A blocking peptide stabilizes lysophosphatidic acid receptor 1 and promotes lysophosphatidic acid-induced cellular responses

Abstract

G protein-coupled receptors regulate a variety of cellular responses and have been considered as therapeutic targets for human diseases. Lysophosphatidic acid receptor 1 (LPA1) is a receptor for bioactive lysophospholipid, LPA. LPA/LPA1-mediated signaling contributes to inflammatory and fibrotic responses in lung diseases; thus understanding regulation of LPA1 stability is important for modulating LPA/LPA1 signaling. Our previous study has shown that LPA1 is degraded in the Nedd4 like (Nedd4L) E3 ubiquitin ligase-mediated ubiquitin-proteasome system. In the current study, we attempt to identify a peptide that stabilizes LPA1 through disrupting LPA1 association with Nedd4L. LPA treatment induces both endogenous and overexpressed LPA1 degradation, which is attenuated by a proteasome inhibitor, suggesting that LPA1 is degraded in the proteasome. LPA increases phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and I-κB kinase in lung epithelial cells, and this effect is promoted by overexpression of a peptide (P1) that mimics C-terminal of LPA1. P1, not a control peptide, attenuates LPA-induced LPA1 ubiquitination and degradation, suggesting that P1 stabilizes LPA1. Further, P1 diminishes Nedd4L-mediated degradation of LPA1 and Nedd4L/LPA1 association. In addition to increasing LPA1 signaling, P1 enhances LPA-induced cell migration and gene expression of Elafin, matrix metallopeptidase 1, and serpin family B member 2 in lung epithelial cells. These data suggest that disruption of LPA1 interaction with Nedd4L by P1 increases LPA1 stability and LPA/LPA1 signaling.

Keywords: E3 ubiquitin ligase; GPCR; LPA1; blocking peptide; degradation; ubiquitination.

FIGURE 1

Lysophosphatidic acid receptor 1 (LPA1) is not stable and is degraded in the ubiquitin-proteasome system in MLE12 cells. (A) MLE12 cells were transfected with LPA1-V5 or LPA1-myc plasmid for 48 h. Cells were then treated with LPA (1 μM) for 0–2 h. LPA1-V5, LPA1-myc, and β-actin levels were examined by immunoblotting. (B) MLE12 cells were treated with MG-132 (20 μM) or leupeptin (100 μM) for 1 h, and then cells were treated with LPA (1 μM) for 0, 0.5, 1, and 2 h. LPA1 and β-actin levels were examined by immunoblotting. LPA1 intensities were analyzed by ImageJ. n = 3, *p < .05, compared to 0 h; **p < .01, compared to 0 h. Shown are representative blots from three independent experiments

FIGURE 2

Peptide P1 promotes LPA-induced phosphorylation of Erk1/2 and I-κB kinase (IKK) in MLE12 cells. MLE12 cells were transfected with P1 plasmid for 48 h, and then cells were treated with LPA (1 μM) for additional 10 min. Phospho-Erk1/2, Erk1/2, p-IKK, and β-actin levels were examined by immunoblotting. Blots were analyzed with ImageJ. n = 3, *p < .05, compared to Veh; **p < .01, compared to Veh. Shown are representative blots from three independent experiments. LPA, lysophosphatidic acid receptor

FIGURE 3

Peptide P1 increases LPA1 stability through mitigating its ubiquitination. (A) MLE12 cells were transfected with P1 or Control peptide (Contp) plasmid for 48 h, and then cells were treated with cycloheximide (CHX, 40 μg/ml) for 2 h. LPA1-V5 and β-actin levels were examined by immunoblotting. LPA1-V5 levels were analyzed by ImageJ. n = 3, **p < .01, compared to untreated cells. Shown are representative blots from three independent experiments. (B) MLE12 cells were transfected with P1 or Control peptide (Contp) plasmid for 48 h, and then cells were collected and subjected to in vivo ubiquitination assay with a modified co-immunoprecipitation protocol. Input lysates were analyzed by immunoblotting with a LPA1 antibody. Shown are representative blots from two independent experiments. IP, immunoprecipitation; LPA, lysophosphatidic acid receptor

FIGURE 4

Peptide P1 attenuates Nedd4L-mediated LPA1 degradation and disrupts LPA1/Nedd4L association. (A) MLE12 cells were transfected with LPA1-V5, Nedd4L-V5, P1, or Contp plasmids for 48 h. LPA1-V5, Nedd4L-V5, and β-actin levels were examined by immunoblotting. LPA1-V5 levels were analyzed by ImageJ. n = 3, **p < .01, compared to untreated cells. Shown are representative blots from three independent experiments. (B) MLE12 cells were transfected with P1 or Control peptide (Contp) plasmid for 48 h, and then cell lysates were subjected to immunoprecipitation with a LPA1 antibody, followed by Nedd4L immunoblotting. Input lysates were analyzed by immunoblotting with Nedd4L and β-actin antibodies. Shown are representative blots from two independent experiments. IP, immunoprecipitation; LPA, lysophosphatidic acid receptor

FIGURE 5

Peptide P1 promotes LPA-induced cell migration in MLE12 cells. MLE12 cells were transfected with P1 plasmid for 48 h, and then cells were scratched using a pipette tip, followed by LPA (1 μM) treatment. Images at 0 and 24 h were taken by microscope. Cell migration was calculated and normalized to untreated cells. n = 6, **p < .01, compared to cells treated with LPA alone. LPA, lysophosphatidic acid receptor

FIGURE 6

Peptide P1 promotes LPA-induced gene expression in MLE12 cells. HBEpCs were transfected with P1 plasmid for 48 h, and then cells were treated with LPA (1 μM) for additional 6 h. Total RNA was extracted and mRNA levels of (A) Elafin, (B) MMP1, (C) and Serpin B2 were analyzed by real-time PCR. n = 2. HBEpC, human bronchial epithelial cell; LPA, lysophosphatidic acid receptor; mRNA, messenger RNA; PCR, polymerase chain reaction

FIGURE 7

Peptide P1 stabilizes LPA1 and promotes LPA/LPA1 signaling and cellular responses through disruption of LPA1/Nedd4L association. LPA1, lysophosphatidic acid receptor 1