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

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.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 (1). Via a receptor-recycling pathway, LRP1 is responsible for the endocytosis of more than 30 different extracellular ligands (2, 3). In neurons, where it is highly expressed and predominantly localized in neuronal cell bodies and dendritic processes (4), LRP1 is a major receptor for apoE/lipoprotein-containing particles and tissue-type plasminogen activator (tPA) 2 (5). 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 (6). 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 (7-9). 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 (5). Further evidence supporting the hypothesis of an involvement in synaptic plasticity results from neuron-specific LRP1 knock-out mice, which...

NR2D-containing NMDA receptors mediate tissue plasminogen activator-promoted neuronal excitotoxicity

“…This data did not indicate an involvement of LRP in this mechanism. A recent report 6 suggests that tPA could promote NMDAR signalling through an earlier recruitment of LPR-1 and a possible post-synaptic density-95 (PSD-95)-dependent interaction of tPA bound-LRP-1 with the NR1 subunit of the NMDAR. Surprisingly, although the tPA-induced potentiation of NMDAR signalling was extensively reported, neither co-application of tPA and NMDA were performed nor were neurotoxicity assays performed by these authors.…”

“…12 The intimate link between tPA and NMDAR is reinforced by other observations, such as the modulation of the phosphorylation state of the NR2B subunit of the NMDAR in conditions of chronic alcohol consumption and contextual fear conditioning 13,14 or the promotion of NMDAR-dependent extracellular signal-regulated kinase (Erk( 1 2 )) signalling in hippocampal neurons. 6,15 Thus, there is now accumulating evidence that tPA must be considered as a positive neuromodulator of NMDAR-mediated glutamatergic transmission. 11,16 NMDAR have a crucial function in brain development, plasticity and survival.…”

“…1,[10][11][12] These interactions mediate several potentially damaging effects of tPA, including potentiation of NMDAR-mediated signalling and excitotoxicity. 6,7,10 During excitotoxic conditions, tPA has been shown to promote NMDA-induced calcium influx in cortical neurons and subsequent neuronal death through the binding to and cleavage of the NR1 subunit of the NMDAR, either directly 10,11 or through the recruitment of LRP. 7 The interaction between tPA and the NR1 subunit was shown in vivo to be involved in both excitotoxic and memory paradigms in mice.…”

“…[1][2][3] However, tPA not only activates plasminogen, but rather acts through several modalities 4 by interacting with the low-density lipoproteinrelated receptor protein (LRP), [5][6][7][8] annexin-II 9 or N-methyl-Daspartate receptors (NMDAR). 1,[10][11][12] These interactions mediate several potentially damaging effects of tPA, including potentiation of NMDAR-mediated signalling and excitotoxicity.…”

The NMDA receptor functions independently and as an LRP1 co-receptor to promote Schwann cell survival and migration

“…In post-synaptic densities in neurons, the NMDA-R is physically and functionally associated with receptors in the low-density lipoprotein (LDL) receptor family, including LDL-receptor-related protein-1 (LRP1) and ApoER2 (also known as LRP8) May et al, 2004;Hoe et al, 2006;Martin et al, 2008). Based on its interaction with LRP1, the NMDA-R functions as an essential signaling co-receptor for LRP1 ligands, including α 2 -macroglobulin (α 2 M), tissue-type plasminogen activator (tPA) and apolipoprotein E (Bacskai et al, 2000;Qiu et al, 2002;Samson et al, 2008;Martin et al, 2008;Sheng et al, 2008;Mantuano et al, 2013).…”