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. 2013 Sep 20;288(38):27434-43.
doi: 10.1074/jbc.M113.497214. Epub 2013 Aug 12.

Identification of the C3a Receptor (C3AR1) as the Target of the VGF-derived Peptide TLQP-21 in Rodent Cells

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Free PMC article

Identification of the C3a Receptor (C3AR1) as the Target of the VGF-derived Peptide TLQP-21 in Rodent Cells

Sebastien Hannedouche et al. J Biol Chem. .
Free PMC article

Abstract

TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation, and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This study describes the use of a series of studies that show G protein-coupled receptor-mediated biological activity of TLQP-21 signaling in CHO-K1 cells. Unbiased genome-wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible G protein-coupled receptors bringing about this activity. Further experiments using a series of defined receptor antagonists and siRNAs led to the identification of complement C3a receptor-1 (C3AR1) as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signaling to pertussis toxin is consistent with the known signaling pathway of C3AR1. The binding of TLQP-21 to C3AR1 not only has effects on signaling but also modulates cellular functions, as TLQP-21 was shown to have a role in directing migration of mouse RAW264.7 cells.

Keywords: C3AR1; Cell Migration; Complement System; G Protein-coupled Receptors (GPCRs); Gene Silencing; Metabolism; RNA-Seq; Receptor Identification; TLQP-21.

Figures

FIGURE 1.
FIGURE 1.
TLQP-21-induced calcium fluxes measured in several cell lines. A, calcium measurements of rat (● and ○) and human (■ and □) TLQP-21 without (● and ■) or with (○ and □) PTX in CHO-K1 cells after ATP priming. B, calcium measurements of positive control (▴; ATP, bradykinin, carbachol, sphingosine 1-phosphate, and lysophosphatidic acid 18:0) and rat (●) TLQP-21-triggered responses after ATP priming in CCL39 cells. C, calcium measurements of rat TLQP-21-triggered responses after ATP priming in O-342 cells. All data are represented as signal − base line/base-line signal (ΔF/F) ± S.D.
FIGURE 2.
FIGURE 2.
Multiple alignment of VGF proteins from selected species. Shown is the hamster VGF complete coding sequence aligned with human (NP_003369), rat (NP_112259), and mouse (NP_001034474) VGF proteins. The TLQP-21 sequence is indicated by a gray bar.
FIGURE 3.
FIGURE 3.
Inhibition of TLQP-21 activity by SB290157. A, calcium measurement after ATP priming of CHO-K1 cells. Shown is the concentration inhibition curve of SB290157 for 1 μm rat TLQP-21 (●) and 1 μm positive control (▴; ATP, bradykinin, carbachol, sphingosine 1-phosphate, and lysophosphatidic acid 18:0). B, concentration inhibition curve of SB290157 for 1 μm rat TLQP-21 generated by Fluo-4-based calcium measurement after ATP priming of O-342 cells. All data are represented as signal − base line/base-line signal (ΔF/F) ± S.D.
FIGURE 4.
FIGURE 4.
Down-modulation of the TLQP-21 response by selected siRNAs. Shown is the calcium response to rat TLQP-21 in CHO-K1 cells upon siRNA knockdown of the 21 selected GPCR candidates (three siRNAs each). Data are presented as a percentage of the rat TLQP-21 response in untransfected CHO-K1 cells. A number is allocated for each GPCR, which corresponds to the values in Table 1.
FIGURE 5.
FIGURE 5.
TLQP-21 activity on C3AR1. A, calcium measurement of the human C3a response in CHO-K1 cells after ATP priming in the presence (○) or absence (●) of PTX. B, calcium measurement of the response to human C3a in HEK293 cells expressing hamster C3AR1 after ATP priming. C, calcium measurement in response to rat TLQP-21 (●) and human TLQP-21 (■) in HEK293 cells expressing hamster C3AR1 after ATP priming. D, calcium measurement in response to human C3a in HEK293 cells expressing rat C3AR1 after ATP priming. E, calcium measurement in response to rat TLQP-21 in HEK293 cells expressing rat C3AR1 after ATP priming. All data are represented as signal − base line/base-line signal (ΔF/F) ± S.D.
FIGURE 6.
FIGURE 6.
TLQP-21 binding studies. A, saturation plot of human 125I-labeled C3a using CHO-K1 membranes. ▵, total; □, nonspecific; ●, specific. B, saturation plot of human 125I-labeled C3a using membranes of HEK293 cells expressing hamster C3AR1. ▵, total; □, nonspecific; ●, specific. C, inhibition-binding isotherms of 125I-labeled C3a in CHO-K1 membrane preparations. D, inhibition-binding isotherms of 125I-labeled C3a in hamster C3AR1-expressing HEK293 membrane preparations. ▴, human C3a; ●, rat TLQP-21; ■, human TLQP-21. Data are represented as means ± S.D.
FIGURE 7.
FIGURE 7.
Effect of TLQP-21 on RAW264.7 cell migration. A, migration of RAW264.7 cells induced by increasing concentrations of human C3a. B, migration of RAW264.7 cells induced by increasing concentrations of rat TLQP-21. C, concentration-dependent inhibition of rat TLQP-21-induced migration by the C3AR1 antagonist SB290157. y axis values correspond to the slope/h.

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