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
View: Text | PDF
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

×

Figure 6

P3 activates ERK1/2 and promotes neurite outgrowth in PC12 cells by a mechanism that requires the NMDA-R and LRP1.

Options: View larger image (or click on image) Download as PowerPoint
P3 activates ERK1/2 and promotes neurite outgrowth in PC12 cells by a me...
(A) PC12 cells were transfected with siRNA specifically targeting Lrp1 or Grin1. Control cells were transfected with NTC siRNA. Expression of the mRNAs encoding LRP1 and the GluN1 NMDA-R subunit was determined 48 hours later by RT-qPCR (n = 4–6; mean ± SEM; 1-way ANOVA: *P < 0.05; ***P < 0.001). (B) PC12 cells were transfected with Lrp1-specific siRNA, Grin1-specific siRNA, or NTC siRNA and then treated with P3 (0.5 μM) or vehicle for 10 minutes. ERK1/2 activation (p-ERK) was determined by immunoblotting. (C) PC12 cells were transfected with Lrp1-specific siRNA, Grin1-specific siRNA, or NTC siRNA, as indicated. The cells were then treated with S-PrP (40 nM), P3 (0.5 μM), or P4 (0.5 μM) for 48 hours. Neurite outgrowth was detected by phase contrast microscopy. Representative images are shown (scale bar, 50 μm). (D) Results are summarized for the studies shown in C and for PC12 cells treated with 20 μM P3. Neurite length was determined in all the cells of ≥5 random fields per treatment, in 3 different experiments (mean ± SEM; 1-way ANOVA: ****P < 0.0001).