An LRP1-binding motif in cellular prion protein replicates cell-signaling activities of the full-length protein
Elisabetta Mantuano, Carlotta Zampieri, Pardis Azmoon, Cory B. Gunner, Kyle R. Heye, Steven L. Gonias
Elisabetta Mantuano, Carlotta Zampieri, Pardis Azmoon, Cory B. Gunner, Kyle R. Heye, Steven L. Gonias
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Research Article Cell biology Inflammation

An LRP1-binding motif in cellular prion protein replicates cell-signaling activities of the full-length protein

Abstract

Low-density lipoprotein receptor-related protein-1 (LRP1) functions as a receptor for nonpathogenic cellular prion protein (PrPC), which is released from cells by ADAM (a disintegrin and metalloproteinase domain) proteases or in extracellular vesicles. This interaction activates cell signaling and attenuates inflammatory responses. We screened 14-mer PrPC-derived peptides and identified a putative LRP1 recognition motif in the PrPC sequence spanning residues 98–111. A synthetic peptide (P3) corresponding to this region replicated the cell-signaling and biological activities of full-length shed PrPC. P3 blocked LPS-elicited cytokine expression in macrophages and microglia and rescued the heightened sensitivity to LPS in mice in which the PrPC gene (Prnp) had been deleted. P3 activated ERK1/2 and induced neurite outgrowth in PC12 cells. The response to P3 required LRP1 and the NMDA receptor and was blocked by the PrPC-specific antibody, POM2. P3 has Lys residues, which are typically necessary for LRP1 binding. Converting Lys100 and Lys103 into Ala eliminated the activity of P3, suggesting that these residues are essential in the LRP1-binding motif. A P3 derivative in which Lys105 and Lys109 were converted into Ala retained activity. We conclude that the biological activities of shed PrPC, attributed to interaction with LRP1, are retained in synthetic peptides, which may be templates for therapeutics development.

Authors

Elisabetta Mantuano, Carlotta Zampieri, Pardis Azmoon, Cory B. Gunner, Kyle R. Heye, Steven L. Gonias

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Figure 3

The NMDA-R is necessary for the response to P3 in macrophages.

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The NMDA-R is necessary for the response to P3 in macrophages. (A) BMDMs...
(A) BMDMs were pretreated with MK-801 (1 μM) or vehicle for 30 minutes. The cells were then treated with LPS (0.1 μg/mL), P2 (0.5 μM), or P3* (0.5 μM), for 6 hours, as indicated. RT-qPCR was performed to compare mRNA levels for Tnf and Il6 (mean ± SEM, n = 3–7, individual points are shown; 1-way ANOVA: ****P < 0.0001). (B) BMDMs were harvested from Grin1fl/fl LysM-Cre+ mice. Grin1 mRNA expression was determined by RT-qPCR and compared with that detected in BMDMs isolated from Grin1fl/fl LysM-Cre– mice (n = 3; mean ± SEM; unpaired 2-tailed t test: ****P < 0.0001). (C) Flow cytometry was performed to detect cell surface GluN1 NMDA-R subunit in BMDMs isolated from Grin1fl/fl LysM-Cre–positive and –negative (wild-type) mice. As a control, cells from LysM-Cre– mice were incubated with secondary antibody only (gray). (D) BMDMs from Grin1fl/fl LysM-Cre+ mice were treated for 6 hours with LPS (0.1 μg/mL), in the presence of S-PrP (40 nM) or increasing concentrations of P3 (1–20 μM), P4 (1–20 μM), or vehicle. RT-qPCR was performed to determine Tnf mRNA (mean ± SEM, n = 3; 1-way ANOVA: ****P < 0.0001). (E) BMDMs from Grin1fl/fl LysM-Cre+ mice were treated for 1 hour with LPS (0.1 μg/mL), in the presence of P1, P2, P3, P3*, and P4, as indicated (each at 0.5 μM). Immunoblot analysis was performed to detect phosphorylated IκBα, total IκBα, and β-actin.