A soluble derivative of PrPC activates cell-signaling and regulates cell physiology through LRP1 and the NMDA receptor
2020; Elsevier BV; Volume: 295; Issue: 41 Linguagem: Inglês
10.1074/jbc.ra120.013779
ISSN1083-351X
AutoresElisabetta Mantuano, Pardis Azmoon, Michael A. Banki, Michael S. Lam, Christina J. Sigurdson, Steven L. Gonias,
Tópico(s)Nuclear Receptors and Signaling
ResumoCellular prion protein (PrPC) is a widely expressed glycosylphosphatidylinositol-anchored membrane protein. Scrapie prion protein is a misfolded and aggregated form of PrPC responsible for prion-induced neurodegenerative diseases. Understanding the function of the nonpathogenic PrPC monomer is an important objective. PrPC may be shed from the cell surface to generate soluble derivatives. Herein, we studied a recombinant derivative of PrPC (soluble cellular prion protein, S-PrP) that corresponds closely in sequence to a soluble form of PrPC shed from the cell surface by proteases in the A Disintegrin And Metalloprotease (ADAM) family. S-PrP activated cell-signaling in PC12 and N2a cells. TrkA was transactivated by Src family kinases and extracellular signal–regulated kinase 1/2 was activated downstream of Trk receptors. These cell-signaling events were dependent on the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1), which functioned as a cell-signaling receptor system in lipid rafts. Membrane-anchored PrPC and neural cell adhesion molecule were not required for S-PrP–initiated cell-signaling. S-PrP promoted PC12 cell neurite outgrowth. This response required the NMDA-R, LRP1, Src family kinases, and Trk receptors. In Schwann cells, S-PrP interacted with the LRP1/NMDA-R system to activate extracellular signal–regulated kinase 1/2 and promote cell migration. The effects of S-PrP on PC12 cell neurite outgrowth and Schwann cell migration were similar to those caused by other proteins that engage the LRP1/NMDA-R system, including activated α2-macroglobulin and tissue-type plasminogen activator. Collectively, these results demonstrate that shed forms of PrPC may exhibit important biological activities in the central nervous system and the peripheral nervous system by serving as ligands for the LRP1/NMDA-R system. Cellular prion protein (PrPC) is a widely expressed glycosylphosphatidylinositol-anchored membrane protein. Scrapie prion protein is a misfolded and aggregated form of PrPC responsible for prion-induced neurodegenerative diseases. Understanding the function of the nonpathogenic PrPC monomer is an important objective. PrPC may be shed from the cell surface to generate soluble derivatives. Herein, we studied a recombinant derivative of PrPC (soluble cellular prion protein, S-PrP) that corresponds closely in sequence to a soluble form of PrPC shed from the cell surface by proteases in the A Disintegrin And Metalloprotease (ADAM) family. S-PrP activated cell-signaling in PC12 and N2a cells. TrkA was transactivated by Src family kinases and extracellular signal–regulated kinase 1/2 was activated downstream of Trk receptors. These cell-signaling events were dependent on the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1), which functioned as a cell-signaling receptor system in lipid rafts. Membrane-anchored PrPC and neural cell adhesion molecule were not required for S-PrP–initiated cell-signaling. S-PrP promoted PC12 cell neurite outgrowth. This response required the NMDA-R, LRP1, Src family kinases, and Trk receptors. In Schwann cells, S-PrP interacted with the LRP1/NMDA-R system to activate extracellular signal–regulated kinase 1/2 and promote cell migration. The effects of S-PrP on PC12 cell neurite outgrowth and Schwann cell migration were similar to those caused by other proteins that engage the LRP1/NMDA-R system, including activated α2-macroglobulin and tissue-type plasminogen activator. Collectively, these results demonstrate that shed forms of PrPC may exhibit important biological activities in the central nervous system and the peripheral nervous system by serving as ligands for the LRP1/NMDA-R system. Host-encoded cellular prion protein (PrPC) is a widely expressed glycosylphosphatidylinositol (GPI)-anchored membrane protein (1Prusiner S.B. Prions.Proc. Natl. Acad. Sci. U. S. A. 1998; 95 (9811807): 13363-1338310.1073/pnas.95.23.13363Crossref PubMed Scopus (5130) Google Scholar, 2Collinge J. Mammalian prions and their wider relevance in neurodegenerative diseases.Nature. 2016; 539 (27830781): 217-22610.1038/nature20415Crossref PubMed Scopus (138) Google Scholar). In prion diseases, scrapie prion protein (PrPSc), a misfolded conformer of PrPC, functions as an infectious agent, recruiting PrPC monomers into multimolecular assemblies, which accumulate to high levels in the central nervous system (CNS). 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Turner A.J. Hooper N.M. Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein.J. Biol. Chem. 2009; 284 (19564338): 22590-2260010.1074/jbc.M109.032599Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). We demonstrate that S-PrP activates ERK1/2 by a pathway that requires LRP1, the NMDA-R, SFKs, and Trk receptors. Membrane-anchored PrPC and NCAM were not required. S-PrP induced neurite outgrowth in PC12 cells and SC migration. Our results support a model in which the LRP1/NMDA-R signaling receptor complex is capable of mediating biological activities of soluble PrPC derivatives and possibly membrane-anchored PrPC expressed by adjacent cells. A recombinant derivative of PrPC (S-PrP), corresponding roughly in sequence (residues 23–231) to the PrPC ectodomain fragment shed by ADAM10 (8Taylor D.R. Parkin E.T. Cocklin S.L. Ault J.R. Ashcroft A.E. Turner A.J. Hooper N.M. 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Recombinant scrapie-like prion protein of 106 amino acids is soluble.Proc. Natl. Acad. Sci. U. S. A. 1996; 93 (8986833): 15457-1546210.1073/pnas.93.26.15457Crossref PubMed Scopus (175) Google Scholar). Neuron-like PC12 pheochromocytoma cells were treated with S-PrP at increasing concentrations for 10 min. ERK1/2 phosphorylation was determined by immunoblot analysis and evident in cells treated with PrPC at concentrations of 30 nm or higher (Fig. 1C). To confirm that the effects of S-PrP on ERK1/2 phosphorylation were not due to endotoxin contamination, S-PrP was boiled at 100 °C for 5 min before addition to PC12 cell cultures. This procedure inactivates most proteins but fails to affect the bioactivity of endotoxin (29Brifault C. Gilder A.S. Laudati E. Banki M. Gonias S.L. Shedding of membrane-associated LDL receptor-related protein-1 from microglia amplifies and sustains neuroinflammation.J. Biol. 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POM3 recognizes an epitope C-terminal to the POM2 epitope and near the center of PrPC. POM1 and POM19 recognize epitopes in the C-terminal globular region of PrPC (30Polymenidou M. Moos R. Scott M. Sigurdson C. Shi Y. Yajima B. Hafner-Bratkovič I. Jerala R. Hornemann S. Wuthrich K. Bellon A. Vey M. Garen G. James M.N.G. Kav N. et al.The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes.PLoS ONE. 2008; 3 (19060956): e387210.1371/journal.pone.0003872Crossref PubMed Scopus (134) Google Scholar). PC12 cells were treated with 40 nm S-PrP in the presence of each antibody (10 µg/ml) for 10 min. Only POM2 blocked ERK1/2 phosphorylation in response to S-PrP (Fig. 1E). The activity of POM2 confirms that the effects of S-PrP are not due to a contaminant such as endotoxin. Furthermore, the selective activity of POM2 suggests that the N-terminal "nonglobular" region of S-PrP is most likely involved in triggering signal transduction. We showed previously that LRP1 localizes transiently to lipid rafts (31Wu L. Gonias S.L. The low-density lipoprotein receptor-related protein-1 associates transiently with lipid rafts.J. Cell. Biochem. 2005; 96 (16149055): 1021-103310.1002/jcb.20596Crossref PubMed Scopus (50) Google Scholar) and that this subpopulation of LRP1 is essential for LRP1-initiated cell-signaling (23Laudati E. Gilder A.S. Lam M.S. Misasi R. Sorice M. Gonias S.L. Mantuano E. The activities of LDL receptor-related protein-1 (LRP1) compartmentalize into distinct plasma membrane microdomains.Mol. Cell. Neurosci. 2016; 76 (27565578): 42-5110.1016/j.mcn.2016.08.006Crossref PubMed Scopus (15) Google Scholar). Because LRP1 and membrane-anchored PrPC apparently function as co-receptors to trigger cell-signaling events initiated by the LRP1 ligand, tPA (27Fernández-Monreal M. López-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. Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-d-aspartate receptor signaling.J. Biol. Chem. 2004; 279 (15448144): 50850-5085610.1074/jbc.M407069200Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), we tested whether LRP1 is necessary for S-PrP-initiated cell-signaling. To begin, we treated PC12 cells with S-PrP together with receptor-associated protein (RAP), which binds to LRP1 and inhibits binding of all other known ligands that activate signal transduction (16Gonias S.L. Campana W.M. LDL receptor-related protein-1: a regulator of inflammation in atherosclerosis, cancer, and injury to the nervous system.Am. J. Pathol. 2014; 184 (24128688): 18-2710.1016/j.ajpath.2013.08.029Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 22Mantuano E. Lam M.S. Gonias S.L. LRP1 assembles unique co-receptor systems to initiate cell signaling in response to tissue-type plasminogen activator and myelin-associated glycoprotein.J. Biol. Chem. 2013; 288 (24129569): 34009-3401810.1074/jbc.M113.509133Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 32Williams S.E. Ashcom J.D. Argraves W.S. Strickland D.K. A novel mechanism for controlling the activity of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Multiple regulatory sites for 39-kDa receptor-associated protein.J. Biol. Chem. 1992; 267 (1374383): 9035-9040Abstract Full Text PDF PubMed Google Scholar). In the absence of RAP, 40 nm S-PrP induced ERK1/2 phosphorylation in PC12 cells; however, this response was entirely blocked by 150 nm RAP (Fig. 2A). ERK1/2 phosphorylation in response to S-PrP also was blocked by methyl-β-cyclodextrin (MβCD), a cholesterol sequestration reagent known to disrupt lipid rafts (33Zidovetzki R. Levitan I. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies.Biochim. Biophys. Acta. 2007; 1768 (17493580): 1311-132410.1016/j.bbamem.2007.03.026Crossref PubMed Scopus (812) Google Scholar). These results suggest that lipid raft-associated LRP1 is involved in S-PrP–initiated cell-signaling. To further test whether intact lipid rafts are required for S-PrP-initiated cell-signaling, we pretreated PC12 cells with fumonisin B1 (FM, 25 µm) for 24 h. FM blocks synthesis of sphingolipids that are key components of lipid rafts (34Merrill A.H. van Echten G. Wang E. Sandhoff K. Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ.J. Biol. Chem. 1993; 268 (8262970): 27299-27306Abstract Full Text PDF PubMed Google Scholar). Fig. 2B shows that FM blocked ERK1/2 phosphorylation in response to enzymatically inactive (EI) tPA, as previously demonstrated (23Laudati E. Gilder A.S. Lam M.S. Misasi R. Sorice M. Gonias S.L. Mantuano E. The activities of LDL receptor-related protein-1 (LRP1) compartmentalize into distinct plasma membrane microdomains.Mol. Cell. Neurosci. 2016; 76 (27565578): 42-5110.1016/j.mcn.2016.08.006Crossref PubMed Scopus (15) Google Scholar), and in response to S-PrP. Similar results were obtained when we studied mouse N2a neuroblastoma cells (Fig. 2C). ERK1/2 was phosphorylated in N2a cells treated with 40 nm S-PrP. The response to S-PrP was blocked by 150 nm RAP and by pre-treating the cells with MβCD. Thus, S-PrP activates cell-signaling in two distinct neuron-like cell lines via a pathway that is inhibited by RAP and by disrupting lipid rafts. In addition to LRP1, RAP blocks binding of ligands to other receptors in the LDL receptor gene family (35Strickland D.K. Gonias S.L. Argraves W.S. Diverse roles for the LDL receptor family.Trends Endocrinol. Metab. 2002; 13 (11854021): 66-7410.1016/S1043-2760(01)00526-4Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). To confirm the importance of LRP1 in S-PrP-activated signaling, we silenced LRP1 gene expression in PC12 cells with siRNA. Fig. 2D shows that LRP1 mRNA expression was significantly decreased in cells transfected with LRP1-specific siRNA compared with cells transfected with nontargeting control (NTC) siRNA. Fig. 2E shows that LRP1 gene-silencing blocked activation of ERK1/2 in response to S-PrP. MβCD inhibited ERK1/2 phosphorylation in cells transfected with LRP1-specific or NTC siRNA. LRP1 activates cell-signaling in response to diverse ligands by functioning as part of a system of receptors, including the NMDA-R, which plays an essential role (21Martin A.M. Kuhlmann C. Trossbach S. Jaeger S. Waldron E. Roebroek A. Luhmann H.J. Laatsch A. Weggen S. Lessmann V. Pietrzik C.U. 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.J. Biol. Chem. 2008; 283 (18321860): 12004-1201310.1074/jbc.M707607200Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 22Mantuano E. Lam M.S. Gonias S.L. LRP1 assembles unique co-receptor systems to initiate cell signaling in response to tissue-type plasminogen activator and myelin-associated glycoprotein.J. Biol. Chem. 2013; 288 (24129569): 34009-3401810.1074/jbc.M113.509133Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 36Sheng Z. Prorok M. Brown B.E. Castellino F.J. N-methyl-d-aspartate receptor inhibition by an apolipoprotein E-derived peptide relies on low-density lipoprotein receptor-associated protein.Neuropharmacology. 2008; 55 (18602124): 204-21410.1016/j.neuropharm.2008.05.016Crossref PubMed Scopus (26) Google Scholar). To test whether S-PrP–activated cell-signaling requires the NMDA-R, we treated PC12 cells with dizocilpine (MK8
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