anti-NR2A/B antibody (rabbit IgG) on the lysates of PTPα−/− fibroblasts co-transfected with cDNAs encoding PSD95 and NR2A (left lane), or PTPα, PSD95 and NR2A (right lane). The symbols beneath the blots show transfection status as −, not transfected; +, transfected. The blots on the right show the immunoprecipitates obtained using non-specific IgG (rabbit). The filters were probed sequentially with antibodies against NR2A (rabbit IgG), PSD95 (mouse IgG) and Src (mouse IgG) as indicated. (B) Western blot analysis of cell lysates used for the immunoprecipitation shown in (A) was conducted with the indicated antibodies (arrows). (C) The bar graph shows the mean ratios (± SE, six experiments) of the band intensity of Src (open bar, detected with the antibody clone327) and C-terminally tyrosine-phosphorylated Src (filled bar, detected with the antibody anti-Src-pTyr529) in cells with PTPα expression (labelled as PTPα+) versus those in cells without PTPα expression (labelled as PTPα−). The dashed line indicates the level of protein detected in cells without PTPα expression. IP: immunoprecipitation. *P < 0.05 (Wilcoxon test). Download figure Download PowerPoint PTPα activity is necessary for initiating and maintaining the regulation of recombinant NMDA receptors by endogenous Src family PTKs in fibroblasts To determine the role of PTPα in the regulation of NMDA receptor functions, we recorded whole-cell currents mediated by the NMDA NR1-1a/NR2A receptor expressed in PTPα−/− fibroblasts with or without the re-introduction of the phosphatase (Figure 5). In all of the patch clamp recording experiments conducted in fibroblasts, cDNA encoding wild-type PSD95 was co-transfected. Whole-cell currents were evoked with L-aspartate or NMDA (250 μM) applied through a double-barrel pipette system. The averaged peak and steady-state amplitudes of whole-cell currents recorded in PTPα−/− cells were 476 ± 69 and 404 ± 52 pA, respectively (n = 28, mean ± SEM). The decay of whole-cell currents during the agonist application was fitted using two exponential components with time constants 185 ± 40 ms (τfast) and 1217 ± 147 ms (τslow), respectively. Compared with the currents recorded in PTPα−/− cells, the re-introduction of PTPα into PTPα−/− cells significantly increased the amplitude of currents mediated by the recombinant NMDA receptors (peak, 839 ± 148 pA; steady state, 663 ± 113 pA; n = 38; also see Figure 5A), but did not significantly change the decay time (τfast, 129 ± 14 ms; τslow, 1094 ± 146 ms). To clarify whether the effects of the re-introduction of PTPα into PTPα−/− cells on recombinant NMDA receptors resulted from the direct modulation of NMDA receptors by PTPα, we transfected cDNAs encoding NMDA NR1-1a, NR2A subunits and wild-type PSD95 into fibroblasts lacking PTKs Src, Fyn and Yes (SYF cells; Klinghoffer et al., 1999) with or without PTPα cDNA co-transfection, and recorded currents evoked by L-aspartate or NMDA. The amplitudes of the peak and steady-state whole-cell currents mediated by NMDA receptors expressed in SYF cells with (n = 39) and without (n = 20) overexpression of PTPα were 674 ± 66 and 458 ± 55 pA, and 758 ± 95 and 425 ± 83 pA, respectively. No statistically significant difference could be found. Thus, it is implied that the enhancement of NMDA receptor-mediated responses in cells in which PTPα was re-introduced may be a result of PTPα-dependent activation of Src family PTKs. Figure 5.Deletion of the PTPα gene abolishes the Src kinase regulation of recombinant NMDA receptors. (A) The peak amplitudes (mean ± SE) of whole-cell responses mediated by recombinant NMDA NR1-1a/NR2A receptors expressed in PTPα−/− fibroblasts with (filled bar, +) and without (open bar, −) re-introduction of PTPα. #, P < 0.05, Mann–Whitney test. (B) An example of whole-cell currents and current–voltage (I/V) relationships mediated by recombinant NMDA NR1-1a/NR2A receptors in PTPα-expressing cells (labelled as PTPα+) before and during the application of PP2 (10 μM, Calbiochem, San Diego, CA). (C) A summary of PP2 or PP3 effects on recombinant NMDA receptors expressed in PTPα−/− cells with PTPα re-introduced. (D) The effects of PP2 on recombinant NMDA receptors expressed in SYF cells or SYF cells co-transfected with cDNA encoding pp60c-Src (labelled as SYF + c-Src). (E) A summary of PP2 effects on the NMDA receptors expressed in PTPα−/− cells without re-introduction of PTPα (labelled as PTPα−) or with re-introducion of catalytically inactive PTPα [labelled as PTPα (C433A)] (Harder et al., 1998). In (C), (D) and (E), the filled and open bars, respectively, indicate the peak and steady-state amplitudes (mean ± SE) normalized to their control responses before application of drugs as indicated. Dashed lines in (C), (D) and (E) indicate the control level of L-aspartate-evoked whole-cell currents before the application of drugs as indicated. Values in parentheses indicate the number of cells tested. *P < 0.05; **P < 0.01 (Wilcoxon test). Download figure Download PowerPoint Then, we examined the effects of the Src family PTK inhibitor, PP2 (0.5–10 μM), on NMDA NR1-1a/NR2A receptors co-expressed in cells in which PTPα was re-introduced. An example presenting the effect of PP2 applied to cells with the re-introduction of PTPα is shown in Figure 5B. We found that PP2 application significantly reduced NMDA NR1-1a/NR2A receptor-mediated whole-cell currents (Figure 5B and C) without changing the reversal potential of currents recorded (Figure 5B). These data indicate that the PP2-induced reduction of current amplitudes recorded in cells in which PTPα was re-introduced results from a decrease in NMDA receptor-mediated whole-cell conductance. The PP2 effect was concentration dependent (Figure 5C), while PP3 (an inactive PP2 isomer) had no effect (Figure 5C). To confirm that the PP2 effects detected in these experiments were produced by the inhibition of Src family PTKs, we examined the effects of PP2 on NMDA NR1-1a/NR2A receptors expressed in SYF cells. We found that PP2 application had no effect on the recombinant NMDA receptors expressed in SYF cells unless Src was re-introduced (Figure 5D). This indicates that the recombinant NMDA NR1-1a/NR2A receptors in cells expressing PTPα may be constitutively regulated by endogenous Src, Fyn and/or Yes. Surprisingly, PP2 (10 μM) application did not produce any inhibition of recombinant NMDA receptors expressed in PTPα−/− cells (Figure 5E). To determine whether the lack of PP2-induced inhibition was related to the absence of PTPα phosphatase activity, we investigated the effects of PP2 on NMDA receptors expressed in PTPα−/− cells co-transfected with cDNA encoding catalytically inactive PTPα (PTPα with a cysteine to alanine mutation at residue 433 in the D1 phosphatase domain) (Harder et al., 1998). PP2 had no effect on NMDA receptors expressed in these cells (Figure 5E), suggesting that PTPα activity may be necessary for initiating and maintaining the NMDA receptor regulation by Src family PTKs. To confirm the role of PTPα activity in the regulation of NMDA receptors by endogenous PTKs, we examined the effects of another PTK inhibitor, lavendustin A (10 μM) (Wang and Salter, 1994; Hemmings, 1997). We found that similarly to PP2, lavendustin A application did not induce any significant reduction of recombinant NMDA receptor-mediated currents recorded from cells without PTPα expression or expressing catalytically inactiv" @default.
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• W2163518851 date "2002-06-17" @default.
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• W2163518851 title "Gain control of N-methyl-D-aspartate receptor activity by receptor-like protein tyrosine phosphatase alpha" @default.
• W2163518851 cites W1532214820 @default.
• W2163518851 cites W1577190501 @default.
• W2163518851 cites W1657182769 @default.
• W2163518851 cites W1829071486 @default.
• W2163518851 cites W1965251357 @default.
• W2163518851 cites W1968054389 @default.
• W2163518851 cites W1977526136 @default.
• W2163518851 cites W1984057909 @default.
• W2163518851 cites W1989572867 @default.
• W2163518851 cites W1989899772 @default.
• W2163518851 cites W1991218955 @default.
• W2163518851 cites W1994157750 @default.
• W2163518851 cites W2008141082 @default.
• W2163518851 cites W2014397920 @default.
• W2163518851 cites W2016954161 @default.
• W2163518851 cites W2024036641 @default.
• W2163518851 cites W2024431713 @default.
• W2163518851 cites W2026715343 @default.
• W2163518851 cites W2030577752 @default.
• W2163518851 cites W2034152669 @default.
• W2163518851 cites W2037291219 @default.
• W2163518851 cites W2039852907 @default.
• W2163518851 cites W2044935554 @default.
• W2163518851 cites W2046929321 @default.
• W2163518851 cites W2053625020 @default.
• W2163518851 cites W2056145172 @default.
• W2163518851 cites W2063714047 @default.
• W2163518851 cites W2066641929 @default.
• W2163518851 cites W2072282121 @default.
• W2163518851 cites W2078897717 @default.
• W2163518851 cites W2079862834 @default.
• W2163518851 cites W2084434864 @default.
• W2163518851 cites W2089815055 @default.
• W2163518851 cites W2090423201 @default.
• W2163518851 cites W2092045031 @default.
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• W2163518851 cites W2115050780 @default.
• W2163518851 cites W2116635659 @default.
• W2163518851 cites W2122643181 @default.
• W2163518851 cites W2127792712 @default.
• W2163518851 cites W2140899588 @default.
• W2163518851 cites W2146578030 @default.
• W2163518851 cites W2167682559 @default.
• W2163518851 cites W963887890 @default.
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