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• W2088396961 abstract "The low density lipoprotein receptor-related protein 1 (LRP1) emerges to play fundamental roles in cellular signaling pathways in the brain. One of its prominent ligands is the serine proteinase tissue-type plasminogen activator (tPA), which has been shown to act as a key activator of neuronal mitogen-activated protein kinase pathways via the N-methyl-d-aspartate (NMDA) receptor. However, here we set out to examine whether LRP1 and the NMDA receptor might eventually act in a combined fashion to mediate tPA downstream signaling. By blocking tPA from binding to LRP1 using the receptor-associated protein, we were able to completely inhibit NMDA receptor activation. Additionally, inhibition of NMDA receptor calcium influx with MK-801 resulted in dramatic reduction of tPA-mediated downstream signaling. This indicates a functional interaction between the two receptors, since both experimental approaches resulted in strongly reduced calcium influx and Erk1/2 phosphorylation. Additionally, we were able to inhibit Erk1/2 activation by competing for the LRP1 C-terminal binding motif with a truncated PSD95 construct resembling its PDZ III domain. Furthermore, we identified the distal NPXY amino acid motif in the C terminus of LRP1 as the crucial element for LRP1-NMDA receptor interaction via the adaptor protein PSD95. These results provide new insights into the mechanism of a tPA-induced, LRP1-mediated gating mechanism for NMDA receptors. The low density lipoprotein receptor-related protein 1 (LRP1) emerges to play fundamental roles in cellular signaling pathways in the brain. One of its prominent ligands is the serine proteinase tissue-type plasminogen activator (tPA), which has been shown to act as a key activator of neuronal mitogen-activated protein kinase pathways via the N-methyl-d-aspartate (NMDA) receptor. However, here we set out to examine whether LRP1 and the NMDA receptor might eventually act in a combined fashion to mediate tPA downstream signaling. By blocking tPA from binding to LRP1 using the receptor-associated protein, we were able to completely inhibit NMDA receptor activation. Additionally, inhibition of NMDA receptor calcium influx with MK-801 resulted in dramatic reduction of tPA-mediated downstream signaling. This indicates a functional interaction between the two receptors, since both experimental approaches resulted in strongly reduced calcium influx and Erk1/2 phosphorylation. Additionally, we were able to inhibit Erk1/2 activation by competing for the LRP1 C-terminal binding motif with a truncated PSD95 construct resembling its PDZ III domain. Furthermore, we identified the distal NPXY amino acid motif in the C terminus of LRP1 as the crucial element for LRP1-NMDA receptor interaction via the adaptor protein PSD95. These results provide new insights into the mechanism of a tPA-induced, LRP1-mediated gating mechanism for NMDA receptors. LRP1 (low density lipoprotein receptor-related protein) is a member of the low density lipoprotein receptor gene family with its highest expression in liver and brain. Following its synthesis in the endoplasmic reticulum as a 600-kDa type I transmembrane glycoprotein, LRP1 is cleaved in the Golgi compartment by furin, producing two subunits, 515 and 85 kDa in size. These two subunits remain noncovalently associated during their transport to the cell surface (1Waldron E. Jaeger S. Pietrzik C.U. Neurodegener. Dis. 2006; 3: 233-238Crossref PubMed Scopus (20) Google Scholar). Via a receptor-recycling pathway, LRP1 is responsible for the endocytosis of more than 30 different extracellular ligands (2Strickland D.K. Ranganathan S. J. Thromb. Haemost. 2003; 1: 1663-1670Crossref PubMed Scopus (102) Google Scholar, 3Bu G. Cam J. Zerbinatti C. Ann. N. Y. Acad. Sci. 2006; 1086: 35-53Crossref PubMed Scopus (112) Google Scholar). In neurons, where it is highly expressed and predominantly localized in neuronal cell bodies and dendritic processes (4May P. Rohlmann A. Bock H.H. Zurhove K. Marth J.D. Schomburg E.D. Noebels J.L. Beffert U. Sweatt J.D. Weeber E.J. Herz J. Mol. Cell. Biol. 2004; 24: 8872-8883Crossref PubMed Scopus (171) Google Scholar), LRP1 is a major receptor for apoE/lipoprotein-containing particles and tissue-type plasminogen activator (tPA) 2The abbreviations used are: tPA, tissue-type plasminogen activator; RAP, receptor-associated protein; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; Erk1/2, extracellular signal-regulated kinase 1 and 2; shRNA, small hairpin RNA; siRNA, small interfering RNA; P-Erk1/2, phospho-Erk1/2; NMDA, N-methyl-d-aspartate; stPA, synthetic recombinant tPA; NPXY(2), distal NPXY motif. (5Zhuo M. Holtzman D.M. Li Y. Osaka H. DeMaro J. Jacquin M. Bu G. J. Neurosci. 2000; 20: 542-549Crossref PubMed Google Scholar). In addition to its role in endocytosis of various ligands, LRP1 has been implicated to play a crucial role in cell signaling. The observation that the LRP1 C terminus undergoes rapid tyrosine phosphorylation after tPA binding corroborates its function as a signal transmitter (6Hu K. Yang J. Tanaka S. Gonias S.L. Mars W.M. Liu Y. J. Biol. Chem. 2006; 281: 2120-2127Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Several adaptor proteins can bind to the LRP1 tail, some of which seem to play a role in neuronal calcium, platelet-derived growth factor, or MAPK signaling (7Bacskai B.J. Xia M.Q. Strickland D.K. Rebeck G.W. Hyman B.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11551-11556Crossref PubMed Scopus (172) Google Scholar, 8Boucher P. Gotthardt M. Trends Cardiovasc. Med. 2004; 14: 55-60Crossref PubMed Scopus (44) Google Scholar, 9Qiu Z. Hyman B.T. Rebeck G.W. J. Biol. Chem. 2004; 279: 34948-34956Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Beyond its known function of lipid uptake, LRP1 has also been attributed to be the mediator of an LTP-enhancing effect of exogenously added tPA in hippocampal slices from tPA-deficient mice (5Zhuo M. Holtzman D.M. Li Y. Osaka H. DeMaro J. Jacquin M. Bu G. J. Neurosci. 2000; 20: 542-549Crossref PubMed Google Scholar). Further evidence supporting the hypothesis of an involvement in synaptic plasticity results from neuron-specific LRP1 knock-out mice, which show motor and coordination defects, muscle tremor, premature aging, and death (4May P. Rohlmann A. Bock H.H. Zurhove K. Marth J.D. Schomburg E.D. Noebels J.L. Beffert U. Sweatt J.D. Weeber E.J. Herz J. Mol. Cell. Biol. 2004; 24: 8872-8883Crossref PubMed Scopus (171) Google Scholar). Given the central role of N-methyl-d-aspartate (NMDA) receptors in synaptic plasticity and the function of LRP1 as a tPA receptor, an interaction between both molecules seems likely. Interestingly, a possible interaction between LRP1 and the NMDA receptor has been deduced from immunohistochemical and co-immunoprecipitation experiments, pointing to an LRP1-NMDA receptor interaction at the synapse (4May P. Rohlmann A. Bock H.H. Zurhove K. Marth J.D. Schomburg E.D. Noebels J.L. Beffert U. Sweatt J.D. Weeber E.J. Herz J. Mol. Cell. Biol. 2004; 24: 8872-8883Crossref PubMed Scopus (171) Google Scholar). In contrast to these findings, it has been reported that tPA binds directly to the NMDA receptor, resulting in the cleavage of the NMDA receptor subunit 1, leading to enhanced NMDA receptor-mediated intracellular calcium accumulation and neuronal degeneration (10Nicole O. Docagne F. Ali C. Margaill I. Carmeliet P. MacKenzie E.T. Vivien D. Buisson A. Nat. Med. 2001; 7: 59-64Crossref PubMed Scopus (621) Google Scholar). Other studies report a proteolysis-independent effect of tPA on NMDA receptor-mediated downstream signaling events through direct interaction of tPA with the NMDA receptor (11Medina M.G. Ledesma M.D. Dominguez J.E. Medina M. Zafra D. Alameda F. Dotti C.G. Navarro P. EMBO J. 2005; 24: 1706-1716Crossref PubMed Scopus (95) Google Scholar). Although the underlying mechanism, how tPA facilitates NMDA receptor activation, remains controversial, the general involvement of tPA in modulating NMDA receptor function is not in doubt (12Pang P.T. Lu B. Ageing Res. Rev. 2004; 3: 407-430Crossref PubMed Scopus (255) Google Scholar, 13Pang P.T. Teng H.K. Zaitsev E. Woo N.T. Sakata K. Zhen S. Teng K.K. Yung W.H. Hempstead B.L. Lu B. Science. 2004; 306: 487-491Crossref PubMed Scopus (917) Google Scholar). In this study, we show that tPA indeed activates the NMDA receptor cascade as hypothesized before (14Samson A.L. Medcalf R.L. Neuron. 2006; 50: 673-678Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). However, we are able to demonstrate that tPA-induced NMDA receptor activation and downstream signaling is exclusively mediated via LRP1. This activation is based on a mechanism involving the distal NPXY motif of LRP1 and the adaptor protein PSD95, which bridges LRP1 to the NMDA receptor. This complex formation is followed by calcium influx into the cell and subsequent activation of the MAPK pathway. Materials—Human recombinant tPA derived from Chinese hamster ovary cells (Actilyse) was purchased from Boehringer Ingelheim, and pure synthetic recombinant human tPA (stPA) was purchased from Biopur (Bubendorf, Switzerland). The NMDA receptor blockers DL-AP5 (Sigma) and MK-801 (Sigma) were dissolved in 0.1 m NaOH solution and in double-distilled H2O, respectively. The MEK inhibitor U0126 (Calbiochem) was diluted from a stock solution of 10 mm in DMSO. We constructed a RAP-GST fusion protein as described before (15Kang D.E. Pietrzik C.U. Baum L. Chevallier N. Merriam D.E. Kounnas M.Z. Wagner S.L. Troncoso J.C. Kawas C.H. Katzman R. Koo E.H. J. Clin. Invest. 2000; 106: 1159-1166Crossref PubMed Scopus (307) Google Scholar). Briefly, the plasmid was transformed into Escherichia coli BL21, and protein expression was induced by 1 mm isopropyl 1-thio-β-d-galactopyranoside (Roth) for 4 h. After bacterial lysis in 3% Sarkosyl buffer, the RAP-GST protein was pulled down with glutathione-Sepharose beads (Amersham Biosciences), eluted with a 10 mm glutathione, 50 mm Tris solution, and dialyzed against phosphate-buffered saline overnight. Antibodies—Primary antibodies used were monoclonal phospho-p44/42 MAPK (E10), polyclonal p44/42 MAP kinase (both from Cell Signaling, Danvers, MA), monoclonal ANTI-FLAG ® M2 antibody, and polyclonal actin 20-33 antibody (both from Sigma). The polyclonal LRP1 antibody (1704), which reacts with the cytoplasmic domain of LRP1, has been described previously (16Pietrzik C.U. Busse T. Merriam D.E. Weggen S. Koo E.H. EMBO J. 2002; 21: 5691-5700Crossref PubMed Scopus (176) Google Scholar). Secondary goat antibodies against mouse and rabbit were both horseradish peroxidase-coupled and purchased from The Jackson Laboratory. Tissue Culture—The immortalized cell line HT22, a subclone of the HT4 cell line, was derived from the mouse hippocampus and therefore has neuronal properties (17Davis J.B. Maher P. Brain Res. 1994; 652: 169-173Crossref PubMed Scopus (302) Google Scholar). The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1 mm minimal essential medium sodium pyruvate (all from Invitrogen), and 100 units/ml penicillin and 100 μg/ml streptomycin (both from Cambrex) in a humidified 5% CO2 incubator at 37 °C. Hippocampal neurons were prepared from either Sprague-Dawley rat embryos at embryonic day 17 or 18 or C57Bl6 wild-type and C57Bl6/129 LRP1 knock-in mouse embryos at embryonic day 15 or 16 (18Roebroek A.J. Reekmans S. Lauwers A. Feyaerts N. Smeijers L. Hartmann D. Mol. Cell. Biol. 2006; 26: 605-616Crossref PubMed Scopus (52) Google Scholar). In brief, hippocampi were collected in Hanks' balanced saline solution (Invitrogen), cut in small pieces, and trypsinized (0.05% trypsin, 0.02% EDTA in phosphate-buffered saline) for 15 min. After mechanical dissociation in Neurobasal (Invitrogen) containing B-27 and 2-fold Glutamax (both from Invitrogen), cells were centrifuged at 500 × g for 4 min and resuspended again in Neurobasal for counting. The cells were then plated onto poly-l-ornithine (100 μg/ml; Sigma)-coated 6-well plates at a density of 5 × 105 cells/well. For calcium-imaging experiments, cells were plated onto poly-l-ornithine-coated glass coverslips in 4-cm dishes. The medium was changed the next day, and the cells were cultured for another 14 days at 37 °C in a humidified 5% CO2 incubator. Drug Treatment—HT22 cells were grown to 80% confluence and serum-starved for 24 h in serum-free Dulbecco's modified Eagle's medium before tPA treatment. tPA (Actilyse) was reconstituted in double-distilled H2O and added to the culture medium for 30 min or 1 h at 37 °C, respectively. Stimulation of MAPK with stPA was detectable at a concentration of 10 μg/ml, whereas 40 μg/ml Actilyse was required to induce the same degree of MAPK stimulation. Therefore, we concluded that 10 μg/ml stPA functionally equals 40 μg/ml Actilyse, which we from now on refer to as tPA. The inhibitors MK-801 (10 μm) and U0126 (50 μm) were preincubated for 15 min, DL-AP5 (100 μm) was preincubated for 5 min, and RAP (500 nm) was preincubated for 1 h, and they were still present during tPA treatment. After 14 days in vitro (DIV), primary hippocampal neurons were treated as HT22 cells, except there was no medium change 24 h prior to stimulation. Calcium Phosphate-mediated Transfection of PSD95 and PDZ Domains—HT22 cells were transfected with three different vector constructs corresponding to the PSD95 PDZ domain I, II, or III. Each single PDZ domain was cloned into the pcDNA 3.1 zeo (+) vector (Invitrogen), bearing a 3× FLAG sequence at its N terminus. The corresponding nucleotide sequences (rat) are as follows: PDZI (nucleotides 178–465), PDZII (nucleotides 463–747), and PDZIII (nucleotides 904–1206) (19Cao J. Viholainen J.I. Dart C. Warwick H.K. Leyland M.L. Courtney M.J. J. Cell Biol. 2005; 168: 117-126Crossref PubMed Scopus (126) Google Scholar). Additionally, a GW1-CMV-vector bearing the full-length PSD95 protein (a kind gift of Dr. Morgan Sheng) (20Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar) was transfected. Cells were seeded in 6-well plates at ∼30% confluence and transfected with 2 μg of DNA/well via a calcium phosphate-mediated transfection protocol (21Sambrook J., and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., pp. 16.14–16.20, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar). 4–6 h post-transfection, the medium was changed to serum-free conditions for 24 h before cells were stimulated with tPA. LRP1 Knockdown with shRNA Vector Transfection—The shRNA expression vector pALsh-LRP1 recognizes the 5′-region of the open reading frame of LRP1 (22Laatsch A. Ragozin S. Grewal T. Beisiegel U. Joerg H. Eur. J. Cell Biol. 2004; 83: 113-120Crossref PubMed Scopus (14) Google Scholar). The generated self-annealing oligodeoxynucleotide targets the following sequence of the human LRP1 coding region: 5′-TAA GAC TTG CAG CCC CAA GCA GTT-3′ (position 72–95 of the human LRP1 open reading frame). pALsh vector alone served as transfection control. The mouse neuronal cell line HT22 was grown in 6-well plates until ∼50% confluence and transfected using Lipofectamine™ 2000 reagent (Invitrogen) at a ratio of 2 μgof plasmid DNA to 4 μl of Lipofectamine. To obtain better transfection efficiency, the cells were retransfected after 48 h with the same amount of DNA for another 24 h in serum-free Dulbecco's modified Eagle's medium. After 72 h in total, cells were stimulated with tPA, lysed, subjected to 10 or 12% SDS-PAGE, and tested for LRP1 knockdown and Erk1/2 activation with Western blotting as described below. siRNA Knockdown—Stealth™/siRNA duplex oligoribo-nucleotides against mouse PSD95, Shc1, and FE65 were purchased from Invitrogen, carrying the following sequences: 5′-GGA GUA UGA GGA GAU CAC AUU GGA A-3′ (PSD95), 5′-CCA GAU GCU CAA GUG CCA CGU GUU U-3′ (FE65), and 5′-GGC UGU GUG GAG GUC UUA CAG UCA A-3′ (Shc1). Transfections in primary hippocampal neurons or HT22 cells were carried out with 40 nm (PSD95), 20 nm (Shc1), and 100 nm (FE65) siRNA duplex with Lipofectamine™ 2000 reagent (Invitrogen) for 48 h. SDS-PAGE and Western Blot Analysis—Primary hippocampal cultures as well as HT22 cells were lysed in radioimmune precipitation buffer (50 mm Tris, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing proteinase inhibitors (complete; Roche Applied Science) and phosphatase inhibitors (1 mm sodium orthovanadate (Sigma) and 10 mm sodium fluoride (AppliChem)). Protein concentration was determined by the BCA method (Pierce), and equal amounts of total protein (20 μg) were resolved on a 12% polyacrylamide gel. Western blot was performed overnight at 30 V and 4 °C. Incubation with the primary antibodies occurred in general overnight, and detection of proteins was carried out with the ECL method (SuperSignal; Pierce). Calcium Imaging—The fluorescent calcium indicator calcium green was used to analyze changes of the [Ca2+]i in cultured neurons and HT22 cells. In detail, after 24 h in serum-free medium, cells cultured on glass coverslips were loaded (60 min at 37 °C) with the fluorescent dye by adding 10 μm calcium green in pluronic acid (both from Molecular Probes, Leiden, Netherlands) to the culture medium. After loading, excessive dye was removed, and the cells were kept in Hanks' balanced saline solution (PAA, Linz, Austria) supplemented with 2 mm CaCl2, 1 mm MgCl2, and 10 mm glucose and transferred to a temperature-controlled recording chamber adapted to an upright microscope (BX51WI, Olympus, Hamburg, Germany), equipped with a Nipkov spinning disk confocal system (QLC10; Visitech, Sunderland, UK) and a krypton/argon laser (Laser Physics, Cheshire, UK). The fluorescence of calcium green was excited at 488 nm. After background subtraction, changes of the emitted fluorescence were analyzed using Metamorph imaging software (Molecular Devices Corp, Downington, PA). Changes of [Ca2+]i in individual cell somata are shown as relative changes of calcium green fluorescence compared with the starting fluorescence intensity (dF/F0). The addition of tPA (40 μg/ml) was conducted 120 s after the recording was started. Inhibitors (10 μm MK-801 or 500 nm RAP) were preincubated for at least 30 min and were present during the whole recording time. In another set of experiments, calcium green was used to determine [Ca2+]i of HT22 cells or primary hippocampal neurons cultured in 96-well plates, and fluorescence intensity was quantified using a fluorescence plate reader (Infinite F200; Tecan, Salzburg, Austria). Dye loading and pretreatment with inhibitors was performed as mentioned above. Fluorescence was excited at 485 nm, and the emitted light was detected at 535 nm. The maximum increase of calcium green fluorescence during a time window of 5 min after the start of the stimulation was normalized to the fluorescence intensity prior to stimulation ((F/F0)max). Glutamate Measurement—Release of l-glutamate from HT22 cells in response to tPA stimulation was examined using an Amplex Red kit (Molecular Probes, Leiden, Netherlands). Cells were cultured in 96-well plates at a density of 16,000 cells/well. 24 h after seeding, culture medium was changed to serum-free medium for another 24 h. Afterward, HT22 cells were incubated for 1 or 5 min with 40 μg/ml tPA in Hanks' balanced saline solution with Ca2+, Mg2+ and glucose. 50-μl samples were used in the assay according to the manufacturer's instructions. The resulting fluorescence was measured using a fluorescence microplate reader (Infinite F200; Tecan). Fluorescence was excited at 540 nm, and the emission was detected at 580 nm. Reverse Transcription-PCR—Total RNA was isolated from either HT22 cells or primary hippocampal neurons (RNeasy minikit; Qiagen). Reverse transcription-PCRs (One-Step reverse transcription-PCR kit; Qiagen) were carried out in 50 μl of reaction solution containing 500 ng of total RNA, 0.4 mm deoxynucleotides, 0.6 μm forward and reverse primer, 5 units of RNase inhibitor, and 2 μl of enzyme mixture. The reaction solution was incubated at 50 °C for 30 min and denatured at 94 °C for 15 min, followed by 45 cycles at 94 °C for 1 min, 53 °C for 30 s, and 72 °C for 2 min. The final extension step was at 72 °C for 10 min. The primers were as follows: forward primer for NMDA receptor subunit 1 (mouse, gi: 6680094) was 5′-AGT GCT GTT ATG GCT TCT GC-3′ (exon 10, positions 1451–1470); reverse primer was 5′-TCG GCC AAA GGG ACT GAA GC-3′ (exon 13, positions 1857–1838). The amplified products (20 μl) were separated by 2% agarose gel and visualized by ethidium bromide staining. Data Analysis—Results were expressed as mean values ± S.E. Statistically significant effects of the calcium-imaging experiments were assessed by two-way analyses of variance followed by post hoc Tukey test for multiple comparisons, and a value of p < 0.05 was considered to indicate statistical significance of the results. Specific Activation of Erk1/2 Kinases by tPA in Neuronal Cells—Previous studies from Medina et al. (11Medina M.G. Ledesma M.D. Dominguez J.E. Medina M. Zafra D. Alameda F. Dotti C.G. Navarro P. EMBO J. 2005; 24: 1706-1716Crossref PubMed Scopus (95) Google Scholar) and Nicole et al. (10Nicole O. Docagne F. Ali C. Margaill I. Carmeliet P. MacKenzie E.T. Vivien D. Buisson A. Nat. Med. 2001; 7: 59-64Crossref PubMed Scopus (621) Google Scholar) have demonstrated that tPA stimulates the MAPK pathway and NMDA receptor-mediated calcium influx into N2a cells and hippocampal neurons. Although both groups postulated a tPA-mediated activation of the NMDA receptor, they proposed different mechanisms of tPA-mediated downstream signaling. Therefore, we set out to determine the tPA-mediated effects using two related model systems: the mouse neuronal hippocampus-derived cell line HT22 (17Davis J.B. Maher P. Brain Res. 1994; 652: 169-173Crossref PubMed Scopus (302) Google Scholar) and rat primary hippocampal neurons. As illustrated in Fig. 1, tPA (Actilyse) is able to induce the phosphorylation of Erk1/2 without altering the total amount of the protein, both in primary hippocampal cells (Fig. 1A) and HT22 cells (Fig. 1B). The amount of Actilyse used in our experiments corresponds to 10 μg/ml pure synthetic tPA (data not shown). Maximal Erk1/2 activation in the two cell systems was observed between 30 min and 1 h, respectively, after the start of stimulation. Shorter stimulations starting from 2 to 15 min did not induce a significant increase in phospho-Erk1/2 in our cell systems and were hence thought to be inappropriate for further studies (data not shown). To further examine the pathway leading to the activation of Erk1/2, we preincubated the cells for 15 min with U0126, an inhibitor of the upstream MAPK kinase (MEK1/2). Subsequent tPA treatment resulted in no Erk1/2 phosphorylation compared with the normal tPA-treated cells, indicating that MEK1/2 is directly responsible for this effect in HT22 cells (Fig. 1C). RAP, an Antagonist for Lipoprotein Receptor Ligand Binding Blocks tPA-mediated Erk1/2 Activation—It has been previously suggested that LRP1 mediates tPA-induced Erk1/2 phosphorylation (6Hu K. Yang J. Tanaka S. Gonias S.L. Mars W.M. Liu Y. J. Biol. Chem. 2006; 281: 2120-2127Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). To verify the involvement of LRP1 in tPA-mediated signaling, we incubated neuronal cells with RAP, thereby inhibiting all ligands from binding to lipoprotein receptors. Preincubation of 500 nm RAP-GST 1 h prior to tPA stimulation resulted in a pronounced decrease of Erk1/2 phosphorylation compared with total Erk1/2 protein in HT22 cells (Fig. 2A, upper bands). Similar effects were detected for primary neurons (Fig. 2B). To investigate a possible proteolytic influence of tPA on LRP1, we measured the protein level of LRP1 β-chain (85 kDa) and C-terminal fragments (∼18 kDa) using the anti-LRP1 polyclonal 1704 antibody (16Pietrzik C.U. Busse T. Merriam D.E. Weggen S. Koo E.H. EMBO J. 2002; 21: 5691-5700Crossref PubMed Scopus (176) Google Scholar). As shown in Fig. 2A (lower bands), tPA treatment did not alter LRP1 protein expression or processing, since no differences in LRP1 β-chain or LRP1 C-terminal fragment protein levels could be detected in tPA-treated compared with untreated control cells. Specific Knockdown of LRP1 with a Short Hairpin RNA Plasmid Construct Results in Decreased Erk1/2 Phosphorylation—To address the question of whether the observed RAP effect on Erk1/2 phosphorylation is actually due to a blockage of LRP1 and not due to any other low density lipoprotein receptor family member, we employed a well documented vector-based shRNA knockdown strategy to specifically block LRP1 protein translation in HT22 cells (22Laatsch A. Ragozin S. Grewal T. Beisiegel U. Joerg H. Eur. J. Cell Biol. 2004; 83: 113-120Crossref PubMed Scopus (14) Google Scholar). As presented in Fig. 3A, Western blot analysis of the short hairpin LRP1 plasmid (shLRP1)-transfected cells confirms the down-regulation of LRP1 β-chain in contrast to the pALsh vector control after 72 h of transfection. The quantitative data on the protein knockdown (Fig. 3B) demonstrate a reduction of LRP1 β-chain by ∼70%. Subsequent tPA stimulation resulted in an almost abolished Erk1/2 phosphorylation signal in the shRNA-transfected cells (Fig. 3C). Taken together, these data indicate that LRP1 is specifically responsible for the tPA-induced Erk1/2 activation and therefore mediates the signal from the cell surface into the cell. NMDA Receptor Open Channel Blocker MK-801 Prevents tPA-induced Erk1/2 Phosphorylation—It has been proposed that tPA might transduce its signal into the cell via direct binding to the NMDA receptor on the cell surface (10Nicole O. Docagne F. Ali C. Margaill I. Carmeliet P. MacKenzie E.T. Vivien D. Buisson A. Nat. Med. 2001; 7: 59-64Crossref PubMed Scopus (621) Google Scholar, 23Fernandez-Monreal M. Lopez-Atalaya J.P. Benchenane K. Cacquevel M. Dulin F. Le Caer J.P. Rossier J. Jarrige A.C. Mackenzie E.T. Colloc'h N. Ali C. Vivien D. J. Biol. Chem. 2004; 279: 50850-50856Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 24Pawlak R. Melchor J.P. Matys T. Skrzypiec A.E. Strickland S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 443-448Crossref PubMed Scopus (121) Google Scholar). Therefore, we wanted to investigate the role of this receptor in our model system by blocking calcium influx with MK-801, a noncompetitive NMDA receptor inhibitor. Consistent with previous findings (11Medina M.G. Ledesma M.D. Dominguez J.E. Medina M. Zafra D. Alameda F. Dotti C.G. Navarro P. EMBO J. 2005; 24: 1706-1716Crossref PubMed Scopus (95) Google Scholar), tPA-induced Erk1/2 phosphorylation in primary neurons and HT22 cells was reduced after preincubation with 10 μm MK-801 (Fig. 4, A and B). Most interestingly, MK-801 reduced the tPA-mediated Erk1/2 phosphorylation in primary hippocampal neurons to similar levels as seen with RAP (Fig. 4B), suggesting that NMDA receptors and LRP1 either act in parallel or in succession in this signaling cascade. DL-AP5, a competitive NMDA receptor antagonist, corroborates the involvement of NMDA receptors in tPA-mediated Erk1/2 activation. To further study the role of the NMDA receptor, both cell types were additionally preincubated with the competitive NMDA receptor antagonist DL-AP5. As expected, the tPA-induced Erk1/2 activation was abolished both in HT22 cells (Fig. 4C) and primary neurons (Fig. 4D), further proving the involvement of NMDA receptors in LRP1-mediated tPA signaling. For control purposes, cells were coincubated with 0.2 mm NaOH and tPA to rule out any solvent effect of DL-AP5 on Erk1/2 phosphorylation. As we used the well established drug Actilyse (Boehringer Ingelheim) instead of pure synthetic tPA, we wanted to rule out any effects of possible low molecular weight contaminants like, for example, glutamate, which would directly activate NMDA receptors, or l-arginine as a prerequisite for NO generation, which could lead to stimulation of MAPK (25Kanterewicz B.I. Knapp L.T. Klann E. J. Neurochem. 1998; 70: 1009-1016Crossref PubMed Scopus (91) Google Scholar). Therefore, we dialyzed the Actilyse against double-distilled H2O with three buffer changes. The subsequent stimulation of HT22 cells with the dialyzed tPA resulted in an Erk1/2 phosphorylation comparable with that seen without dialysis (Fig. 4E). This implies that the observed Erk1/2 activation through the NMDA receptor pathway is due to tPA itself and not to any other component in the tPA solution. These results strongly suggest a pivotal role of tPA in LRP1-mediated NMDA receptor activation. The Functional Cooperation of LRP1 and NMDA Receptor Can Be Certified by Calcium-imaging Experiments—To provide further evidence for an LRP1-mediated effect on NMDA receptor activation, we measured calcium influx into the cells in response to tPA stimulation. The addition of tPA resulted in a rapid increase of [Ca2+]i in both HT22 cells (Fig. 5, A and C) and primary hippocampal neurons (Fig. 5, B and D). The pattern of the calcium signal was similar in both cell types, showing an initial peak and a subsequent plateau phase. This calcium response to tPA was abolished if the cells were pretreated with the NMDA receptor antagonist MK-801 (Fig. 5, A and B) or the LRP1 binding inhibitor RAP (Fig. 5, C and D). For statistical evaluation, we compared the maximum calcium increase after the addition of tPA. As shown in Fig. 5E, the calcium signal evoked by tPA was significantly reduced by MK-801 or RAP treatment (* or #, p < 0.05, n = 43–91 single cells from at least three individual coverslips). These experiments suggest that tPA-LRP1 signaling can activate NMDA receptors within several sec" @default.
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• W2088396961 title "The Functional Role of the Second NPXY Motif of the LRP1 β-Chain in Tissue-type Plasminogen Activator-mediated Activation of N-Methyl-D-aspartate Receptors" @default.
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